Identification of Genes for Spontaneous Neuropathic Pain in Mice: Whole Genome and Candidate Gene Approaches

by

Merav Yarkoni-Abitbul

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Faculty of Dentistry University of Toronto

© Copyright by Merav Yarkoni-Abitbul 2014

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Identification of Genes for Spontaneous Neuropathic Pain in Mice: Whole Genome and Candidate Gene Approaches

Merav Yarkoni-Abitbul

Doctor of Philosophy

Faculty of Dentistry

University of Toronto

2014

Abstract

Chronic neuropathic pain (NP) affects many people worldwide; causing suffering that is difficult

to treat, incurable and not preventable. There is growing hope that pain genetics may identify

novel treatment targets. In this dissertation we report on candidate NP genes using a mouse NP

model produced by hindpaw denervation. Previous research showed that inbred A/J (A-mice)

but not C57BL6/J (B-mice) express highly variable levels of self-mutilation of the denervated

hindpaw (‘autotomy’, a behaviour related to NP). This suggested that genetic and environmental

factors interact (GXE) in controlling this variance. Using this NP model in recombinant inbred

mice, a region on chromosome-15 (‘Pain1’) was identified as harbouring autotomy gene(s).

Here we report that Csf2rb1, a gene encoding the colony stimulating factor-2β1 common

receptor of GM-CSF (granulocyte-macrophage colony stimulating factor), and interleukins 3 and

5, is a candidate autotomy gene in Pain1. Up-regulation in Csf2rb1 expression levels in the

lumbar spinal cord correlated autotomy levels in denervated A and B mice vs. their naïve or

sham groups. Csf2rb1-expressing cells were labelled immunohistologically in several CNS

structures known to process pain inputs, including spinal dorsal horn, central canal, and select

spinal white matter regions, hippocampal dentate gyrus, ventricle linings and periventricular and

arcuate hypothalamus nuclei. CSF2RB1 protein levels were increased in spinal cord and brain of

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denervated A mice expressing autotomy vs. non-autotomizing A and B mice, and vs. their

control groups (naïve and sham A and B mice). Based on cyto-morphology and co-localization

with Vimentin, but not GFAP (astrocytes), OX42 (microglia), NeuN (neurons), MAP2 (neurons),

and NG2 (oligodendrocytes) markers, Csf2rb1-expressing cells were identified as ependymal

cells/radial glia/tanycytes. Previous studies showed that C3H/HeN mice express significantly

more pain behaviour than C3H/HeJ mice in several models. This contrast has been attributed to

a mutation in Tlr4 encoding Toll-like receptor-4 in C3H/HeJ mice. We show here that

denervated C3H/HeN mice express higher autotomy levels than C3H/HeJ mice. Spinal Csf2rb1

expression levels increased significantly post-denervation in C3H/HeN but not C3H/HeJ mice.

Thus, we propose that spontaneous NP behaviour in mice is associated with up-regulated

Csf2rb1 and CSF2RB1 levels in the CNS and associated with TLR4 signalling.

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Acknowledgments

I would like to thank first and foremost my supervisor Prof. Ze’ev Seltzer for his

excellent supervision, and for guiding me every step of the way throughout my PhD program.

His scientific expertise and superb research ideas are the great outcome of this research work. I

especially thank Prof. Seltzer for allowing me to discover the fascinating combination of genetics

and neuroscience and to become a true scientific investigator. Without him this PhD

achievement would not be possible.

I thank the members of my lab for their moral support, team-work, and for assisting me in

certain parts of my research: Drs. Elaheh Soleimannejad, Shihong Zhang, and Tina Elahipanah,

and David Tichauer, and Mariam Mashregi.

I thank the members of my committee, Prof Siew-Ging Gong, Prof. Michael Salter, and

Prof. Lei Sun for all their amazing support throughout my PhD program.

I am especially thankful for Dr. Simon Beggs and Prof. Michael Salter for allowing me to

carry out my immunohistochemical experiments in their laboratory at the Hospital for Sick

Children. I thank Dr. Beggs for assisting me in all the immunohistochemical experiments, for

his thorough supervision and technical support.

I am very grateful to Dr. Natalia Kraeva (Department of Anesthesiology, Toronto

General Hospital) for her wonderful technical support, guidance and supervision in my real time

PCR experiments that I carried out in her lab.

Special thanks to Dr. Ruslan Dorfman for his scientific expertise and for expertly and

kindly guiding me throughout my PhD program.

Thank you so much to my beautiful family, my husband Yaniv, and my sons Eli, Erez

and Yagel. I could not have done this without you. You have been so supportive and so patient

with me throughout the years and I owe you the world. I love you all and I thank you for

allowing me to achieve this goal in my life.

Thank you Mom, Yaniv and Alon. Thank you for your moral support and for believing

in me. Thank you for being there every moment of the day and throughout all the difficult

moments. You really helped me make this happen! This one is for Dad!!! I know he would have

been so proud.

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Table of Contents

Abstract ii

Acknowledgments iv

Table of Contents v

List of Tables xi

List of Figures xii

List of schemes xiv

List of Appendices xv

List of abbreviations xvi

Chapter 1: Introduction and general aims 1

1.1 Chronic pain and its clinical manifestations 3

1.2 Pathophysiology of neuropathic pain 4

1.2.1 Spontaneous ectopic activity 4

1.2.2 Peripheral sensitization 6

1.2.3 Central sensitization 7

1.2.4 Sympathetically maintained pain 9

1.2.4.1 Direct coupling between the sympathetic neurons and

sensory neurons in the DRG 10

1.2.4.2 Chemically mediated coupling between the sympathetic

efferents and sensory neurons in skin 10

1.2.4.3 α -adrenoceptor-mediated super sensitivity of nociceptive

fibers 11

1.2.4.4 Nerve degeneration 11

1.3 The memory of pain and pre-emptive analgesia 12

1.4 Modeling chronic pain in animals 12

1.4.1 The Neuroma Model for spontaneous pain 14

1.4.2 Contrasting autotomy behaviour in mouse and rat strains 16

1.4.2.1 Additional mouse strains used to study autotomy behaviour 17

1.5 Mini-review of the genetics of neuropathic pain 18

1.5.1 Neuropathic pain genes in partial nerve injury models 20

1.6 Epigenetics and chronic pain 20

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1.7 Sex and gender differences in chronic pain 21

1.8 Rationale for comparing mouse to human genomes 21

1.9 Hypotheses and Aims 24

Chapter 2: Materials and methods 25

2.1 Induction and assessment of the pain phenotype (autotomy) 25

2.1.1 Mice 25

2.1.2 Surgical procedures 27

2.1.3 Phenotyping autotomy behaviour 28

2.2 Tissue acquisition 28

2.3 RNA extraction 29

2.4 Microarraying and data analysis 30

2.4.1 Study design 30

2.4.2 Agilent Two-Color Microarray Protocol 33

2.4.3 Expression array analyses 33

2.4.4 Gene interaction networks analysis 35

2.4.5 SNP variation in inbred mouse strains with known levels of

autotomy behaviour 36

2.4.6 Interrogating published Csf2rb1-immunohistochemically labelled

spinal cord and brain slices of naïve B mice 37

2.4.7 Methodological aspects of literature survey of the biological

relevance of candidate genes 37

2.5 Gene follow-up assays 37

2.5.1 Quantitative real-time PCR 37

2.5.2 Immunocytochemistry 38

2.5.3 Quantitation of CSF2RB1 labelled cells in the spinal cord and brain 40

2.5.4 Co-localization analyses 40

2.5.5 Counting pStat3 labelled cells in the spinal cord and brain 41

2.6 Statistical analysis 41

Chapter 3: Study I: Regulation of autotomy levels by gene expression changes:

Whole genome study of the DRGs and spinal cord 42

3.1 Introduction 42

3.2 Results 45

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3.2.1 Autotomy phenotypes in denervated A and B mice 45

3.2.2 Criteria for candidate gene selection 48

3.2.3 Group comparisons of genome-wide gene expression data 49

3.2.3.1 Constitutive gene expression levels of autotomy-related genes 50

3.2.3.2 Expression of DRG genes in denervated A and B mice 53

3.2.3.3 Spinal cord genes whose expression is associated with

autotomy levels 57

3.2.4 Network analysis 58

3.2.4.1 Network analysis for DRG genes in denervated A and B mice 58

3.2.4.2 Network analysis of genes expressed in the spinal cord of

denervated A and B mice 61

3.2.5 Pain1 DRG genes in denervated A and B mice 64

3.2.6 Pain1 genes associated with autotomy in spinal cord 68

3.2.7 In Situ hybridization (ISH) labelling of Csf2rb2 in the spinal cord

and brain of the mouse 70

3.2.8 Csf2rb1 gene network 76

3.2.9 SNP analysis in candidate autotomy genes – Pain1 genes in intact

A and B mice 78

3.2.10 Other candidate genes related to autotomy 82

3.2.11 Selecting the best candidate gene in Pain1 83

3.3 Discussion 85

3.3.1 Csf2rb1 as a candidate autotomy gene in Pain1 85

3.3.2 Cacng2 in Pain1 as a candidate autotomy gene 86

3.4 Conclusions 88

Chapter 4: Study II: Expression of CSF2RB1 by spinal central canal ependymal

cells/radial glia/tanycytes correlates with autotomy levels in

Mice 89

4.1 Introduction 89

4.2 Results 90

4.2.1 Autotomy behaviour in denervated A and B mice 90

4.2.2 Csf2rb1 pattern of expression correlates with autotomy behaviour

in denervated A mice 94

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4.2.3 Autotomy behaviour in denervated C3H/HeJ and AKR/J mice 97

4.2.4 Csf2rb1 expression levels in C3H/HeJ vs. AKR/J mice 101

4.2.5 CSF2RB1 protein is differentially expressed in spinal cords of A

and B mice 103

4.2.6 Evidence that CSF2RB1 is localized in ependymal cells/radial

glia/tanycytes of the central canal 110

4.2.7 CSF2RB1 in the brain is associated with high autotomy levels in

denervated A mice 112

4.2.8 CSF2RB1 in ependymal cells/radial glia/tanycytes in other brain

regions 117

4.3 Discussion 119

4.3.1 Csf2rb1 is correlated with pain levels in denervated A mice 119

4.3.2 CSF2RB1 is expressed in ependymal cells/radial glia/tanycytes

of the central canal 120

4.3.3 CSF2RB1 cells in the hippocampal dentate gyrus 123

4.3.4 CSF2RB1 cells in the hypothalamic peri-ventricular and arcuate

nuclei 124

4.3.5 Autotomy behaviour in denervated C3H/HeJ mice 125

4.4 Conclusions 125

Chapter 5: Study III: Tlr4 and pStat3 as candidate genes controlling autotomy

behaviour in mice 127

5.1 Introduction 127

5.1.1 Tlr4 127

5.1.2 Tlr4 up-regulation in pain behaviour 130

5.1.3 Tlr4 blockade reverses established neuropathic pain behaviour 133

5.1.4 C3H/HeJ vs. C3H/HeN mice 133

5.1.5 Tlr4 and Csf2rb1 in the inflammatory process 136

5.1.6 pStat3 136

5.1.7 C3H/HeJ and C3H/HeN mice in the Neuroma Model 138

5.2 Results 139

5.2.1 Autotomy behaviour in denervated C3H/HeN (Tlr4 wild-type) and

C3H/HeJ (Tlr4-deficient) mice 139

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5.2.2 Csf2rb1 expression patterns in C3H/HeN and C3H/HeJ mice 141

5.2.3 CSF2RB1+ cells in the central canal of C3H/HeN mice 144

5.2.4 Tlr4 gene regulation in A vs. B mice 144

5.2.5 Tlr4 gene polymorphisms in MHS vs. NLS mice 146

5.2.6 pStat3 in dorsal horn of A vs. B mice 147

5.2.7 pStat3 in the dentate gyrus of A vs. B mice 149

5.2.8 pStat3 in the peri-ventricular nucleus of A mice 149

5.3 Discussion 151

5.3.1 Tlr4 contributes to chronic neuropathic pain 151

5.3.2 Tlr4 gene is differentially expressed in A vs. B mice 152

5.3.3 Proposed mechanisms for Tlr4 in NP 154

5.3.4 Csf2rb1 gene expression levels in wild-type and TLR4-deficient

Mice 155

5.3.5 pStat3 expression in the spinal cord of A vs. B mice 156

5.3.6 Proposed mechanism for pStat3 and CSF2RB1 in the spinal cord 157

5.3.7 Hippocampal pStat3 expression is associated with autotomy 158

5.4 Conclusions 160

Chapter 6: General Discussion and Conclusions 161

6.1 Mechanisms involving the Colony-Stimulating Factor 2 Receptor Beta 1,

CSF2RB1 162

6.1.1 The GM-CSF cytokine and its receptors in the nervous system 162

6.2 CSF2RB1 is expressed in Ependymal cells/radial glia/tanycytes in

the CNS 167

6.2.1 CSF2RB1+ ependymal cells/radial glia/tanycytes surrounding the

spinal central canal and in spinal lamina X 168

6.2.2 Metabotropic glutamate receptor 1 alpha (mGluR1α) is expressed

in ependymal cells/tanycytes/radial glia of the spinal central canal 172

6.2.3 Endothelin B receptor is expressed in ependymal

cells/tanycytes/radial glia of the spinal central canal 172

6.3 Co-localization of CSF2RB1 and the neural stem cell marker Vimentin

in the spinal cord 173

6.4 The hippocampus in processing nociceptive input 175

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6.4.1 Processing chronic pain inputs in the hippocampus 177

6.5 Which cell types express CSF2RB1 in the hippocampus? 178

6.6 The involvement of CSF2RB1 in neuroprotection 180

6.6.1 Cytoprotection involves CSF2RB1 operating in a complex with

the erythropoietin receptor (EpoR) 180

6.6.2 The neuroprotective cytokine erythropoietin is a ligand for

CSF2RB1 180

6.7 CSF2RB1 operating with Receptor d’origine nantais (RON) 182

6.8 pStat3 in the spinal cord 184

6.10 pStat3 in the peripheral nervous system (PNS) contributing to neuronal

hyperexcitablility may be associated with CSF2RB1 in A mice 186

6.11 The pain-related genes induced by pStat3 in the PNS and CNS 187

6.12 pStat3 is expressed in the subventricular zone of the brain but not

around the spinal central canal 187

6.13 Study design considerations in gene expression studies 189

6.13.1 Pooled vs. individualized arrays 189

6.13.2 The number of arrays per group 190

6.13.3 Type of group comparisons and choice of the reference (control)

group 191

6.13.4 Arrayed neural tissues 193

6.13.5 Statistical consideration 194

6.13.6 Group comparisons 194

6.14 Limitations of the studies 195

6.15 Future direction of research 197

6.16 Conclusions 199

References 201

Appendices 238

Copyright acknowledgements 294

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List of Tables

Table 1: Strains, group types and the number of animals per group 26

Table 2: Comaprison types and genes associated with each comparison type 34

Table 3: Probes and genes with contrasting expression levels between naïve A vs.

B mice are presented 51

Table 4: Pain1 genes in lumbar DRGs showing a significant contrast in the expression

level between intact A and B mice at P<0.05 and a fold change of more than 1.5 or less

than -1.5 (listed in alphabetical order) 52

Table 5: Pain1 genes in lumbar spinal cord showing a significant contrast in the

expression level between intact A and B mice at P<0.05 and a fold change of more than

1.5 or less than -1.5 (listed in alphabetical order) 53

Table 6: Probes and genes that are regulated with autotomy behaviour in A and B mice 55

Table 7: Candidate autotomy genes in the DRGs and biological processes in which they

operate 60

Table 8: Candidate autotomy genes in the spinal cord and biological processes in

which they operate 62-3

Table 9: Pain1 genes in naïve, sham-operated and denervated A and B mice that

reacted to the treatments by changed expression levels in the DRG 65-7

Table 10: Pain1 genes in the individual 3 group comparisons that yielded in the spinal

cord (ADH vs. AS; ADH vs. BD; ADH vs. ADL) 69

Table 11: SNPs in Pain1 candidate autotomy genes regulated constitutively between

A and B mice and between other MHS (A/HeJ, C3H/HeJ, BALBc/ByJ, BALB/cJ) vs.

NLS (AKR/J, C57BL/10J) strains 81

Table 12: Candidate autotomy genes in Pain1 and their known relevance to pain 83

Table 13: Documented phenotypic differences between Tlr4 wild-type and mutant mice 135

Table 14: Tlr4 SNPs that contrast between A and B mice and are located in coding

regions (synonymous and non-synonymous SNPs), intronic and un-translated regions 147

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List of Figures

Figure 1: Study design for microarray experiment 32

Figure 2: Course of autotomy behaviour in denervated A and B mice 47

Figure 3: Average autotomy onset day of denervated A and B mice 48

Figure 4: Numbers of DRG genes throughout the genome whose expression level is

regulated by the effects of: (1) denervation in A mice expressing high autotomy vs. sham

operated A mice, (2) denervation in different strains expressing contrasting autotomy

levels and (3) the effect of different environmental controls 57

Figure 5: Number of spinal cord genes throughout the genome whose expression level is

regulated by the effects of: (1) denervation in A mice expressing high autotomy vs. sham

operated A mice, (2) denervation in different strains expressing contrasting autotomy

levels and (3) the effect of different environmental controls 58

Figure 6: Physical genetic map of Csf2rb1 and Csf2rb2 70

Figure 7: Photomicrographs of Csf2rb2 ISH in select regions of mouse brain 72

Figure 8: Photomicrographs from the Allen Brain Atlas showing a strong labelling of in situ

hybridization of Csf2rb2 in the lumbar spinal cord of a 56 weeks old intact B male mouse 73

Figure 9: Photomicrographs from the Allen Brain Atlas showing a strong labelling of

in situ hybridized reaction product to Csf2rb2 in nuclei of certain cells clustering in

specific brain nuclei and regions of an intact 56 weeks male old B mouse 74-5

Figure 10: Gene networks of Csf2rb1 76-7

Figure 11: Postoperative course of average autotomy scores in denervated, sham and

naive A and B mice 92

Figure 12: Average autotomy onset day in denervated A and B mice 93

Figure 13: Autotomy scores at onset day in denervated A and B mice 93

Figure 14: Correlation of Csf2rb1 gene expression levels with autotomy behaviour on

PO day 14 in denervated A and B mice 96

Figure 15: Csf2rb1 gene expression levels correlation with onset day of autotomy and

autotomy onset scores in denervated A and B mice 97

Figure 16: Postoperative course of average autotomy scores in C3H/HeJ and AKR/J mice 99

Figure 17: Autotomy average onset day in C3H/HeJ and AKR/J mice 100

Figure 18: Score of autotomy at onset day of denervated C3H/HeJ and AKR/J mice 100

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Figure 19: Csf2rb1 gene expression levels in the spinal cord of C3H/HeJ and AKR/J mice 102

Figure 20: Csf2rb1 gene expression levels in denervated CeH/HeJ and AKR/J mice

sub-grouped by autotomy onset day 102

Figure 21: Immunohistochemical analysis of CSF2RB1 expressing cells in the spinal

cord 106

Figure 22: CSF2RB1 expression levels in the spinal dorsal horn 107

Figure 23: The number of long CSF2RB1+ processes in the spinal cord of A and B mice 108

Figure 24: The number of long CSF2RB1+ processes extending laterally in A and B mice 109

Figure 25: Co-localization of CSF2RB1 and Vimentin in ependymal cells/tanycytes/

radial glia surrounding the central canal in control and denervated A and B mice 111

Figure 26: Photomicrographs of hippocampal dentate gyrus sections labelled immuno-

histologically for CSF2RB1, expressed in pyramidal and granule layers 114

Figure 27: Correlation of the number of CSF2RB1+ long processes per 200μm of the

dentate gyrus with autotomy behaviour 115

Figure 28: CSF2RB1 expression in the dentate gyrus 116

Figure 29: CSF2RB1 immunoreactive cells in select brain regions 117-8

Figure 30: Course of autotomy levels in denervated C3H/HeN mice carrying the

wild-type Tlr4 gene sequence and C3H/HeJ mice carrying the mutant gene 140

Figure 31: Average autotomy onset day in denervated C3H/HeN and C3H/HeJ mice 140

Figure 32: The average score of autotomy at its onset in denervated C3H/HeN and

C3H/HeJ mice 141

Figure 33: Csf2rb1 gene expression levels in denervated C3H/HeN and C3H/HeJ mice 142

Figure 34: Correlation of Csf2rb1 gene expression levels (in denervated C3H/HeN and

C3H/HeJ mice) with the onset day of autotomy and autotomy onset scores 143

Figure 35: CSF2RB1 immunostaining in the central canal of C3H/HeN mice 144

Figure 36: Tlr4 gene expression levels in DRG and spinal cord of A vs. B mice 146

Figure 37: Photomicrographs of the spinal cord of denervated and sham operated A

mice labelled immunohistologically for pStat3 148

Figure 38: Photomicrographs of the dentate gyrus of denervated and sham operated A

mice labelled immunohistologically for pStat3 150

Figure 39: Photomicrographs of the brain ventricles of denervated and sham operated A

mice labelled immunohistologically for pStat3 151

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List of Schemes

Scheme 1: A model for TLRs in peripheral nerve injury and neuropathic pain

(reprinted with permission from RightsLink, License Number 3378941306268) 129

Scheme 2: TLR4 is a potential master switch in pain regulation after nerve injury

(reprinted with permission from RightsLink, License Number 3378941061859) 131-2

Scheme 3: Normal expression of GM-CSF/ IL-3/IL-5, CSF2RA and CSF2RB1

receptor complex in the intact peripheral nervous system and spinal dorsal horn 165

Scheme 4: Expression of CSF2RB1 and CSF2RA and their ligands following

peripheral nerve injury (PNI) in the Neuroma Model 166

Scheme 5: Ependymal and neuroglial cells surrounding the central canal of the spinal

cord (Gray, 1918) 169

Scheme 6: Adapted from (Qiagen): CSF2RB intracellular signalling through Jak/Stat3,

PI3K and Src 185-6

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List of Appendices

Appendix I: Genome-wide candidate genes associated with autotomy in the DRGs 238

Appendix II: Genome-wide candidate genes associated with autotomy in the spinal cord 246

Appendix III: SNP variations in A vs. B mice, and other high- (C3H/HeJ, BALBc/cByJ),

low- (C57BL/10J, AKR/J, C58/J) and intermediate-autotomy score strains (129S1/SvImJ,

129x1/SvJ, DBA/2J) 249

Appendix IV: mRNA levels of the reference gene Hprt across treatment groups and

strains following real-time PCR 291

Appendix V: Number of CSF2RB1+ cell extensions around the central canal in naïve and

sham-operated A and B mice, and in denervated A high autotomy and low autotomy mice 292

Appendix VI: Number of CSF2RB1+ cell processes crossing the dentate gyrus per 200 μm

unit length in naïve and sham-operated A and B mice 293

Appendix VII: Number of CSF2RB1+ cells in the polymorph dentate gyrus per ROI

of 2500 μm2 of naïve and sham-operated A and B mice 293

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List of Abbreviations

A

A A/J

ABA Allen Brain Atlas

ADH Denervated A/J high-autotomy mouse

ADL Denervated A/J no/low-autotomy mouse

AFS Autotomy final score

AI Intact A/J mouse

AOS Autotomy onset score

AS Sham-operated A/J mouse

AMPA Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ANOVA Analysis of variance

ARH Hypothalamic arcuate nucleus

ATP Adenosine triphosphate

B

B C57BL6/J

BD Denervated C57BL6/J mouse

BI Intact C57BL6/J mouse

BS Sham-operated C57BL6/J mouse

BDNF Brain-derived neurotrophic factor

C

CaMKII Calmodulin-Dependent Protein Kinase II

CCI Chronic constriction injury

cDNA Complementary DNA

CGRP Calcitonin gene-related peptide

CNS Central nervous system

CREB cAMP response element-binding protein

cRNA Complementary RNA

CSF2RB1 Colony stimulating factor 2 receptor beta

CTP Cytidine 5'-triphosphate

D

DEPC Diethyl pyrocarbonate

DNA Deoxyribonucleic acid

DRG Dorsal root ganglia

E

ECM Extracellular matrix

EGF-BP Epidermal growth factor-binding protein

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ENU N-ethyl-N-nitrosourea

ERK1 Extracellular signal-regulated kinase 1

ERK2 Extracellular signal-regulated kinase 2

F

FAS Final autotomy score

G

GDNF Glial cell-derived neurotrophic factor

GFAP Glial fibrillary acidic protein

GITC Guanidine isothiocyanate

GM-CSF Granulocyte-macrophage colony stimulating factor

GPCR G protein-coupled receptors

H

Hapmap Haplotype map

Hprt1 Hypoxanthine guanine phosphoribosyl transferase 1

5HT 5-hydroxytryptamine, also known as Serotonin

I

IASP International Association for the Study of Pain

IB4 Isolectin-B4

IL-1β Interleukin 1 beta

IL-3 Interleukin 3

IL-5 Interleukin 5

IL-6 Interleukin 6

IL-3Rβ Interleukin-3 receptor beta

INF-γ Interferon gamma

i.p. Intraperitoneal

ITGAM Integrin alpha M

J

Jak Janus kinase

JNK c-Jun N-terminal kinase

M

MAP-2 Microtubule-associated protein 2

MAPK Mitogen-activated protein kinase, also known as ERK

MCP-1 Monocyte chemoattractant protein 1

MGI Mouse Genome Informatics

mGluR Metabotropic glutamate receptor

MHS-A Moderate/High scores of autotomy in A mice

xviii

MHS- C3H/HeJ Moderate/High scores of autotomy in C3H/HeJ mice

MHS- C3H/HeN Moderate/High scores of autotomy in C3H/HeN mice

MMLV-RT Moloney murine leukemia virus reverse transcriptase

MMP9 Matrix metallopeptidase 9

mRNA Messenger RNA

MSP Macrophage Stimulating Protein

N

Nav 1.8 Sodium channel, voltage-gated, type X, alpha subunit

NFκB Nuclear factor kappa B

NeuN Neuronal nuclei

NGF Nerve growth factor

NIH National Institute of Health

NLS-A No/low scores of autotomy in A mice

NLS-AKR/J No/low scores of autotomy in AKR/J mice

NLS-B No/low scores of autotomy in B mice

NLS-C3H/HeJ No/low scores of autotomy in C3H/HeJ mice

NLS-C3H/HeN No/low scores of autotomy in C3H/HeN mice

NMDA N-methyl-D-aspartate

NO Nitric Oxide

O

OD Optical density

Oligo Oligonucleotide

OX42 Anti-Integrin αM [CD11b]

P

PAG Peri-aqueductal gray

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PGE2 Prostaglandin E2

PI3K Phosphatidylinositol-3-kinase

PKA Protein kinase A

PKC Protein kinase C

PLC Phosphoinositide phospholipase C

PMPS Post-mastectomy pain syndrome

PNS Peripheral nervous system

PO Post-operative

PSL Partial sciatic nerve ligation

PVi Periventricular nucleus pars internal

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Q

qPCR Quantitative PCR

QTL Quantitative trait loci

R

RI Recombinant inbred lines

RNA Ribonucleic acid

ROI Region of Interest

RON Receptor d’origine nantais (macrophage stimulating 1 receptor)

ROS Reactive Oxygen Species

rRNA Ribosomal RNA

RT Reverse transcriptase

RT-PCR Real time PCR

S

siRNA Small interference RNA

SMP Sympathetically maintained pain

SNL Spinal nerve ligation

SNP Single nucleotide polymorphism

Stat Signal-transducer and activator of transcription protein

STK Stem-cell derived Tyrosine Kinase

T

TNFα Tumor necrosis factor α

TRAF6 TNF receptor-associated factor 6

TRPV1 Transient receptor potential cation channel, subfamily V, member 1

U

UTR Un-translated region

UV Ultraviolet

V

V3 Third ventricle

VMAB Ventromedial afferent bundle

VTA Ventral tegmental area

Chapter 1 Introduction and general aims

Chronic neuropathic pain is a complex disease that follows nerve damage and affects nearly 5%

of the human population with no effective cure, causing suffering and emotional, motor, mental,

sleep dysfunctions and social participation problems to the pain patients and their families. It

also involves major costs to the economy. Over the past two decades there has been a substantial

advancement in our understanding of neuropathic pain. For example, we now know that a

genetic pre-disposition to develop neuropathic pain exists in humans and animals alike, and that

certain individuals will suffer much more pain than others, following the same inciting injury,

due to a genetic pre-disposition interacting with environmental risk factors. In this respect,

phantom and spontaneous pains are felt in certain limb amputees and in some women following

breast mastectomies and in individuals who lose an organ such as an eye, tooth, uterus, etc.

Likewise, spontaneous pain in certain animal rodent models is an outcome of total and partial

nerve transections, and its extent depends on their genetic background, interacting with the

environment.

The purpose of this study was to identify genes that are responsible for initiating and/or

maintaining spontaneous neuropathic pain in an animal model, the Neuroma Model (Wall et al.,

1979) that mimics the pathophysiology of human leg amputees. In this model, total hind paw

denervation is performed in inbred mice of certain genetic backgrounds, and pain is assessed by

self-mutilation behaviour (‘autotomy’). The study used a whole genome approach to interrogate

differential gene expression levels following hind paw denervation that triggers autotomy

behaviour in a few neural structures known to be involved in pain processing, i.e., ipsilateral

lumbosacral dorsal root ganglia (DRGs) associated with the injured nerves and the lumbar spinal

cord where these nerves terminate and synapse with 2nd

-order neurons (Chapter 3). Both the

DRGs and the spinal cord are neuroplastic tissues in which changes in gene and protein

expression levels occur following peripheral nerve injury and both serve as candidate tissues for

investigators to study pain mechanisms. The study focused on mouse lines that are known from

previous experiments by us and others to genetically contrast in the levels of autotomy behaviour

following hind paw denervation (i.e., A vs. B) to seek candidate pain genes. The focus of our

search for such genes was on a previously identified quantitative trait locus (QTL) on

1

2

chromosome 15, named Pain1, that was linked with autotomy in A and B mice (Seltzer et al.,

2001).

The whole genome gene expression profiling study identified in the spinal cord a candidate gene

in Pain1, named Csf2rb1 that we associated with autotomy behaviour in this dissertation using

genetic and immunohistochemical techniques (Chapter 4). The Csf2rb1 gene and its expressed

CSF2RB1 protein were correlated with autotomy behaviour in the spinal cord and brain. Two

additional genes that were studied in the context of Csf2rb1 are Tlr4 and the phosphorylated

protein Stat3 product of Stat3 that correlated with autotomy behaviour (Chapter 5). In the final

chapter of this dissertation (Chapter 6) we discuss possible mechanisms that combine Csf2rb1,

Tlr4, and Stat3, together and as separate entities in the context of the mechanisms of chronic

neuropathic pain.

The following experimental procedures for this thesis were performed by the author (MYA):

RNA extractions, RNA quantifications, gel electrophoresis, cDNA syntheses, primer designs,

RT-PCRs, immunohistochemistry, fluorescence microscopy and imaging, cell counts, in-silico

studies, autotomy phenotyping, some animal surgeries, perfusions and tissue isolation, as well as

statistical analysis, graphs and tables. Surgery, perfusion and tissue extraction of animals for

gene expression profiling and immunohistochemistry were performed by Drs. Shihong Zhang,

Elaheh Solemannejad, and Simon Beggs. Gene expression profiling using microarrays was

performed by outsourcing at the Microarray Centre (ACTG, Toronto, ON). Statistical analysis

for the microarray gene expression data was performed by the author (MYA) and David

Tichauer.

The following sections of the Introduction provide an overview of chronic neuropathic pain, its

clinical manifestations and pathophysiology, including the changes that occur in the nervous

system following nerve damage, such as spontaneous ectopic activity, peripheral and central

sensitization, sympathetically-maintained pain, collateral sprouting, nerve degeneration and

regeneration, disinhibition, excitotoxic cell death, and role of glia cells. Next, follows an

introduction to modeling chronic pain in animals and the Neuroma Model that was used in this

study. The Introduction concludes with a mini-review of the genetics of neuropathic pain, and

hence the rationale of comparing mouse to human genomes in the present study.

3

1.1 Chronic pain and its clinical manifestations

Chronic pain is defined as pain experienced over a period of 3 (some propose 6) months or more

following nerve injury, certain diseases, consumption of drugs and exposure to certain

chemicals. In addition it may be produced by other causes such as inheriting specific

polymorphisms on certain sodium channel genes (reviewed by Suzuki and Dickenson, 2000).

Neuropathic pain is distinguished from other chronic pain syndromes in that it was originally

defined as ‘pain initiated or caused by a primary lesion or dysfunction in the nervous system’

(International Association for the Study of Pain (‘IASP’) terminology) (Merskey and Bogduk,

1994). A more precise definition was developed by Treede et al (2008) stating it as ‘pain arising

as a direct consequence of a lesion or disease affecting the somatosensory system’ (Treede et al.,

2008). It can be subdivided into peripheral and central neuropathy, depending on where the

primary lesion or dysfunction is situated. In the periphery, neuropathic pain can result from

diseases (e.g., diabetes mellitus, uremia, herpes zoster, HIV and cancer) or due to

complete/partial nerve injury following surgery/trauma (e.g., after limb amputation, total nerve

transection by a shrapnel/knife, and partial trauma to the nerves/other tissues), or nerve

compression by tumors, entrapment, and nerve crush, burn or freezing, etc. Central pain

syndromes, however, result from damage/diseases to various structures in the brain or the spinal

cord (e.g., stroke, multiple sclerosis, amyotropic lateral sclerosis, cancer, etc). Despite these

diverse causes, the symptoms of neuropathic pain are similar and often include spontaneous pain,

pain in response to non-noxious stimuli (known as tactile and thermal allodynia), increased

responsiveness to noxious stimuli (known as mechanical and thermal hyperalgesia), sensory

deficits (e.g., anesthesia, hypoesthesia, analgesia and hypoalgesia), and sensory abnormalities

(e.g., dysesthesia and paresthesia), sympathetic dependence, spread to body parts not innervated

by affected nerves or central nervous system (CNS) structures, and exacerbation by movements

(Merskey and Bogduk, 1994)(Woolf and Mannion, 1999). However, not everyone undergoing

such inciting events develops neuropathic pain. For instance, the incidence of phantom limb

pain in Danish limb amputees is about 80% (Jensen and Nikolajsen, 1999), and of post-

mastectomy pain syndrome (PMPS) in women with breast cancer is up to 65% (Livneh-Fuchs,

2005). Moreover, pain intensity, frequency and duration in these patients also varies widely

from individual to individual, even when the inciting injury was the same (Jensen and

Nikolajsen, 1999). This variability suggests that chronic pain is a complex trait, controlled by

4

variations in many genes. Moreover, this genetic control is further modified by non-genetic

situational, emotional, cultural, social and other environmental factors. While the identity of

most genes for neuropathic pain is still unknown, much is known about the pathophysiological

outcomes controlled by these genes. The next section describes the pathophysiology of

neuropathic pain.

1.2 Pathophysiology of neuropathic pain

Looking at neuropathic pain from the perspective of its natural history, the earliest signal of

occurred injury is ‘injury discharge’, an immediate discharge of a barrage of action potentials in

many injured sensory fibbers at the time of nerve injury (Cohn and Seltzer, 1991; Seltzer et al.,

1991a, 1991b; Wall et al., 1974). Injury discharge may induce changes in the peripheral nervous

system (PNS) and CNS, thought to be responsible for stimulus-evoked pain (i.e., allodynia and

hyperalgesia), and spontaneous pain (Devor and seltzer, 1999). Nerve injury is also associated

with an interruption of nerve conduction from the receptive field to the CNS, followed by

Wallerian degeneration of the axons distal to the lesion. After a few days new sprouts grow out

in an attempt to regenerate and reinnervate the original targets. Even under the best conditions,

when the targets are present and not amputated, this attempt will only be partially successful

(Devor et al., 1979). Apart from the local effects at the site of nerve injury, there are several

changes that manifest in sites far beyond the lesion. These include chromatolysis of the nucleus

and other changes in the cell body of the injured primary afferent neurons in dorsal root ganglia

including dramatic changes to its profile of gene expression levels (Costigan et al., 2002; Cragg,

1970; Kim et al., 2009b, 2012; Liu et al., 2012a; Méchaly et al., 2006; Persson et al., 2009a;

Rodriguez Parkitna et al., 2006; Stam et al., 2007; Valder et al., 2003; Vega-Avelaira et al.,

2009; Wang et al., 2002a; Xiao et al., 2002). Even though the injury occurred in the PNS, there

are numerous additional changes in the CNS that contribute to neuropathic pain states, including

the activation of glia cells, accentuation of excitatory transmission and reduced inhibitory

transmission (see below).

1.2.1 Spontaneous ectopic activity

Following nerve injury, a neuroma forms at the cut end, comprising blood vessels, fibrotic scar

tissue and sprouts of severed axons. Sprouts emitted from damaged primary afferents develop

5

hyperexcitability that is so pronounced that it manifests in ongoing, ectopic activity, which

contributes an afferent drive for spontaneous pain but also a background sensitizing input for

allodynia, hyperalgesia, and spread of pain, and pain exacerbated by movement and by

sympathetic activity. Nerves damaged partially also develop a neuroma, termed ‘in-continuity’

or ‘partial neuroma’. The ectopic spontaneous firing develops not only in the injured nerve end

where sprouts in nerve-end neuromas are located (Burchiel, 1984; Devor and Raber, 1990;

Govrin-Lippmann and Devor, 1978; Wall and Devor, 1983) but also in the DRGs where the

soma of injured afferents are located (Burchiel, 1984; Devor et al., 1994; Govrin-Lippmann and

Devor, 1978; Kajander and Bennett, 1992; Wall and Devor, 1983). These two ectopic input

sources result from changes in the expression of ion channels in the genome located at the

nucleus of cell bodies of the injured peripheral afferents in the DRGs (Devor et al., 1993). Under

normal conditions certain Na+, K

+, Ca

2+ and Cl

- channels are constantly synthesized in the soma

and conveyed by rapid axonal transport to be assembled in the axolemma along the proximal and

distal axon and its ends, where they play a role in impulse generation, conduction, and synaptic

transmission including self-modulation of excitability by autoreceptors (Cummins and Waxman,

1997). Following nerve injury there is a change in the expression levels of these ion channel

subunits, including down-regulation of some channels and up-regulation of others that are not

expressed under normal conditions (Costigan et al., 2002; Cragg, 1970; Kim et al., 2009b, 2012;

Liu et al., 2012a; Méchaly et al., 2006; Persson et al., 2009a; Rodriguez Parkitna et al., 2006;

Stam et al., 2007; Valder et al., 2003; Vega-Avelaira et al., 2009; Wang et al., 2002a; Xiao et al.,

2002). In addition, there is redistribution of Na+ channels and their clustering in pathological

membrane regions (Devor et al., 1993), such as the sprouts in the neuroma and the membrane of

the afferent’s soma in the DRG. As a result, there is local hyperexcitability manifesting in

spontaneous firing and evoked firing in the sprouts of the neuroma as well as in the somata (Wall

and Devor, 1983). Thus, changes in the expression and function of ion channels may contribute

to the development of certain chronic pain states (Suzuki and Dickenson, 2000). The increase in

production, transport, and membrane insertion of transducer channels and voltage-gated ion

channels in nociceptors is primarily due to the activation of intracellular signalling transduction

pathways activated by inflammatory mediators. This so-called sensitization of nociceptors is a

factor of peripheral sensitization (Suzuki and Dickenson, 2000).

6

1.2.2 Peripheral sensitization

Peripheral nerve injury, like any other significant tissue injury, produces inflammation, a process

clinically characterized by redness, warmth, swelling, and pain. Inflammation is a complex

interaction between damaged endothelial cells, sympathetic efferents, and primary sensory

afferent neurons, and inflammatory cells such as macrophages, neutrophils, lymphocytes and

mast cells (Scholz and Woolf, 2007). In response to tissue damage, all of these cellular players

release inflammatory mediators, such as H+ and K

+ ions, nitric oxide, norepinephrine, serotonin

(5-HT), bradykinin, endothelin, prostaglandins (i.e., PGE2), cytokines, platelet activating factor,

histamine, glutamate, nerve growth factor (NGF) (Julius and Basbaum, 2001). From

sympathetic efferent fibbers, there is a release of neuropeptides, and catecholamines (Raja,

1995). From peripheral terminals of nociceptive afferent fibbers, there is a release of substance

P, calcitonin gene-related peptide CGRP, neurokinin A (Hudspith et al., 2006). These peptides

modify the excitability of sensory and sympathetic nerve fibbers, induce vasodilation, and

promote the release of further chemical mediators by inflammatory cells. The resulting ‘soup’ of

inflammatory mediators sensitizes further the nociceptive neurons and produces the phenomenon

of peripheral sensitization (Hudspith et al., 2006). In the event of partial nerve injury, where the

original targets are still innervated, the effect of peripheral sensitization is such that non-noxious,

low-intensity mechanical and thermal stimuli that would not normally cause pain are now

perceived as painful (hence the concept of allodynia). There is also an increased responsiveness

to noxious thermal and mechanical stimuli at the site of innervation, known as hyperalgesia

(Hudspith et al., 2006).

Another signal triggering cascades that result in chronic pain is not electrical but a chemical

signal, manifests in neurotrophic factors (i.e., NGF, BDNF, NT3 and GDNF) that are released

under normal conditions from healthy tissues, permeating in between the cells of the tissues of

the body to eventually reach the axolemma of primary afferent terminals in those tissues and get

bound to neurotrophic factor receptors on those membranes (i.e., trkA-C and ret, respectively).

The complex of trophin-receptor then gets internalized into the protoplasm of primary afferents,

uploaded onto the axonal transportation machinery, and get transferred retrogradely to the cell

bodies in the DRGs, to inform the genome that the terminals in the periphery are connected to

7

healthy tissues. But when these afferents are injured, transport of those trophic signals is

arrested, triggering the cascade of changes that lead to chronicity of pain (Fitzgerald et al., 1985).

Other than changes occurring at the injured nerve, there are transneuronal neuroplastic changes

in the CNS, that manifest as abnormal processing of sensory inputs (Hama et al., 1996). These

changes appear as ‘central sensitization’, discussed in the next section.

1.2.3 Central sensitization

Neuroplasticity in the CNS involves many processes, including activation of glial cells

(microglia and astrocytes), disinhibition by excitotoxic destruction of dorsal horn inhibitory

interneurons, reorganization of neuronal circuits in the dorsal horn, and changes in descending

pain pathways (Suzuki and Dickenson, 2000; Woolf and Salter, 2000; Zimmermann, 2001).

Under normal conditions proinflammatory mediators, cytokines, neuropeptides and transmitters

excite or inhibit pre- and post-synaptic cells in the spinal cord and its corollary trigeminal

brainstem complex. Ca2+

, Na+, and Cl

- influxes into post-synaptic cells and K

+ outfluxes,

depolarize and hyperpolarize neurons in the dorsal horn as a summated result of synaptic

activities that integrate excitatory and inhibitory synapses (reviewed in Latremoliere and Woolf,

2009). An increase in intracellular Ca2+

in post-synaptic cells beyond a certain level appears to

be the key trigger for initiating activity-dependent ‘central sensitization’. There is Ca2+

induced

activation of intracellular kinases PKC, PKA, CaMKII on the post-synaptic neurons. These

kinases phosphorylate NMDA and AMPA receptors on their C-terminus and change their

activity and their trafficking to or from the membrane of post-synaptic cells, leading to ongoing

excitation and central sensitization (Latremoliere and Woolf, 2009). Activated PKC contributes

both to hyperexcitability by activating NMDA receptors and to disinhibition, by reducing

inhibitor transmission [apoptosis of inhibitory interneurons and inhibition of descending peri-

aqueductal grey (PAG)] (Latremoliere and Woolf, 2009). Other intracellular pathways that

sustain central sensitization include the phosphatidylinositol-3-kinase (PI3K) pathway and the

mitogen-activated protein kinase (MAPK) pathway that involves the extracellular signal-

regulated kinases ERK1 and ERK2 (reviewed in (Latremoliere and Woolf, 2009). Post synaptic

neurons in the dorsal horn also retain several metabotropic (G-protein coupled) glutamate

receptor subtypes (mGluR), which are either Ca2+

permeable (i.e., GluR1 and GluR3) or Ca2+

impermeable (i.e. mGluR2). The mGluR family is composed of 8 receptors that form 3 groups,

8

based on their sequence similarities and their coupling with specific Gα-proteins (reviewed in

(Latremoliere and Woolf, 2009).

Peripheral nerve injury initiates proliferation and activation of glial cells in the spinal cord as

well as infiltration of immune cells from the periphery, especially macrophages and T-cells (Cao

and DeLeo, 2008; Tanga et al., 2005; Watkins et al., 2007). Some immune cells may be related

to clearing debris of dying terminals of primary afferents and to apoptotically and excitotoxically

dying second order dorsal horn neurons. Microglia proliferate and become activated, whereby

they produce and release trophic factors, neurotransmitters, cytokines, and reactive oxygen

species into the dorsal horn (Romero-Sandoval et al., 2008; Watkins and Maier, 2002), and these

factors interact with neurons and trigger central sensitization and pain following nerve injury (Ji

and Suter, 2007; Latrémolière et al., 2008; Ledeboer et al., 2005; Meunier et al., 2007; Milligan

et al., 2004; Raghavendra et al., 2003a, 2003b; Verge et al., 2004). The signals that trigger

microglial activation and recruitment include ATP and NO (Davalos et al., 2005; Duan et al.,

2009; Nimmerjahn et al., 2005), cytokines, and chemokines, some of which are released by

injured sensory neurons and others by microglial cells themselves or by astorcytes and T-cells

(Abbadie et al., 2003, 2009; DeLeo and Yezierski, 2001; Dominguez et al., 2008; Milligan et al.,

2008; van Rossum and Hanisch, 2004; Watkins and Maier, 2002; Watkins et al., 2001). Release

of cytokines by microglia increases neuronal excitability through activation of ERK and CREB

(Ji et al., 2009; Kawasaki et al., 2008). Activated microglia also release BDNF and NO (Coull et

al., 2003; Horvath et al., 2008) promoting segmental disinhibition (Coull et al., 2005). Finally,

microglia can also provoke neuronal death by ROS, pro-apoptotic cytokines such as TNF (Huang

et al., 2005), and by a diminished glutamate uptake (Chéret et al., 2008; Tawfik et al., 2008;

Tilleux and Hermans, 2007). T-cells produce specific cytokines such as INF-γ, which reduce

GABAergic currents in the dorsal horn (Vikman et al., 2003) through activation of IFN- γ

receptors (Vikman et al., 1998) and also activate and recruit microglia. Astrocytes also become

activated after peripheral nerve injury (Garrison et al., 1994; Ji et al., 2009; Milligan and

Watkins, 2009) with a slower onset and more prolonged time course than microglia, and may

play more of a role in the maintenance of neuropathic pain hypersensitivity than microglia (Gao

et al., 2009; Zhang et al., 2006; Zhuang et al., 2005). Overall, these mechanisms that follow

peripheral nerve injury either increase excitability or reduce inhibition (Latremoliere and Woolf,

2009).

9

These changes may explain most sensory abnormalities manifesting as neuropathic pain,

including spontaneous pain, hypersensitivity to stimuli and the spread of pain to regions extra-

territorial to the field originally innervated by the injured nerve.

1.2.4 Sympathetically-maintained pain

In addition to neuroplasticity of the somatosensory nervous system there are plastic changes in

the autonomic nervous system that act on sensory nerves after nerve insult, contributing to

sympathetically-maintained pain (SMP). This manifests in the somatosensory and sympathetic

nervous systems interacting , manifesting in one of the symptoms of neuropathic pain

syndromes such as neuroma pain, which may be aggravated or maintained by activity of the

sympathetic nervous system (Chabal et al., 1992).

Normally, sympathetic-sensory direct coupling between sympathetic and nociceptive neurons

does not exist in peripheral nerves or DRGs. Such an abnormal coupling typically develops after

peripheral nerve injury or inflammation, manifesting in a chemical interaction between

sympathetic and nociceptive neuron terminals in skin (i.e., catecholamines and neuropeptides

released by sympathetic nerve endings), and via the development of α-adrenoceptor-mediated

supersensitivity in nociceptors in the skin in the presence of released inflammatory mediators.

Evidence from human studies have shown the direct association of noradrenaline in the

development of hyperalgesia in sensitized normal skin, either through direct binding or

sensitization of α-adrenoceptors (Drummond, 1998, 1999; Fuchs et al., 2001; Lipnicki and

Drummond, 2001). Similarly, in patients with SMP, exogenous noradrenaline evoked

spontaneous pain and hyperalgesia in affected skin, but not in unaffected contralateral skin in

these patients or in control subjects (Ali et al., 2000). Evidence suggests that the noradrenaline

role in nociception in SMP is mediated by α 1-adrenoreceptors,which are more abundant in these

patients (Davis et al., 1991; Drummond et al., 1996; Fuchs et al., 2001).

Evidence from animal models of neuropathic pain have pointed to an increased sensitivity of

sensory nerves to noradrenaline, mediated by α-adrenoceptor subtypes 1 or 2, depending on the

species, site of injury and coexisting inflammation (Ali et al., 1999; Baik et al., 2003; Hong and

Abbott, 1996; Lee et al., 1999a; Sato and Perl, 1991). The excitatory effects of noradrenaline, as

well as other α-adrenoceptor agonists and sympathetic chain stimulation have been observed in

10

various nerve injury models. Nociceptive nerve fibbers in the neuromas of complete sciatic

nerve transected rats can be activated both by stimulation of the sympathetic chain and by local

injection of α -adrenoceptor agonists (Chen et al., 1996). Similarly, nociceptive fibbers that

survive partial injury of a peripheral nerve can be stimulated by alpha-adrenoceptor agonists in

rabbits (Sato and Perl, 1991) and primates (Ali et al., 1999), and can increase hyperalgesia and

activation of nociceptive afferents (Sato and Perl, 1991).

1.2.4.1 Direct coupling between the sympathetic neurons and sensory

neurons in the DRG

In animal models, peripheral nerve insult has been shown to trigger sympathetic nerve sprouting

into the DRG. New post-ganglionic sympathetic fibbers normally associated with blood vessels

and involved in vasoconstrictor activity, sprout and form basket-like structures around primary

neuronal cell bodies that survive the nerve injury (McLachlan et al., 1993). Studies suggest that

very few α-adrenoceptors are present on the cell bodies of primary afferent neurons under

normal conditions but that these increase following nerve injury in parallel with an increase in

regional sympathetic nerve sprouting (Birder and Perl, 1999; Ongioco et al., 2000; Petersen et

al., 1996; Xie et al., 2001). In some models of chronic pain, a pre-emptive sympathectomy

prevented the expression of chronic pain (Coderre et al., 1986). However, pain behaviour has

not yet been shown to increase with increased sympathetic sprouting in the CNS (Kim et al.,

2001). Moreover, the time of appearance of those basket-like terminals of sympathetic efferents

around somata of primary afferents in the DRGs is many weeks after chronic pain behaviour has

started or even long gone, suggesting that they are an epiphenomenon unrelated to the

appearance of chronic pain.

1.2.4.2. Chemical coupling between sympathetic efferents and sensory

neurons in skin

Two groups have reported abnormal migration and sprouting of non-peri-vascular sympathetic

fibbers in rat skin of the hindpaw (Yen et al., 2006) and lower lip (Grelik et al., 2005a, 2005b)

into the upper dermis following partial nerve injury to the sciatic and trigeminal nerve,

respectively. In each study, an increase in sensory innervation to the upper dermis was also

11

observed. Newly sprouted abnormal sympathetic fibbers were found wrapped around sensory

fibbers, forming new associations that potentially provide a histological setting for chemically

mediated coupling between sympathetic nerve terminals and sensory receptors. Peak

sympathetic innervation overlapped with an increase in spontaneous grooming of the affected

area that was not observed in the sham animals, implying a relationship between the cutaneous

changes and pain (Grelik et al., 2005a). The exact mechanism of sympathetic-sensory coupling

in these rats is yet to be established, but may involve activation of α-adrenoceptors, which may

be expressed on nociceptive fibbers and directly activate firing or they may be expressed on cells

closely associated with nerve fibbers and trigger nociceptive fibbers indirectly.

1.2.4.3. α-adrenoceptor-mediated super sensitivity of nociceptive fibbers

It seems that adrenergic responsiveness is not due to exposure of α-adrenoceptors to increased

concentrations of ligand, but rather that decreased availability of the ligand results in increased

sensitivity and/or over-expression of genes encoding the adrenergic receptor (Drummond, 2004).

Receptor sensitization and changes in expression may be associated with tissue injury and

inflammation (Gibbs et al., 2008). Noradrenaline may contribute to pain and inflammation by

enhancing the turnover of inflammatory mediators and algogenic substances, such as nerve

growth factor and prostaglandins (Gonzales et al., 1991) as part of peripheral sensitization, or

may directly trigger antidromic firing of nociceptive neurons by activating α-adrenoreceptors on

nociceptive afferents (Lin et al., 2003).

1.2.5 Nerve degeneration

In addition to peripheral and central mechanisms that contribute to neuroplasticity that occur

following nerve injury, some injured primary afferents begin to degenerate. This manifests in

the ‘dying back’ phenomenon where the proximal segments of some of the axons in an injured

nerve undergo shrinkage on the axon diameter, slowing down of conduction velocity in the

shrunk segment. In addition, some primary afferents show transganglionic degeneration of the

injured nerve terminals in the spinal dorsal horn (in the case of a peripheral and spinal nerve

injury), and in the trigeminal brainstem nuclei (in case of a trigeminal nerve injury) (Arvidsson,

1979).

12

1.3 The memory of pain and preemptive analgesia

The memory of pain exists in humans and lower animals, and frequently dominates the primary

experience leaving an impact on pathophysiology and suffering. Memory and learning are

characteristics processed by the hippocampal region of the brain. Many animal studies have

demonstrated that an analgesic given prior to a noxious stimulus or injury is more effective at

preventing central sensitization than the same analgesic given after the stimulus. The key

concept behind pre-emptive analgesia is that painful stimuli establish the memory of pain. The

hypothesis of pre-emptive analgesia is that analgesia administered before the painful stimulus

will prevent or reduce subsequent pain analgesic requirements in comparison to the identical

analgesic administered after the painful stimulus, by preventing or reducing the memory of pain

in the nervous system. For example, Dickenson and Sullivan (Dickenson and Sullivan, 1987)

showed in rats that intrathecal opiods administered before intracutaneous formalin injection

inhibit central sensitization. Opiods given just after the formalin injection had much less effect.

In another electrophysiologic study, Woolf demonstrated that the systemic morphine dose

needed to prevent central hyperexcitability, given before brief noxious electrical stimulation of

the gastrocnemious-soleus nerve in rats, was one-tenth the dose required to abolish prolonged

activity after it had developed (Woolf, 1983). Other reports suggest that the severity of acute

pain, such as surgery, influences the development of chronic pain. Wilkins et al. compared

phantom sensation and phantom pain in 60 children and adolescents with congenital limb

deficiency or amputation after surgery or trauma (Wilkins et al., 1998). Phantom sensation was

present in 7.4% of the congenital group and 69.7% of the surgical group. Data from a large

series by Sherman likewise indicate that loss of an existing limb is associated with a nearly ten-

fold greater likelihood of phantom sensation than agenesis of a limb (Sherman, 1997). Arnstein

reported that if severe pain is allowed to persist for more than 24 hours, neuroplastic changes

associated with the development of intractable chronic pain syndromes are evident (Arnstein,

1997).

1.4 Modeling chronic pain in animals

Dissecting the complex experience of chronic pain in humans necessitated invasive research in

animal models dedicated to the study of painful neuropathies. The rationale for using such

13

models is based on the expectations that mechanisms underlying chronic pain-like behaviours in

animals contribute to the same behaviours in humans with chronic pain. The genes associated

with these mechanisms could become targets of potential analgesic treatments. This highlights

the need for animal models that mimic human pain, as described below.

Earlier models of injury-induced neuropathic pain involved total denervation of a paw or a tooth,

as well as deafferenting the CNS by a few or multiple dorsal rhizotomies (i.e., by transection of

dorsal roots that provide the CNS with afferent input from all the body), or total or partial nerve

section(s), or cut-and-resuture, or crush, or freezing, or burning, or pulpectomies or pulpotomies

of the teeth (Lombard et al., 1979; Seltzer et al., 1991a). More recent models concentrated on

partial denervations of the sciatic nerve and trigeminal tributaries. Unlike models of total

denervation, these models preserve some of the sensory information passing from the hindpaw or

face to the spinal cord or brainstem nuclei, respectively. The three mostly used models are the

chronic constriction injury (CCI) model of the sciatic nerve (Bennett and Xie, 1988), the partial

sciatic nerve ligation (PSL) model (Seltzer et al., 1990), and the L5 and L6 spinal nerve ligation

(SNL) model (Kim and Chung, 1992). Much of what modern pain research has taught us has

been gained from using these models. Several additional animal models for chronic pain,

primarily for inflammatory and neuropathic pain following nerve injury, have been developed in

recent years (reviewed in (Bennett et al., 2003; Zeltser and Seltzer, 1994).

In addition to the above animal models for chronic pain that are produced by peripheral nerve

injury, other models for chronic pain caused by spinal cord injuries (SCI) are also available. SCI

pain in humans is typically perceived in several body part including regions that are insensate to

external stimuli, and is usually bilateral. It is referred to as deafferentation pain, dysesthetic pain

or central dysesthesia syndrome (Davidoff et al., 1991; Nashold, 1991; Siddall et al., 2002;

Yezierski, 1996). “Below-level” pain is the most common. This type is perceived below the

level of the injury. Other types of SCI pain include “at-level” and “above-level” pain syndromes

(Hicken, 2002; Siddall et al., 2002). “At-level” pain is associated with damage at, or near the

site of injury, and is modeled by ischemic, excitotoxic, and contusion injury to the spinal cord

(Hulsebosch et al., 2000; Siddall et al., 1995; Wiesenfeld-Hallin et al., 1994; Yezierski et al.,

1998). Excitotoxic SCI models produce excessive grooming behaviour, and autotomy, and is

associated with neuronal loss that includes the neck of the dorsal horn with sparing of the

superficial laminae (Devroede et al., 1989; Krupina et al., 2010; Speiser et al., 1991; Yezierski,

14

2005; Yezierski et al., 1998). “Below-level” neuropathic pain is modeled by the contusion

model (Christensen and Hulsebosch, 1997; Hulsebosch, 2002; Vierck and Light, 2000) and is

dependent upon partial deafferentation of rostral targets of the spinothalamic and associated

pathways (Reviewed by Yezierski, 2005). Behavioral measures include licking, guarding,

orientation, vocalizations, (Yezierski, 2005).

The Neuroma Model of neuropathic pain was the first to be studied in detail and is still one of

the few available models for spontaneous human pain syndromes triggered by nerve injury

(Coderre et al., 1986; Wall et al., 1979; Zeltser and Seltzer, 1994). This model is described in

greater details below, for it was used in the present research study.

1.4.1 The Neuroma Model for spontaneous pain

This model is expressed by self-mutilation (‘autotomy’) of a denervated paw. Transection of the

sciatic and saphenous nerves leads within couple weeks (in rats) or a few days (in mice) to

abnormally excessive licking, scratching and biting of the denervated portions of the hindpaw

(Wall et al., 1979). This behaviour lasts up to 2 months (in rats) and about 1 month (in mice).

Wall et al. (1979) developed a scoring system for this behaviour that is still widely accepted (see

Methods). The accepted explanation offered as a rationale underlying the expression of self-

mutilation in this model is that the ectopic inputs from nerve-end neuromas and the DRGs

associated with the injured nerve, combined with abnormally sensitized processing of this input

in the CNS, leads to the perception of pain that is referred to the denervated limb. In an attempt

to rid itself of the pain, the animal licks, scratches and injures the painful paw, which is the

behavioural endpoint of this model. Onset, duration and final score on the last day of the

behavioural follow up period provide as quantifiable spatio-temporal parameters of this model,

serving as indicators of the onset, duration and intensity of the pain.

There have been controversies on whether autotomy behaviour is a sign of pain (see for example

Rodin and Kruger, 1984), since similar behaviours can be induced in animals by certain skin

irritations or diseases (assumed to induce itching) and injection of excitatory agents into the CNS

(Asada et al., 1996; Nojima et al., 2004). In addition, it has been argued that the absence of

sensory input from the denervated limb may in itself result in self-mutilation because the animal

may not recognize the anesthetic limb as part of the body and attempts to remove it as if a

15

foreign body. However, although such factors may contribute to the behaviour under specific

circumstances, it is widely accepted now that autotomy following nerve lesion reflects chronic

neuropathic pain (for reviews, see Coderre et al., 1986; Devor and seltzer, 1999; Kauppila,

1998).

Autotomy behaviour can be prevented or suppressed by drugs, surgical procedures that offer pain

relief in humans, such as anticonvulsants, tricyclic antidepressants, NMDA receptor antagonists,

dorsal column stimulation and dorsal root lesions. On the other hand, analgesic drugs such as

nonsteroidal anti-inflammatory drugs, which do not offer relief for neuropathic pain patients, do

not affect autotomy (Coderre et al., 1986; Gao et al., 1996; Kauppila, 1998; Levitt, 1985; Seltzer,

1995; Wiesenfeld-Hallin and Hallin, 1984; Zeltser et al., 2000). Social manipulations also affect

autotomy levels significantly (e.g., single vs. communal housing, housing with animals that

concurrently express vs. do not express chronic pain, or housing with the opposite vs. same sex)

(Devor et al., 2007; Nissenbaum et al., 2010).

Finding genes that control both autotomy in rodents and chronic pain in humans could provide

strong evidence for this behaviour being an expression of pain in rodents. This, in fact, has

happened in part by way of work presented in this thesis dissertation for Csf2rb1 (so far only in

mice), and previously for Cacng2, a report to which we contributed (Nissenbaum et al., 2010).

Although autotomy is not a normal response of humans to chronic pain, it occurs in many

species ranging from rodents to primates. Onset and course of autotomy are closely correlated

with the timing and amount of ectopic firing of neural impulses from sensory nerve fibbers at the

severed nerve end and from the DRGs (Devor and seltzer, 1999). Treatments that suppress

chronic pain in humans had the same effect on this behaviour in operated rodents, and vice versa,

drugs that were not effective in human neuropathic pain were ineffective in blunting autotomy in

denervated rodents. The Neuroma Model was chosen for this study because it mimics the clinical

condition of phantom limb pain, anesthesia dolorosa and post-plexus avulsion syndromes that

can develop in susceptible individuals after an amputation or denervation of a limb.

16

1.4.2 Contrasting autotomy behaviour in mouse and rat strains

Autotomy levels vary greatly among different inbred, outbred and selected lines of mice and rats

raised, operated and maintained under identical environmental conditions (Defrin et al., 1996;

Devor et al., 1982; Mogil et al., 1999a; Shir et al., 2001; Wall et al., 1979; Wiesenfeld and

Hallin, 1981), demonstrating that genetic factors play a major role in controlling the contrasting

levels of autotomy in these rodents. Lines of rats have been selected for high versus low

autotomy levels from a common outbred strain of Sabra rats (Devor and Raber, 1990; Devor et

al., 2005a), further confirming the genetic basis of this trait. Since this thesis focuses on the

genetics of autotomy as a model of spontaneous neuropathic pain, two strains of mice that

contrast on the autotomy phenotype, A/J (‘A’; a high-autotomy inbred strain) and C57BL6/J

(‘B’; a low-autotomy inbred strain) mice were chosen to compare their gene expression levels in

key neural tissues that control pain chronicity – the peripheral injured nerve and the spinal cord.

A few studies documented the contrast in autotomy levels between A and B mice, showing the

high susceptibility in A mice (Devor et al., 2007; Minert et al., 2007a; Mogil et al., 1999a;

Seltzer et al., 2001), and the resistance to autotomy behaviour in B mice, following a period of

35-36 days post-hindpaw denervation (Devor et al., 2007; Minert et al., 2007b; Seltzer et al.,

2001). In addition to studies on spontaneous pain, A and B mice were also shown to contrast in

stimulus-evoked pain behaviours following PNI and SNL (Mogil et al., 1999a; Persson et al.,

2009b).

The A inbred strain is widely used in cancer and immunology and has been developed by LC

Strong in 1921 from a cross between a Cold Spring Harbour albino and a Bagg albino

(http://jaxmice.jax.org/strain/000646.html). It is highly susceptible to cortisone-induced

congenital cleft palate. It has a high incidence of spontaneous lung adenomas, and lung tumors

readily develop in response to carcinogens. In addition to atherosclerosis resistance, A mice are

resistant to diabetes, obesity, insulin resistance and glucose intolerance. On either chow or high

fat diet, A mice maintain low glucose and insulin levels. A mice develop cigarette smoke-

induced emphysema in approximately half the time when compared with B mice. Structural lung

damage caused by induced asthma mimics the phenotype found in asthma patients more closely

than does the induced damage in BALB/cJ mice.

17

The B inbred strain is commonly used as a general purpose strain and background strain for the

generation of congenics carrying both spontaneous and induced mutations

(http://jaxmice.jax.org/strain/000664.html). B mice are used in a wide variety of research areas

including cardiovascular biology, developmental biology, diabetes and obesity, genetics,

immunology, neurobiology, and sensorineural research. B mice have a low susceptibility to

tumors and delayed senescence relative to BALB/cJ and DBA/2J. Other characteristics include

among others 1) a high susceptibility to diet-induced obesity, type 2 diabetes, and

atherosclerosis; 2) resistance to audiogenic seizures and 3) late-onset hearing loss.

1.4.2.1 Additional mouse strains used to study autotomy

behaviour

In addition to A and B mice, many other inbred mouse strains are used to study autotomy

behaviour, such as 129/J, AKR/J, BALB/cJ, C3H/HeJ, C3H/HeN, C58/J, CBA/J, RIII/J and

SM/J. These strains were previously phenotyped by us and others to determine their

susceptibility to develop autotomy following hind paw denervation (Blech-Hermoni, 2005;

Mogil et al., 1999a). Here we introduce and describe the additional strains that contrast in

autotomy behaviour which we used in our follow-up assays for seeking candidate pain genes.

The C3H mouse strain is susceptible for the development of autotomy behaviour

following hind paw denervation. It was developed in 1920 by LC Strong from a cross of

a Bagg albino female with a DBA male. A spontaneous mutation occurred in C3H/HeJ

strain sometime between 1960 and 1968 at the Tlr4 gene locus (Tlr4lps

). This is a

dominant-negative point mutation Pro→His at position 712 in the third exon of the gene

(Poltorak et al., 1998). These mice are therefore considered TLR4-deficient mice.

C3H/HeJ mice are non-responsive to lipopolysaccharide and thus endotoxin resistant

(Morrison and Ryan, 1979). They are highly susceptible to infection by Gram-negative

bacteria, and once infected with Salmonella exhibit a delayed chemokine production,

impaired nitric oxide generation, and attenuated cellular immune responses.

Macrophages from C3H/HeJ mice fail to induce inflammatory cytokines such as TNFα,

IL-1 and IL-6 (Morrison and Ryan, 1979). These mice have been maintained at the

Jackson laboratory and are distinct from the C3H/HeN mice, maintained by Harlan and

18

lack the mutation in the Tlr4 locus. C3H/HeN mice are the respective wild type controls.

This thesis contains studies using both C3H/HeJ and C3H/HeN mice (Chapters 4 & 5).

More description on these mice and their phenotypic variability is discussed in Chapter

5.1.

The AKR/J inbred mouse strain is resistant to autotomy behaviour following hind paw

denervation. AKR mice were originally inbred at the Rockefeller Institute, and are

widely used in cancer research for their high leukemia incidence (60-90%) and in

immunology as a source of the Thy1.1 (theta AKR) antigen. AKR/J mice are viremic

from birth, and express the ecotropic retrovirus AKV in all tissues. Other mutations that

characterise this strain include alteration in hair development, adrenocortical lipid

depletion, resistance to aortic lesion formation on a semi-synthetic high fat diet and hypo

responsiveness to diets containing high levels of fat and cholesterol.

1.5 Mini-review of the genetics of neuropathic pain

It is generally believed that behavioural traits related to chronic pain reflect altered gene

expression levels in the cell body of the injured primary afferent neurons in DRGs associated

with the injured nerve, in their intact neighbours, as well as in neurons and glia in the CNS

(Coyle, 2007; Griffin et al., 2007; Kim et al., 2009b; Ko et al., 2002; Liu et al., 2012a; Nesic et

al., 2005; Persson et al., 2009b; Rodriguez Parkitna et al., 2006; Sun et al., 2002; Valder et al.,

2003; Wang et al., 2002a). Both peripheral and central mechanisms contribute to neuroplasticity

that depends on the expression of specific genes in the PNS and CNS. There have been a few

genome-wide studies to identify genes regulated by nerve injury and correlated with chronic pain

levels in animal models (Coyle, 2007; Griffin et al., 2007; Kim et al., 2009b; Ko et al., 2002;

Lacroix-Fralish et al., 2006; Liu et al., 2012a; Nesic et al., 2005; Persson et al., 2009b; Rodriguez

Parkitna et al., 2006; Sun et al., 2002; Valder et al., 2003; Wang et al., 2002a). Most of the

studies on gene expression profiling after nerve injury have been modeled in rats, and fewer

studies were performed in mice. The present study used the Neuroma Model for spontaneous

neuropathic pain in mice, which mimics the pathophysiology of pain in human leg amputees, to

study novel genes and decipher new mechanisms within peripheral and central neural tissues.

We hypothesize that both mice and humans carry the same genes and have similar mechanisms

19

by which neuropathic pain is established and maintained. The rationale for comparing human to

mouse genomes is discussed in Section 1.6.

The following identified genes include members of several classes, which operate to produce

chronic pain in numerous mechanisms. These are G protein-coupled receptors (‘GPCR’), ligand-

and voltage-gated ion channels, receptor tyrosine kinases, growth factors and neurotrophic

factors, cytokines are inflammatory mediators, neuropeptides, cell cytoskeletal genes, cell

surface/extracellular matrix genes, and more. GPCRs are neurotransmitter receptors and they

produce chronic pain by increasing intracellular Ca2+

and by exciting or inhibiting neuronal

activity; Ligand- and voltage-gated ion channels include Na+, K

+ and Ca

2+ types and they

produce chronic pain by increasing or diminishing electrical excitability. Receptor tyrosine

kinases include PKA, PKC and ERK and they produce chronic pain by phosphorylating

membrane bound receptors and activating them. Several growth and neurotrophic factors

contribute to chronic pain, and these include NGF, EGF, GDNF and BDNF. They can change

the expression levels of some ion channels in the DRG when they are no longer transported to

the cell body, in occurrence with the disconnected nerve from the periphery. Other ways by

which neurotrophic factors can contribute to chronic pain is by axonal sprouting and by

decreasing inhibition (BDNF). Cytokines are inflammatory mediators that contribute to glial

activation and to neuronal hyperexcitability, provoke cell death and reduce GABAergic currents

in the dorsal horn thus producing chronic pain. Neuropeptides such as substance P and

neurokinin A function as neurotransmitters and contribute to the development of chronic pain by

modifying the excitability of sensory and sympathetic nerve fibbers, inducing vasodilation, and

promoting the release of further chemical mediators by inflammatory cells.

The proteins encoded by these genes have helped forming, in part, the current dogma that

neuropathic pain involves the cross talk of many cell types in the nervous system, namely

various types of neurons and glial cells. In this respect, neurons fire impulses but also respond to

activated glial cells (i.e., microglia and astrocytes) that release factors to which neurons respond

and change their excitability. In addition to the changes in the levels of specific gene expression,

changes in protein structure by way of post-translational protein modifications may also play a

role after nerve injury in DRGs, trigeminal ganglion, spinal cord, brainstem and brain

(Latremoliere and Woolf, 2009).

20

1.5.1 Neuropathic pain genes in partial nerve injury models

A few genes for neuropathic pain behaviour were previously identified by others, in

collaboration with our group. An example is the Cacng2 gene, a gene for spontaneous

neuropathic pain, which is discussed in greater detail in the Discussion of this dissertation. Other

genes of stimulus-evoked neuropathic pain that have been identified using partial nerve injury

models include Kcns1, Trpa1 and P2rx7. The potassium channel alpha subunit KCNS1,

involved in neuronal excitability, is constitutively expressed in sensory neurons and markedly

downregulated in rats following SNI, CCI and SNL relative to their naïve controls (Costigan et

al., 2010). The same study also identified a single SNP within the Kcns1 gene in humans that

correlated with neuropathic pain in five of six independent patient cohorts (total of 1359

subjects). These cohorts included patients with lumbar back pain with disc herniation and limb

amputation pain subjects. Another study by the same group indicated that TRPA1, a

nonselective cation channel expressed by nociceptors, contributes to cold hypersensitivity in rats

following SNI (del Camino et al., 2010). Finally, variation within the coding sequence of the

gene encoding the P2X7 receptor, a member of the family of ionotropic ATP-gated receptors,

affects chronic pain sensitivity in both mice and humans (Sorge et al., 2012).

1.6 Epigenetics and chronic pain

Epigenetics refers to genetic control by factors other than the DNA sequence that alters gene

expression levels. Among these are environmental factors that play a critical role in pain

phenomics and genetics. Gene regulation is modified in part by DNA methylation and

unmethylation processes. When DNA is unmethylated, it is exposed in a manner that allows

gene transcription machinery to bind to it and begin transcription. During gestation, different

tissues acquire specific patterns of DNA methylation. These patterns confer cell type identity by

regulating genome function (Razin and Szyf, 1984). DNA methylation is a reversible biological

signal (Ramchandani et al., 1999) that occurs in response to external signals. We now know that

DNA methylation is also involved in chronic pain (Doehring et al., 2013; Qi et al., 2013). This

is important because DNA methylation is not only a mechanism underlying inter-individual

differences in the susceptibility for developing chronic pain, but it is also potentially reversible

by pharmacological or therapeutic interventions (Stone and Szyf, 2013). Wang et al. recently

demonstrated a role for DNA methylation in the spinal cord following nerve injury in rats (Wang

21

et al., 2011) and Tajerian et al. reported that methylation of the SPARC promoter in

intervertebral discs is associated with chronic back pain in both pre-clinical models and in human

patients(Tajerian et al., 2011). Thus, studies exploring the relationship between epigenetics and

chronic pain will identify differentially regulated genes and pathways that can be more directly

targeted pharmacologically.

1.7 Sex and gender differences in chronic pain

Documented evidence indicates that the prevalence of many common chronic pain conditions is

higher in females compared to males (Fillingim et al., 2009). For example, females show higher

frequencies of many musculoskeletal pain conditions, migraines, and irritable bowel syndrome

(Fillingim et al., 2009). Neuropathic pain-like behaviours in rodents following partial nerve

injury or spinal cord injury also demonstrates strain-dependent sex differences (DeLeo and

Rutkowski, 2000; Gorman et al., 2001). In rodents, sex differences can also be influenced

significantly by genotype (Mogil et al., 2000), such that within the same strain males express

more pain than females, or vice versa. Examples include 129/J, A/J, BALB/cJ, CBA/J and CM/J

mouse strains. Differences also exist between the sexes in response to analgesics, especially

opiods. Male rodents show robustly more opioid antinociception compared to their female

counterparts (Craft, 2003); however, sex differences in opiod analgesia depend on the specific

opioid, the way a drug is administered, and the pain model used (Niesters et al., 2010). Some

documented a slightly more analgesic effect of morphine among women than men (Niesters et

al., 2010).

1.8 Rationale for comparing mouse to human genomes

The mouse model is a powerful tool to investigate the mechanisms underlying a complex trait

using invasive methodologies that are impossible in humans. Mouse genomes provide robust

bioinformatic tools to identify human genes by offering a first screen that identifies quantitative

trait loci (QTLs) for a trait under investigation, thereby directing attention to specific

chromosomal regions along the human genome where candidate pain genes may be sought. A

comprehensive understanding of gene regulation in animal models of ongoing pain is greatly

needed in order to better understand the molecular mechanisms of neuropathic pain and to

identify novel targets for the development of better pain treatments.

22

Identifying genetic variations in human subjects with/out chronic pain could highlight genes that

control pain via sequence variations in coding regions that could result in variable protein

expression and function operating in pain pathways. There are, however, other genetic

mechanisms that could affect the pain phenotype, such as gene expression levels that result in the

abundance of the gene products, thereby affecting neural functions associated with processing

pain inputs. Since the tissues associated with pain processing (i.e., brain, spinal cord and DRGs)

are not available for study in living human subjects, and suitable post-mortem material is not

available, we must rely on animal models for studying tissue-specific gene expression levels or

sequence variations. The close homology in genomes of human vs. other mammalian species,

and the similarity between humans and rodents in brain pathways and some pain phenotypes,

justify a comparative whole-genome approach (Vallender and Lahn, 2004). Mice are used for

genetics since the murine genome is the most studied and there are powerful assays available for

genetic manipulation. There are readily available inbred mice lines of different genetic

background. In fact, four of these lines (DBA/2J, 129/J, A and B) have already been fully-

sequenced by Celera:

(http://findarticles.com/p/articles/mi_m0EIN/is_2000_Oct_12/ai_66001567) and many others

genotyped in high density (Frazer et al. 2007). These lines contrast on several pain phenotypes,

one of which is autotomy (Mogil et al., 1999a). There are additional resources available for

mapping the murine genome for QTLs, including recombinant inbred lines (RIs) that are

descendants of parental lines that contrast on pain phenotypes, i.e., the 23 lines of the AXB-BXA

RI set that were derived as recombinant inbred daughter lines by crossing the A and B strains,

and that express highly contrasting line-specific autotomy levels following the same hindpaw

denervation (Seltzer et al., 2001). Furthermore, haplotype maps (‘Hapmaps’) of many inbred

mouse strains are now available from Perlegen (Frazer et al., 2007);

(http://www.ncbi.nlm.nih.gov/SNP/), Roche (Liao et al., 2004), and MIT

(http://www.broad.mit.edu/mouse/hapmap/). Consomic lines (Nadeau et al., 2000; Singer et al.,

2004); http://jaxmice.jax.org/type/consomic.html), advanced intercrossed lines (AILs) (Darvasi

and Soller, 1995) and congenic lines

(http://www.informatics.jax.org/morsebook/frames/frame11.shtml), are now available for in-

silico QTL mapping. Additionally, genetic manipulations such as knock-outs, knock-ins, small

interference RNA (siRNA), antisense treatments, and ENU-induced mutations, are all powerful

23

methodological approaches that enable investigators to study the genetics of pain in mice. These

make mice the best animal model to identify genetic determinants controlling chronic pain. No

less important is the fact that there are a number of mouse models for neuropathic pain, of which

the Neuroma Model (autotomy) was chosen for this study. Since work done by others and our

group have already identified the A and B mice as strains contrasting on this trait, part of the

present study was to compare gene expression levels in neural structures of these two lines.

Mouse strains expressing variable phenotypes under the same experimental conditions are often

used to positionally map genetic loci associated with that phenotype. These assays are also

known as QTL mapping assays. In a previous QTL mapping study that used 23 AXB and BXA

recombinant inbred (RI) lines, and their parental A and B inbred strains, from which the AXB

and BXA lines were derived by repeated crossings and inbreeding, our group identified Pain1, a

QTL on mouse chromosome 15 having a major effect on autotomy behaviour (Seltzer et al.,

2001). This study was based on the documented contrast in autotomy levels expressed by A and

B mice following the same hindpaw denervation procedure. The presence of a QTL in Pain1 has

been since confirmed by another study that used a different genetic assay and parental strains of

a different genetic background than those used by our group (Devor et al., 2005b). QTL

mapping is an unbiased approach to seek for the causative gene(s) in Pain1, which has already

come to completion. Another unbiased approach to seeking for Pain1 genes is a whole genome

expression profiling array which is used in this study (Veltman and de Vries, 2006). The

rationale for using a whole-genome array to seek for Pain1 genes in the DRGs and the spinal

cord is that both of these tissues are targets of neuroplasticity that may contribute to the contrast

in autotomy behaviour in A vs. B mice. We were interested to isolate the target tissue for Pain1.

We reasoned that if Pain1 controls autotomy in the PNS it would be regulated in the DRGs, and

if Pain1 plays role in the CNS it would be regulated in the spinal cord or the brain. We chose to

look for Pain1 in the spinal cord because of the numerous changes that occur both in neurons

and in glia cells following the nerve denervation which may be attributed by alterations in gene

expression levels.

24

1.9 Hypotheses and Aims

On the basis of this introduction, we posit that:

1. Contrasting spontaneous chronic neuropathic pain (CNP) behaviour following hindpaw

denervation in the mouse is associated with differential gene expression levels in DRGs

associated with the injured nerves, and in lumbar spinal cord segments that process

afferent inputs from these nerves.

2. The environment can influence the levels of spontaneous CNP behaviour of mice

following hindpaw denervation in the mouse by way of regulating the expression level of

some of the genes in the DRGs associated with injured nerves, and/or in the lumbar

spinal cord.

3. One or more genes in mouse Pain1 control the variability in autotomy levels across

strains of mice undergoing the same hindpaw denervation in the DRG, the spinal cord or

the brain.

To address these hypotheses, the aims of this dissertation are:

1. To use whole genome expression microarrays to identify CNP genes in lumbar DRGs

and spinal cords of inbred mice expressing contrasting autotomy levels following

hindpaw denervation by peripheral neurectomy.

2. To identify in these whole genome expression microarrays CNP genes in lumbar DRGs

and spinal cords that regulate their expression level by interaction with the environment,

thereby affecting the levels of autotomy following hindpaw denervation by peripheral

neurectomy.

3. To prioritize the best gene for autotomy in Pain1 using molecular and

immunohistochemical techniques.

25

Chapter 2 Materials and Methods

This project has been approved by the Ethics Committee of the University of Toronto (protocols

numbers: 20006329, 20007793, 20008876 and 20008322). This study also adhered to the

guidelines of the International Association for the Study of Pain and those of the National

Institutes of Health (NIH), which funded this project.

2.1. Induction and assessment of the pain phenotype (autotomy)

2.1.1 Mice

Mouse strains with contrasting autotomy behaviour were used herein to study gene and protein

expression levels that correlate with the phenotype. A total of 233 male mice (79 A/J; 60

C57BL6/J; 39 C3H/HeJ; 38 C3H/HeN; 17 AKR/J) ages 8-12 weeks were used in the studies

reported here (Table 1). For the purpose of gene expression, immunohistochemistry and

behavioural studies, these mice were denervated, sham-operated (see below Surgical Procedures)

or left intact. For the genome wide expression profiling study, spinal cords and DRGs from the

same mice were profiled on individual arrays except for one spinal cord and one DRG isolated

from 2 different mice.

Study I: Genome-wide expression profiling of A vs. B mice: This study was used as a non-

biased approach to seek for candidate genes, particularly in Pain1, associated with autotomy

behaviour. For this study we used 7 groups of mice (detailed below) to seek for contrasts in gene

expression levels within high autotomy mice and low autotomy mouse groups of the A and B

strains. A total of 89 male mice (45 A/J; 44 C57BL6/J) ages 8-9 weeks comprised this study,

including 8 intact mice per strain, 12 sham-operated mice per strain and 24 and 25 mice that

underwent the denervation procedure. For the purpose of gene expression profiling, 5 mice per

group were selected; the groups, naïve (AI, BI), sham (AS, BS), denervated low-autotomy

(ADL, BD) and denervated high-autotomy (ADH).

Study II: Follow-up assays for Csf2rb1 gene: This study was used in order to validate, in part,

the findings of Pain1 candidate gene, Csf2rb1, resulting from the gene expression profiling.

Mice were denervated, sham-operated or were left intact for further qPCR and

26

immunohistological experiments. Additional to the A and B strains, other mouse strains with

contrasting autotomy behaviour were used in this study to validate the up regulation in Csf2rb1

gene expression in high autotomy mice compared to low autotomy mouse groups. A total of 148

male mice (62 A/J; 30 C57BL6/J; 39 C3H/HeJ; and 17 AKR/J) ages 8-12 weeks were included

in this study.

Study III: Studying Csf2rb1-associated genes, Tlr4 and Stat3, with autotomy behaviour: This

study was used to correlate Tlr4 and Stat3 genes with autotomy behaviour and with Csf2rb1 in

mice. Autotomy behaviour and Csf2rb1 gene expression levels were compared between Tlr4

wild-type and mutant mice. pStat3 was compared immunohistologically in the spinal cord and

brain tissues of high autotomy A mice and low autotomy A and B mouse groups. A total of 88

male mice (8 A/J; 3 C57BL6/J; 39 C3H/HeJ; and 38 C3H/HeN) ages 8-12 weeks were used in

this study.

Table 1: Strains, group types and the number of animals per group

Intact = naïve mice, not operated; Sham = transection of the skin and muscles of hindpaw;

Denervated = transection of the sciatic nerve as well as skin and gluteal muscles, transection of

the saphenous nerves involved additional cut of overlaying skin; No/Low = autotomy scores of

0-1; Moderate = autotomy scores of 2-7; High = autotomy scores of 8-11.

Strains A B C3H/HeJ AKR C3H/HeN

Groups

Autotomy levels

No/Low High No/ Low No/Low High No/Low No/Low High

Intact 16 12 6 6 6

16

Sham 20 16 6 6 12

Denervated 18 25 32 17 10 5 4

SUBTOTAL 54 25 60 29 10 17 22 16

TOTAL=233 79 60 39 17 38

27

Autotomy levels by strain:

NLS-A No/low scores of autotomy in A mice

NLS-B No/low scores of autotomy in B mice

NLS-AKR/J No/low scores of autotomy in AKR/J mice

NLS-C3H/HeJ No/low scores of autotomy in C3H/HeJ mice

NLS-C3H/HeN No/low scores of autotomy in C3H/HeN mice

MHS-A Moderate/High scores of autotomy in A mice

MHS- C3H/HeJ Moderate/High scores of autotomy in C3H/HeJ mice

MHS- C3H/HeN Moderate/High scores of autotomy in C3H/HeN mice

2.1.2 Surgical procedures

Animals were deeply anesthetized by an induction dose of 4% halothane inhalation, switched

down to 2% for a maintenance dose. When the animal became irresponsive to noxious stimuli

(pinching the paw or tail), the fur on the left leg was clipped, and under sterile conditions the

sciatic nerve innervating the left hindpaw was exposed unilaterally by cutting the skin and the

overlaying gluteal muscles. The nerve was then tightly ligated with 2 5-0 silk sutures with the

proximal suture being first, and transected in between the ligatures. This order ensured that the

injury introduces only one injury discharge to the nervous system by way of the proximal suture.

The skin was closed with Michel metal clips (#9), which were removed on PO day 14. The

saphenous nerve was exposed on the same side through a skin incision on the medial thigh,

ligated (as above), and cut. The wounds were closed in layers using 4-0 vicryl sutures. For mice

undergoing sham operation, the skin and overlying muscles on the left hindpaw were cut and re-

sutured (as above), leaving the sciatic and saphenous nerves intact. After recovery from the

anesthesia in a holding cage (kept at 300C) the animals were returned to their original cage

groupings, 4 per cage. Mice of the same strain and treatment were caged together and kept in the

animal facility of the Faculty of Dentistry at the University of Toronto under normal husbandry

conditions [i.e., water and chow pellets (2018 Teklad global 18% protein rodent diet that is based

in part on soy protein [www.harlan.com/products])] were supplied ad libitum; a 12 hr light/dark

cycle, lights switched on at 07:00]. Mice were sacrificed on day 14 PO (A/J, C57BL6/J and

C3H/HeN mice), 21 PO (C3H/HeJ and AKR/J mice) or upon reaching the maximal ethically

permissible score of autotomy score of 11 (see 2.1.3, below).

28

2.1.3 Phenotyping autotomy behaviour

Autotomy was scored daily by a single trained observer (MYA) using the following acceptable

scale (Wall et al., 1979). Removal of one nail was not scored as part of the autotomy behaviour

because it could have been caused by a traumatic accident to an insensate nail. Removal of two

or more nails was considered an intentional act driven by the animal’s wish to rid itself of the

pain referred to the spontaneously painful paw. One point was assigned for two or more injured

nails, and for each injured half toe, up to a maximal ethically permitted score of 11 if all 5 toes

were self-injured. If an animal reached this score prior to the end of the study on days 14 or 21,

it was euthanized and its score was added in subsequent days as if it is still alive. These scores

were used in data analyses together with scores of other animals in its group. There is no way to

avoid bias of the experimenter to the strain under study because A mice are albino whereas B

mice are black. However, experimenters were partially blinded to the experimental group within

each strain because both sham operated and denervated mice had skin wounds and metal clips.

Nevertheless, it is impossible to avoid some biasing because denervated mice are paralyzed and

disclose their neurological status by dragging behind the paralyzed leg; whereas sham operated

mice are not. See Table 1 for a summary of strains, experimental groups, treatment types and

phenotypic levels.

2.2 Tissue acquisition

Mice were deeply anesthetised by an i.p. injection of a mixture of urethane (1.125 mg/kg) and

alpha chloralose (75 mg/kg) prior to trans-cardiac perfusion-fixation. The cardiac cavity was

exposed, a metal cannula connected to 2 20ml syringes was driven through the apex to the

ascending aorta and clamped in situ. The right atrium was cut and the circulatory system flushed

with diethyl pyrocarbonate (DEPC)-treated saline (to limit the entry of exogenous RNAases from

the perfusate). Then the body tissues were fixed by perfusion with RNAlater (Ambion,

Burlington, ON, Canada) following the protocol of LeDoux et al. (2006) to maintain RNA

integrity post-mortem. The lumbosacral spinal cord and the ipsilateral L3-L6 DRGs were

extracted under direct vision using a stereomicroscope (at X25), dripping RNAlater during the

entire procedure to maintain RNA integrity and prevent the tissues from drying. For

immunohistochemical staining, mice were flushed with 1% Phosphate Buffered Saline (PBS, 20

29

ml) followed by 4% paraformaldehyde (50 ml), spinal cord segments L3-L6 and whole brains

were carefully removed and stored at 30% sucrose until sectioning.

2.3 RNA extraction

DRG tissues were disrupted and lysed with GITC-containing buffer (buffer RLT; Qiagen,

Mississauga, ON, Canada) using the disposable polypropylene pellet pestle (Sigma, Oakville,

ON, Canada). Next, the samples were homogenized by passing them through a syringe fitted

with a 20-gauge needle. High-molecular weight DNA was sheared by passing the lysate though

the needle at least 20 times or until a homogeneous lysate was achieved. RNA extraction was

then followed using the RNeasy micro-kit (Qiagen, Mississauga, ON, Canada). Briefly, ethanol

was added to the samples to adjust binding conditions, and then applied to RNeasy MiniElute

Spin Columns for adsorption of RNA to membrane. Contaminants were removed with simple

wash steps, and RNA was eluted from the column with RNase-free water.

Spinal cord tissues were homogenized in QIAzol (Qiagen, Mississauga, ON, Canada) using a

tissue rupture (Qiagen, Mississauga, ON, Canada) with plastic disposable probes. RNA

extraction followed thereafter, using the lipid tissue mini-kit (Qiagen, Mississauga, ON, Canada).

Briefly, Chloroform was added to the samples to separate the phases of the lysate, and then

ethanol was added to the aqueous phase to adjust binding conditions. Samples were applied to

RNeasy Spin Columns for adsorption of RNA to membrane. Contaminants were removed with

simple wash spins of buffers RW1 and RPE, and RNA was eluted from the column with RNase-

free water.

RNA purity and concentration was confirmed from the OD readings at wavelengths of 260/280

nm using a dual beam UV spectrophotometer (Biochrom, Holliston, MA, USA) then by running

1.5% agarose gel. For microarray, RNA integrity was determined by capillary electrophoresis

using the RNA 6000 Nano LabChip and the Agilent Bioanalyzer 2100. RNA samples were

stored at -80°C until further analysis.

30

2.4 Microarraying and data analysis

2.4.1 Study design

In this study we arrayed DRG and spinal cord tissue specimen from individual mice, 5

mice per group, to seek for autotomy candidate genes. We discuss our rationale for

choosing this design over other previously published studies in the field in the following

section. In the majority of gene studies, pooled RNA, rather than individual samples

from spinal cord or DRGs, was used for array analyses. On the one hand, group

comparisons, based on whole genome gene expression data that use pooled samples, is

economical because it involves fewer expression arrays. On the other hand, however,

pooling carries an unavoidable disadvantage that one cannot exclude a-priori individual

outliers from the arrayed group, because their RNA is pre-mixed with that of non-

outliers. Having outliers in the group is bound to skew the group average and increase its

variance, as it is impossible to determine which animal sets off the mean of a group,

skewing the group average either to a falsely higher or lower value from the true average

value. Unlike arithmetic averaging that seeks the mean value of a group after a careful

examination of the values contributed by individuals in the group, allowing pre-averaging

exclusion of outliers, such studies produce inherently imprecise data, as opposed to the

use of individual animals. Thus, the pooled approach is bound to be less sensitive in

identifying true findings by increasing false positive and false negative candidate gene

identifications.

In neurobiology studies especially, particular genes that encode for stress-response

proteins and immunoglobulins, can be affected by many factors unrelated to the

experimental treatment. As a result, some individual samples could potentially have

levels 5-10 fold higher than levels typical of the group in some of these genes (Hardiman,

2009). For example, individual animals may be infected or some tissue samples may be

anoxic for relatively long periods before they are preserved, which allows cells to

respond to stress. It is easier to detect and compensate for such aberrations, if individual

samples are hybridized. A comparison of samples from pooled and non-pooled design

found that most identified genes that differentially regulated between the two groups in

31

the pooled study resulted as extreme outliers in only one individual in the non-pooled

study (Hardiman, 2009). Furthermore, examining individual samples separately allows

one to estimate variation between individuals which is sometimes important and often

interesting. Therefore, it is generally recommended against pooling from only a small

number of individuals (n<20) (Hardiman, 2009). For these reasons, this study focused on

individualized array analysis.

Previous gene expression studies on mice, mostly using the pooled approach, focused their

attention either on one tissue, such as DRG, spinal cord or brain, or on one gene, such as using

knockout mice, or on disease causing neuropathic pain. However, no report has appeared in

press on a study in mice after peripheral nerve injury, used the single animal arraying study

design to seek neuropathic pain-related genes in both the DRGs and spinal cord in the same

study. Thus, the significance of the present study is that it is the first to use single animal

arraying to detect altered gene expression levels both in the DRGs and spinal cord in an attempt

to find mechanisms involved in the contrasting neuropathic pain-related behaviour.

In this study, we arrayed RNA extracted from DRGs of individual mice, one mouse specimen

per array, in 7 groups, for a total of 35 arrays. In a different and parallel experiment, we arrayed

RNA extracted from spinal cords of the same individual mice, one mouse specimen per array, in

7 groups, for a total of 35 arrays. The total number of arrays used in our study was 70. Figure 1

shows the study design for this experiment.

32

Figure 1: Study design for microarray experiment. Showing in (A) the 35 arrays divided into 7

mouse groups and corresponding to DRG RNA extracted from 35 individual mice, and in (B) the

35 arrays divided into 7 mouse groups and corresponding to spinal cord RNA extracted from the

same 35 individual mice. Group comparisons are demonstrated by green (Naive A vs. Naïve B)

red (MHS-A vs. Sham A; MHS-A vs. NLS-A; MHS-A vs. NLS-B) and blue (NLS-B vs. Sham B)

arrows. The B sham group was excluded from the main data analysis, but served as an internal

control to isolate genes in the B strain that are involved with denervation, Wallerian

degeneration, nerve regeneration and apoptosis.

A A

1 2 3 4

5 6 7

Mouse 4

Mouse 1 Mouse 2

Mouse 5

Mouse 3

Mouse 9

Mouse 6 Mouse 7

Mouse 10

Mouse 8

Mouse 14

Mouse 11 Mouse 12

Mouse 15

Mouse 13

Mouse 19

Mouse 16 Mouse 17

Mouse 20

Mouse 18

Mouse 24

Mouse 21 Mouse 22

Mouse 25

Mouse 23

Mouse 29

Mouse 26 Mouse 27

Mouse 30

Mouse 28

Mouse 34

Mouse 31 Mouse 32

Mouse 35

Mouse 33

Naïve Sham A

NLSA

MHS

BNaive

BSham

BNLS

= Array; A = A mice; B = B mice; MHS = Moderate/High autotomy scores

NLS = No/Low autotomyscores

B. Spinal Cord

A A

1 2 3 4

5 6 7

Mouse 4

Mouse 1 Mouse 2

Mouse 5

Mouse 3

Mouse 9

Mouse 6 Mouse 7

Mouse 10

Mouse 8

Mouse 14

Mouse 11 Mouse 12

Mouse 15

Mouse 13

Mouse 19

Mouse 16 Mouse 17

Mouse 20

Mouse 18

Mouse 24

Mouse 21 Mouse 22

Mouse 25

Mouse 23

Mouse 29

Mouse 26 Mouse 27

Mouse 30

Mouse 28

Mouse 34

Mouse 31 Mouse 32

Mouse 35

Mouse 33

Naïve Sham A

NLSA

MHS

BNaive

BSham

BNLS

= Array; A = A mice; B = B mice; MHS = Moderate/High autotomy scores

NLS = No/Low autotomyscores

A. Dorsal Root Ganglia

33

2.4.2 Agilent Two-Color Microarray Protocol

Microarray-based gene expression analysis was performed using the Agilent 4x44K gene chip

Microarray Centre, UHN, Toronto, ON, Canada). Complementary RNA (cRNA) was amplified

for a two-color microarray gene expression analysis. Labelled cRNA was synthesized using

double-stranded cDNA (Quick Amp Labeling protocol, Agilent): first and second strand cDNA

was synthesized from 200 ng or more total RNA using an MMLV-RT Oligo dT promoter Primer

containing a T7 RNA polymerase promoter site (Agilent Quick Amp Kit, Two-Color,

Mississauga, ON, Canada); the cRNA was synthesized and labelled with Cy5-CTP or Cy3-CTP

NPTs by in vitro transcription using T7 promoter-coupled double stranded cDNA as template;

and the amplified cRNA product was purified using an RNeasy mini spin column (Qiagen,

Mississauga, ON, Canada) with buffer RPE. The yield of the in vitro transcription reaction was

determined by product absorbance at 260 nm measured using NanoDrop ND-1000 UV-VIS

Spectrophotometer (Agilent, version 3.2.1, Mississauga, ON, Canada). Following labelling,

samples were hybridized to the Agilent 4x44K gene chip, using the Agilent Gene Expression

Hybridization Kit (65ºC, 17 hrs, 10 rpm). Arrays were washed and scanned immediately to

minimize the impact of environmental oxidants on signal intensity (GenePix 4000B scanner,

Molecular Devices, Sunnyvale, CA, USA). The microarray scanned data was extracted to

images using Agilent Feature Extraction Software, producing output files of the ‘*.tiff’ format,

for determination of gene expression levels. To avoid bias, samples were blinded and treated on

the microarray in a randomized fashion.

2.4.3 Expression array analyses

Log ratio data from the Agilent 2-color microarray were imported into and analyzed with Partek

Genomic Suite software version 6.4. Two independent gene expression analyses were performed

for mRNA extracted from: (i) the DRGs, and (ii) spinal cords. For each microarray analysis,

gene expression levels were compared within- and between-strains of the intact/naive, sham-

operated and denervated A and B strains, for a total of 4 comparisons. Each group comprised

five animals that were selected for meeting the criteria of expression of certain levels of

autotomy (or the lack thereof) following hindpaw denervation, and satisfactory perfusion

34

fixation, judged the by hardness of the perfused material. Total RNA from each animal

represented one biological sample for arraying.

Prior to running the statistical tests, samples were controlled for the following factors in a four-

way ANOVA: (i) Batch Day, (ii) Strain (A vs. B), (iii) Operation Type (naïve vs. sham vs.

denervated), and (iv) Autotomy Phenotype (NLS vs. MHS of 2-11). The outcome for controlling

the samples yielded 2 outliers that were excluded from the statistical analysis (one denervated

NLS-A mouse was excluded from DRG data; one B sham mouse was excluded from spinal cord

data). For those 2 groups that had an outlier removed, the statistical tests were performed on 4

mice per that group, instead of 5 mice per group.

Following correction, each two selected comparison groups were analyzed by ANOVA, and

gene lists were then generated according to the following criteria: p-value<0.05 and -1.5<fold

change>1.5. An independent comparison between naïve/intact A vs. B mice (AI vs. BI) was

performed. Three additional independent comparisons between groups of denervated mice

expressing high-autotomy vs. low- autotomy scores were performed and were later used in a

VENN analysis. The three comparisons were: (i) between denervated MHS-A mice having

moderate-to-high autotomy scores (ADH) vs. sham-operated A mice who never expressed

autotomy (AS), (ii) ADH vs. denervated NLS-A mice expressing no or low autotomy scores

(ADL), and (iii) ADH vs. denervated NLS-B mice who typically express no or low autotomy

scores (BD). VENN analysis of all 3 comparisons yielded a shortlisted number of candidate

genes in the DRGs and spinal cords associated only with the expression of MHS in denervated A

mice, but not in sham operated A mice and not in B mice. This analysis excluded genes whose

expressions levels were regulated with the denervation. A final independent comparison within

the B strain (NLS-B vs. sham-operated B mice) was used as an internal control to seek genes in

B mice that associate with nerve degeneration, regeneration and apoptosis, and genes for

resistance to autotomy (See Table 2).

Contrast type Yield genes associated with:

AI vs. BI Injury discharge, genes differential between strains A and B ADH vs. ADL Autotomy behaviour (GXE) ADH vs. AS Nerve degeneration, regeneration, apoptosis in A mice ADH vs. BD Autotomy behaviour, genes differential between strains A and B BD vs. BS Nerve degeneration, regeneration, apoptosis and autotomy resistance in B mice

Table 2: Comaprison types and genes associated with each comparison type

35

2.4.4 Gene interaction network analysis

The products of candidate genes that were considered in this study are expected to operate each

in its tissue target. However, genes interact within genetic networks where their products affect

the expression of one or more genes thereby forming a biochemical pathway underlying

physiological processes. Moreover, sometime a gene is pleiotropic, where its product operates in

more than one such network or process. Identifying such networks and processes places a

candidate gene within a functional context that is of great interest to unravel. Functionally

related genes can be found using network analysis. For the purpose of our work we made use of

GeneMANIA, a website that searches publicly available biological datasets to find functionally

related genes (http://www.genemania.org/). These include protein-protein, protein-DNA and

genetic interactions, pathways, reactions, gene and protein expression data, protein domains and

phenotypic screening profiles. The names of candidate genes for autotomy in DRGs and spinal

cord (identified from the genome-wide gene expression results) were fed by us into

GeneMANIA to seek for gene interaction networks. The nodes connecting between every two

genes were denoted in a specific color/category, depending on the type of interaction between

the two genes.

For example, an interaction denoting co-expression means that two genes were linked if their

expression levels were similar across conditions and time series. Data used for such an analysis

originated in gene expression studies in previously published gene expression articles.

Similarly, an interaction denoting physical interaction means that two gene products were

linked over time in reported protein-protein interaction studies.

Genetic interaction means that two genes were functionally associated if the reported effects of

perturbing one gene were found to be modified by perturbations to a second gene over time.

Shared protein domains means that two gene products were linked if published reports

indicated that they have the same protein domain.

Co-localization means that two genes were linked if they were both expressed in the same tissue

or if their gene products were both reportedly identified in the same cellular location.

36

Pathway denotes instances where two gene products were reportedly linked in the same reaction

within the same pathway.

Predicted denotes any predicted functional relationships between reported gene products, often

protein-protein interactions. A major source of data for predicting a gene’s function originates

from comparative analysis of its functional relationships in other organisms via orthology. For

example, two proteins can be predicted to interact if their orthologs in another organism are

known to interact. In such cases, network names describe the original data source of

experimentally measured interactions and from which organism(s) the proposed interactions

derive.

Other denotes any networks that did not fit into any of the above categories. Examples include

phenotype correlations from Ensembl (www.ensembl.org) disease information from OMIM

(www.omim.org) and chemical genomics data (Bredel and Jacoby, 2004).

2.4.5 SNP variation in inbred mouse strains with known levels of

autotomy behaviour

The MGI SNP Variation Database

(http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=snpQF) includes mapped data on

SNPs of tens of inbred mouse strains. We used it in this dissertation to search for SNPs in the

Pain1 region that differed across autotomy contrasting strains and could be implicated as

causative variants in candidate autotomy genes). To this end, SNPs were searched in the genes

shortlisted as candidate genes for autotomy in the Pain1 QTL. For each gene, SNPs were

screened in the following genetic regions: mRNA un-translated regions (UTRs), synonymous

coding regions, non-synonymous coding regions, and introns, using the following criteria. SNPs

were considered as candidate autotomy related SNPs if they differed between: (1) A and B mice

and (2) Strains known from previous studies to express MHS following hindpaw denervation

(i.e., C3H/HeJ, BALB/CByJ, or CBA/J) vs. strains known to express NLS following the same

denervation (i.e., AKR/J). SNP data was not available for C58/J mice.

37

2.4.6 Interrogating published Csf2rb1-immunohistochemically

labelled spinal cord and brain slices of naïve B mice

The Allen Brain Atlas (http://www.brain-map.org/) is an internet-based resource that published

histological photomicrographs of brain and spinal cord slices from a 56 days old naïve C57BL/6J

male mouse, using the in situ hybridization ISH method to label various murine genes with

radiocatively labelled riboprobes. Regrettably, this resource did not include micrographs of

Csf2rb1, but of Csf2rb2, and representative slides are included in this dissertation and their

relevance is discussed herein.

2.4.7 Mythological aspects of literature survey of the biological

relevance of candidate genes

A Pubmed search was performed for each of the candidate genes in Pain1 with the following

keywords: ‘brain’, ‘nerve’, ‘CNS’, ‘spinal cord’, ‘nerve injury’, ‘inflammation’ (‘inflamm*’),

’neurodegenerat*’, ‘analges*’, ‘pain’, ‘nocicept*’, ‘neuropath*’, ‘hyperalges*’, and ‘allodyn*’.

Any gene that was co-notated in 3 or more keywords was considered a candidate gene.

2.5 Gene follow-up assays

2.5.1 Quantitative real-time PCR

Since our results identified in the gene expression microarray study that up-regulation in spinal

cord, but not DRGs, levels of Csf2rb1 is the only candidate autotomy gene in Pain1, in the

follow up study we only validated the expression of this gene in the spinal cord. This was

carried out on A and B mice of the same 7 groups (at least 3 mice/group) but using a new batch

of mice that did not participate in the gene expression microarray study. cDNA was synthesized

from 1 μg total RNA (QuantiTect Reverse Transcription Kit, Qiagen) as follows. RNA

purification with gDNA wipe out buffer for 2 min at 42°C preceded the reverse transcription

reaction with Quantiscript Reverse transcriptase (1µl), Quantiscript RT Buffer x4 (4µl) and RT

Primer Mix (1µl) in a 20 µl reaction (Qiagen) for 15 min at 42°C. Reverse Transcriptase was

38

immediately inactivated by incubation for 3 min at 95°C. Gene-specific primers were designed

using the Integrated DNA Technologies website (www.idtdna.com). To reduce the contribution

of contaminating genomic DNA, primers were designed to span exon junctions. The primers: for

Csf2rb1 were mCSF2RB1_F TCGCTTTGGCTGTGTCTCTGTATACAG and mCSF2RB1_R

CATGCTGCCAGGAGGCCAG, and for Hprt mHPRT_F GTTGTTGGATATGCCCTTGA, and

mHPRT_R AGATTCAACTTGCGCTCATC.

Templates were amplified at the following PCR conditions: An initial incubation for 3 min at

95°C and 44 cycles of 15 sec at 95°C, 30 s at 66°C, and 10 sec at 95°C. The gene encoding for

the enzyme hypoxanthine guanine phosphoribosyl transferase 1 (Hprt) was chosen as the

reference gene for normalization because it is a constitutively and stably expressed in the

mammalian brain regardless of treatments [for example, in a murine model of epilepsy (Pernot et

al., 2010)], and therefore, it is frequently used as a reference gene (de Kok et al., 2005; Kõks et

al., 2008; Korostynski et al., 2007). Other reference genes that we checked in the spinal cord

(i.e., S18 rRNA and Gapdh) using temperature gradient RT-PCR, were not used in our following

experiments because they did not produce a stable PCR product (i.e. fragment was detected

following agarose gel electrophoresis). Reactions were performed in quadruplets on a

MiniOpticon (BioRad) cycler using SsoFast EvaGreen Supermix (10 μl, Biorad), cDNA

template (1 μg), and gene-specific primers (2.5 pmol) in a 20 µl reaction volume.

qPCR levels were compared in the new batch of A and B mice (i.e., mice that did not participate

in the gene expression microarray study) and mice of other strains with known contrasting

autotomy levels (i.e., C3H/HeJ and C3H/HeN), known from previous studies, including from our

lab, to express MHS following hindpaw denervation vs. AKR/J that are known to express NLS

following the same procedure. For the purpose of qPCR analysis, mice were denervated, sham-

operated, or left intact as described above. All denervated mice were followed up PO and scored

for autotomy prior to qPCR analysis as described above.

2.5.2 Immunohistochemistry

Using a cryotome, frozen tissues were sectioned into 50 μm sections. Floating sections were

washed with 1% PBS three times for 15 min at room temperature, and were then blocked with

10% donkey serum (in 3% triton) for 50 min at room temperature. The side of the spinal cord

39

ipsilateral to the surgical procedure was marked by a small indent in the ventral horn. The

sections were incubated for 48 hrs at 4°C with primary antibodies prepared in 1% PBS solution

containing 3% donkey serum and 3% triton, including the following antibodies:

1. Mouse NeuN (1:2,000, Chemicon, Temecula, CA, USA) was used as a neuronal marker.

2. Mouse OX-42 (1:2,000, Abcam, Toronto, ON, Canada) was used as a microglial marker.

3. Mouse GFAP (1:2,000, Sigma, Oakville, ON, Canada) was used as an astrocyte marker.

4. Mouse NG2 (1:1,000, Millipore, Burlington, MA, USA) was used as an oligodendrocyte

marker.

5. Mouse MAP2 (1:2,000, Chemicon, Temecula, CA, USA) was used as a neuronal marker.

6. Mouse anti-human Vimentin (1:200, Millipore, Burlington, MA, USA) was used as an

ependymal cells/ radial glia/tanycytes marker.

7. Rabbit IL3Rβ (1:500, Santa Cruz, Dallas, TX, USA) was used to label CSF2RB1.

8. Rabbit pStat3 (1:500, Cell Signalling Technology, Burlington, MA, USA) was used to label

phosphorylated STAT3 (on Tyr705).

Sections were washed with 1% PBS 3 times for 15 min at room temperature and were then

incubated for 3 hrs at room temperature with light-protected secondary antibodies prepared in

1% PBS solution containing 3% donkey serum and 3% triton (donkey anti-rabbit Cy3 1:1,000

Jackson ImmunoResearch Laboratories, INC., West Grove, PA, USA; donkey anti-mouse Cy5

1:1,000 Jackson ImmunoResearch Laboratories, INC., West Grove, PA, USA. Following

incubation, sections were washed again with PBS 3 times for 15 min at room temperature and

remained protected from light to prevent bleaching and fading of the fluorescence signal.

Sections were mounted on glass slides and then cover-slipped with a mounting medium (Sigma,

Oakville, ON, Canada). Labelled tissue slides were observed using a Zeiss epi-fluorescent

microscope. Select slides of representative tissues were used for Z-stack photographic

recordings.

Immunohistochemistry controls: Spleen tissue is known to express CSF2RB1

(http://www.biorbyt.com/csf2rb-antibody-5). Therefore, in a pilot experiment that was carried

out prior to using the IL3Rβ antibody against spinal cord CSF2RB1, we used murine spleen

tissue as a positive control to verify that the antibody is indeed specific. As a negative control, 2

spinal cord sections were labelled with only secondary antibodies, to determine antibody

40

specificity with the primary antibody sections. To prevent background staining, solutions of the

antibodies were diluted to the optimal concentration, as well, blocking, reaction times and

temperature of incubation, were optimized. To exclude other effects such as autofluorescence,

optimal tissue fixation processes were adopted.

2.5.3 Quantitation of CSF2RB1 labelled cells in the spinal cord

and brain

In spinal cord transverse sections, CSF2RB1-positive cells having a soma located around the

central canal and extending processes dorsally, ventrally or laterally were counted if their

processes extended for a distance from the soma that was at least 20 μm in length. CSF2RB1-

positive labelling was also found in coronal brain sections, mainly throughout the dentate gyrus

of the hippocampus and in the hypothalamus (mostly in the arcuate nucleus). Each cell extended

a thinner process than those of the spinal cord around the central canal. Labelled processes were

arranged in a parallel manner, traversing the dentate gyrus and the arcuate nucleus perpendicular

to their layers. This parallel arrangement and uniform labelling enabled us to count the number

of labelled processes in the dentate gyrus as follows. An arbitrary region of interest (ROI) was

selected and a 200 μm line was drawn on the photomicrograph, perpendicular to the processes.

The number of CSF2RB1-positively labelled processes that crossed the line was counted per a

standard unit length (200 μm). To avoid bias by the experimenter, images were coded to conceal

the identity of the mouse strain and treatment group. ROIs were selected in a sequential manner

for the randomized sections: upper right aspect of the dentate gyrus, lower right aspect, upper left

aspect and lower left aspect.

2.5.4 Co-localization analyses

All images showing co-localization of CSF2RB1 with Vimentin positive cells were first z-

stacked by the Volocity 3D Image Analysis Software (version 6.1.1, PerkinElmer, Woodbridge,

ON, Canada) using the DE convolution feature prior to co-localization analysis. A region of

interest around the central canal was chosen for setting the background fluorescence threshold,

and then the software assesses the level of co-localization of CSF2RB1 with Vimentin by

calculation of the Pearson Correlation Coefficient and p value that determined how many

41

profiles containing one label also contained the other label. To avoid bias, images were coded to

conceal the mouse strain and treatment.

2.5.5 Counting pStat3 labelled cells in the spinal cord and brain

In the spinal dorsal horn, pStat3+ cells appeared as small dots indicating labelling of the nuclei.

Such cells were present in laminae I and II of the dorsal horn as well as in the ventral horn, but

devoid of the central canal region. Here we only counted those located in lamina I and the inner

part of lamina II, since this region is associated with pain processing. The region of interest

where cells were counted was a square 0.01mm2 (100μm x 100μm). pStat3+ cells were counted

both in the ipsilateral and contralateral sides of the dorsal horn. In the polymorph layer of the

hippocampal dentate gyrus a region of interest measured 2500 μm2

was used for counting

labelled cells. pStat3+ cells were counted bilaterally. Avoiding bias was accomplished as

described above. ROIs in all slides were selected in the same region in the middle of spinal

dorsal horn, in laminae I and II.

2.5 Statistical analysis

Analysis of genome wide gene expression microarray data was carried out by the Partek

Genomic Suite (Version 6.4; Partek Inc.). All other data (behaviour, qPCR and

immunocytochemistry) were analyzed using SPSS (version 13.0). Normality tests determined

distribution type prior to data analysis. One-way or two-way ANOVA tests and an independent

2-tailed T-test were used, where appropriate, to determine the level of significance of the

difference in the means of tested groups. Groups included MHS, NLS, or the combination of the

two (Denervated), Sham-operated, Naïve, or the combination of the two (Control) in the

following strains: A, B, AKR, C3H/HeJ and C3H/HeN. Tukey or LSD post hoc tests were used

to identify which groups contrasted significantly in the ANOVA tests. Independent variables:

Group and Day PO. Dependent variables: Autotomy score, gene expression levels, number of

cells. P<0.05 was considered significant in all tests. When the same set of data was used in

more than one statistical comparison, inflation of the alpha level was adjusted with the

Bonferroni factor. FDR corrections were used for P<0.05, P<0.01 and P<0.001. All averages

appear ± SEM.

42

Chapter 3

Study I: Regulation of autotomy levels by gene expression

changes: Whole genome study of the DRGs and spinal cord

3.1 Introduction

The complexity of neuropathic pain remains a challenge for researchers trying to decipher the

genetic factors that contribute to understanding the mechanisms involved and identifying novel

targets for analgesics. Neuropathic pain is a complex trait resulting from various types of insults

to the nervous system. We modelled neuropathic pain in response to peripheral nerve injury, as

is seen following trauma and surgery (e.g., in leg amputees and women post-mastectomy). To

this end, we used the Neuroma Model (Wall et al., 1979) in the mouse to seek genes involved in

the development and maintenance of spontaneous neuropathic pain. We selected this rodent

model because it closely mimics spontaneous neuropathic pain following peripheral denervation

in humans, for which there is still a tremendous need for analgesics that confer an effective

alleviation of pain without adverse side effects. The Neuroma Model was developed by our

group in 1979, has since been published in hundreds of reports including many reports by our

group, and much is known about the pathophysiology involved.

While humans have a diverse genetic makeup, that of inbred mice is strain-specific, making the

latter a good animal model advantageous toward studying complex traits such as neuropathic

pain. Like individual humans, strains of inbred mice of different genetic backgrounds are

susceptible or resistant in various degrees to develop chronic pain in a model-specific manner

(Mogil et al., 1999a, 1999b), which marks an advantage starting point in associating genotype to

phenotype, as well as the impact of the environment given an identical genome (Seltzer and

Dorfman, 2004). For example, certain inbred mouse strains show high autotomy scores, while

others show moderate, low or very low scores, even though the inciting injury (i.e. denervation

of the hindpaw) was the same (Devor et al., 2007; Mogil et al., 1999a). Based on this phenotypic

variation we previously mapped a QTL on mouse chromosome 15 associated with autotomy,

which we named Pain1, using the contrasting autotomy strains A and B and their 23 recombinant

inbred strains (Seltzer et al., 2001). In the present study, we used the same contrasting strains in

43

a non-biased approach to carry out a genome-wide gene expression to seek for candidate genes

for autotomy in the DRGs associated with the injured nerves, and the spinal cord, where these

original nerves terminate and where pain processing is altered due to the injury.

It is noteworthy that sexual dimorphism exists in pain phenotypes, including autotomy, such that

the same mouse strain may be highly susceptible to autotomy in one sex and less susceptible to

autotomy in another sex (Devor et al., 2007; Mogil et al., 1999a). Therefore, this study focused

on male mice only. Autotomy expressing male mouse strains include the A/J, BALB/cJ,

C3H/HeJ and C3H/HeN, CBA/J and SM/J (‘autotomy susceptible’ strains), while the strains

C57BL6/J, C57BL10/J, C58/J and AKR/J express relatively low autotomy following the same

denervation procedure (‘autotomy resistant’ strains). The intermediate strains that develop

autotomy at medium scores include 129/J, RIIIS/J and DBA/2J. These levels depend on the lab

where the study was carried out and the environmental conditions at the time of the study

including diet, type of anesthetic agent used, and other intra-operative variables including the

way the nerves and tissues were handled by the experimenter, and housing conditions (e.g.,

social “cage effects”). This information is important because the availability of inbred strains of

mice and rats is informative not only in the context of genetic variability but also for unraveling

epigenetic factors; both aspects are analyzed in the present study. Since most mouse strains were

already densely mapped for SNP variations across the genome, based in part on the full sequence

map produced by Celera, knowing their autotomy levels (under specific environmental

conditions) is a powerful tool that enables investigators to identify candidate genes associated

with this trait, a feature that will be used in the present study to prioritize candidate genes based

on having highly polymorphic regions between autotomy A, B and other susceptible and

autotomy resistant strains.

Numerous studies already used genome-wide expression profiling as an initial method to seek

candidate genes and pathways contributing to neuropathic pain. Some used peripheral nerve

injury rodent models (Coyle, 2007; Griffin et al., 2007; Kim et al., 2009b; Ko et al., 2002; Kõks

et al., 2008; Lacroix-Fralish et al., 2006; Liu et al., 2012a; Nesic et al., 2005; Persson et al.,

2009b; Rodriguez Parkitna et al., 2006; Sun et al., 2002; Valder et al., 2003; Wang et al., 2002a),

while others focused on other types of insult, such as inflammation pain models (Géranton et al.,

2007) or disease-caused pain models (Takasaki et al., 2012). Other studies used mice with

44

specific knocked out genes to study the role of a specific gene in relation to pain behaviour

(Kõks et al. 2008; Wang et al. 2010). In one study the injury was to the CNS (Nesic et al.,

2005). Studies profiled genes from DRGs, spinal cord or brain tissues, mainly from the rat, and

very few studies were performed in mice models. Two studies on rats profiled genes in both

DRG and spinal cord associated with neuropathic and inflammatory pain (Rodriguez Parkitna et

al., 2006; Wang et al., 2002a). In the present dissertation study we profiled genes in key PNS

(i.e., DRGs) and CNS (i.e., spinal cord) tissues in the mouse that are known to process normal

daily pain, and change following injury to produce neuropathic pain. The rationale for studying

genes in both tissues in described in the next section.

Some triggers of the neural mechanisms that produce chronic pain are believed to operate very

early after injury. As described in the Introduction (Chapter 1) these signals originate in injured

tissues and nerves, informing the soma of injured primary afferents (and perhaps trans-

synaptically also the CNS) that an injury has just occurred. One of these signals is an electrical

neural message (‘injury discharge’, (Cohn and Seltzer, 1991; Devor and seltzer, 1999; Seltzer et

al., 1991b, 1991c; Vatine et al., 1998; Wall et al., 1974; Zeltser et al., 2000). Thus, genes

involved in determining the excitability of primary afferents to injury resulting in the emission of

an injury discharge at the time of nerve injury, as well as genes that operate in the CNS and deal

with the impact that injury discharge has on the CNS are important determinants of chronicity,

because difference in the type and abundance (i.e., constitutive expression levels) of genes

encoding these signals may explain the contrast in autotomy between the studied groups. These

autotomy genes may encode for a higher excitability level of primary afferent neurons when, for

instance, A mice are injured and a lower excitability in B mice when they are similarly injured.

We reason that higher excitability in A mice at the time of insult would manifest in more injured

afferents firing an injury discharge or firing at a higher frequency than B mice undergoing the

same injury. In addition, other genes may control the dynamic balance of the excitations and

inhibitions in the CNS that process the impact of injury discharge when it floods the spinal (or

trigeminal) dorsal horn with synaptic inputs. For example, the expectation is that B mice, having

genes encoding inhibitions expressed at considerably higher levels than A mice, are posed to

better cope with the injury discharge than A mice, by inhibiting the injury discharge before it

impacts the CNS pain pathways and triggering the cascades of chronic pain. To this end, we

sought genes in the DRGs and spinal cord having significantly different expression levels in

45

intact A (‘AI’) vs. intact B (‘BI’) mice (at a genome-wide significance level and at a 1.5<fold

change <-1.5) .

Another approach we used in the present study represents the traditional study design, seeking

autotomy genes by contrasting expression levels after the nerve injury has taken place in MHS

vs. NLS mouse groups, adjusted by the expression levels in groups of sham-operated mice to

exclude genes related to surgery and anesthesia. Specifically, we compared gene expression

levels in MHS mice to 3 independent groups of NLS, to produce a candidate gene list of

autotomy-causing and alternatively autotomy-protecting genes. Some of these genes are thought

to be regulated in the PNS and others in the CNS because both of these sites contribute to the

development of chronic neuropathic pain by mechanisms of peripheral and central sensitization.

To this end we sought autotomy candidate genes in the DRGs, where potential gene regulation of

the injured nerves occurs and in the spinal cord, where gene regulation of 2nd

order neurons,

interneurons and glial cells, all affecting neuropathic pain neurotransmission, proceeds. Our

main focus was to detect the Pain1 candidate gene that is regulated either in the DRGs or in the

spinal cord.

3.2 Results

3.2.1 Autotomy phenotypes in denervated A and B mice

Prior to the whole-genome microarray experiment that represents this study, we phenotyped our

mice and selected 5 mice per group for tissue extraction. Seven groups of mice comprised this

study: 4 groups of A mice and 3 groups of B mice. Two groups of naïve mice of the A (AI) and

B (BI) strains were not operated and as expected, none expressed autotomy behaviour. Two

groups of sham-operated A (AS) and B (BS) mice were subjected to general anesthesia and

tissue injury, but not nerve injury, and as expected, they also did not express autotomy. B mice

that underwent complete hindpaw denervation did not express autotomy or expressed very low

levels of this behaviour (i.e., final scores of 0-1; BD). This was also expected from the literature,

except for the report by Mogil et al. (Mogil et al., 1999a) who found that denervated B mice

expressed the highest levels of autotomy compared to 10 other inbred lines. However, following

our results (Seltzer et al., 2001) that reported that B mice expressed no- or very low autotomy

46

levels, in a follow up study Mogil et al. retracted from this oversight (Devor et al. 2005) . By the

end of the behavioural follow up on day 14 PO, the group of A mice that underwent the same

denervation procedure as B mice, expressed a wide range of autotomy scores varying from 1 to

11 (Figure 2b). The first day, in which any sign of autotomy was observed (Autotomy Onset

Day; ‘AOD’) in these mice, also varied widely from day 1-14. Five of the denervated A mice

that expressed the highest autotomy scores on PO day 14 (ADH) were selected for the gene

expression study along with 5 denervated A mice that expressed the lowest levels of autotomy on

the same PO day (ADL). Figures 2 and 3 indicate the average daily autotomy score and the

AOD of the groups included in the gene expression study (respectively). Figures 2 and 3 show

that differences between ADH vs. ADL and BD mice is highly significant.

47

Figure 2: Course of autotomy behaviour in denervated A and B mice. Following sciatic and

saphenous nerve section, A mice were followed up daily and their autotomy behaviour was

scored up to day 14. (A) Average autotomy scores (± SEM) are presented for A mice (N=25) vs.

B mice (N=24). ANOVA for repeated measurements with Tukey’s post hoc test were performed

for all 14 days; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (B) Final autotomy scores on

post-operative day 14 are shown for all 49 A and B mice. Based on these scores, mice of the A

strain were sub-divided into 3 groups, NLS-A group with final autotomy scores of 0 or 1, N=10),

a moderate autotomy group having on day 14 PO scores ranging from 2-7 (N=7), and a group of

high autotomy scores ranging from 8-11 (N=8). All 24 B mice showed NLS of 0-1.

0123456789

1011

0 5 10 15 20 25

Fin

al a

uto

tom

y sc

ore

Mouse number

Autotomy in A and B mice on day 14 PO

A high-autotomy

A moderate-autotomy

A low-autotomy

B low-autotomy

B

0

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Au

toto

my

sco

re

Post operative day

Autotomy in A and B mice

A mice

B mice

A

*

***

**

****

****** ***

****

********

********

48

Figure 3: Average autotomy onset day of denervated A and B mice. Group averages of the first

day any score of autotomy was observed (Autotomy onset day) are presented for A mice with

high autotomy scores (N=15) and of the 2 groups that expressed NLS throughout the 14 days of

PO follow up period: A mice (N=10) and B mice (N=24). A one-way ANOVA with the

Tukey’s post hoc test revealed that denervated MHS-A mice started to express autotomy at a

significantly shorter delay than the 2 other NLS groups.

3.2.2 Criteria for candidate gene selection

In order to select for candidate genes in the DRG and the spinal cord, we followed a specific set

of criteria. First, we performed one-one group comparisons that yielded long lists of genes

responsible for inducing ‘injury discharge’ and ‘autotomy’ before or after denervation in MHS-A

mice. Next, we considered statistical corrections for multiple testing to reduce the number of

candidate genes. Using a VENN analysis strategy, we were able to look closer at shared

autotomy genes between different group comparisons and to exclude genes associated with the

nerve injury which were not relevant to autotomy. Using the GeneMANIA website that

integrates a given set of genes into gene network interactions, we distinguished candidate

autotomy genes further by acquiring the biological processes and cellular pathways in which

they are engaged. Since this analysis yielded a series of candidate genes, we chose to focus

particularly on the Pain1 region, for which we had previous knowledge for controlling autotomy

behaviour in A vs. B mice.

0

2

4

6

8

10

12

14A

uto

tom

y o

nse

t d

ay

Autotomy onset day in A and B mice

A high-autotomy

A low-autotomy

B low-autotomy

P<0.0001P<0.0001

49

It should be noted here that meanwhile we performed our individual group comparisons in a non-

biased approach of selecting candidate autotomy genes throughout the genome, we found the

Pain1 gene, Csf2rb1, as the second most significant (P<10-05

) genome-wide gene in the group

comparison ADH vs. ADL in spinal cord, as well as the 7th most significant genome-wide gene

in the group comparison ADH vs. AS (P<10-04

) within this tissue. This gene was also

significantly different in the genome-wide comparison ADH vs. BDL (P<10-05

) in the spinal

cord. We discuss its candidacy further in later sections and show why we selected it our primary

candidate gene for further study.

The following additional criteria were used to select for the best candidate gene in Pain1.

Candidate Pain1 genes that yielded from the VENN analysis following nerve injury were sought

out in the Allen brain atlas for their in situ expression in the murine spinal cord. Candidate

Pain1 genes that differentially regulated between autotomy susceptible A mice vs. autotomy

resistant B mice before the nerve injury were analyzed in the following manner. Polymorphisms

that may explain the differences between A and B autotomy levels were sought in these genes.

The pain relevance of candidate genes was determined using a Pubmed search for key pain

words. The next sections describe each criterion in full detail.

3.2.3 Group comparisons of genome-wide gene expression data

Our analysis for the genome-wide expression data consisted of several one-one group

comparisons using the 7 studied groups (Section 2.4.1 Study design, Figure 1). Separate

analyses were applied to the DRG microarray dataset and for the spinal cord dataset. For each

target tissue, four separate group comparisons were carried out. The first group comparison

detected mRNA differences in intact A (AI) vs. intact B (BI) mice. This comparison followed

the natural history of chronic pain chronologically, seeking potential differential mRNA levels

between these two strains at the time of nerve injury. Such variations may affect the injury

discharge and its impact on the CNS in A vs. B mice. The dataset resulting from comparing

spinal cord genes in AI vs. BI is expected to emerge constitutively expressed genes contributing

to alterations in apoptosis and excitotoxicity of pain inhibitory interneurons in spinal cords of A

vs. B mice. This potential difference may trigger central sensitization in A mice that could drive

the nervous system to produce more chronic pain in these mice compared to B mice, and may

50

explain, in part, why A mice express higher autotomy levels. The dataset resulting from

comparing DRG genomes of AI vs. BI is expected to yield genes associated with the immediate-

early response of primary afferents to injury and initiation of Wallerian degeneration and

regeneration.

The next three group comparisons provided autotomy associated genes. Our strategy for

selecting candidate genes for autotomy was to compare the ADH group (i.e., A mice expressing

MHS of 2-11) with other groups of mice that express NLS (i.e., A sham, NLS-B mice, and NLS-

A mice), and then use VENN analysis to select genes that were significantly regulated in all

three of these group comparisons (see Section 2.4.2 for detailed methods; see Figures 4 and 5 in

Results).

3.2.3.1 Constitutive gene expression levels of autotomy-related

genes

Genes showing a significant fold change in the constitutive expression levels in the DRGs and

spinal cords of intact A (‘AI’) vs. B mice (‘BI’), of more than 1.5, or less than -1.5 (at P<0.05)

were listed. A summary of their numbers in these tissues and strains is shown in Table 3

indicating that more than 5000 out of 44,000 probes on the array (corresponding to ~4500 genes

in either tissue) were differentially expressed throughout the genome in mice of the two strains at

P<0.05. However, many of these are false positive determinations that were included as an

artefact of the multiple comparisons (i.e., 44,000 probes). To compensate for the outcome of the

inflation of the alpha level, a False Discovery Rate (FDR) analysis (at various levels) was carried

out, and as expected, at a level of FDR<0.05 the number of probes differentially expressed in the

DRG and spinal cord of the 2 strains dropped to 1296 and 973 probes, respectively. Reducing

the significance level to FDR<0.01 caused an additional reduction in the number of probes to

760 and 594 probes, respectively, and at FDR<0.001 the numbers dropped further to 439 and

321 probes, respectively. Thus, the number of probes with contrasting constitutive expression

levels across the two strains remained high. It is conceivable that some of these genes may

confer a susceptibility or resistance to the development of autotomy post- hindpaw denervation.

However, in addition to these genes, many other genes are likely involved in other contrasting

phenotypes between intact A and B mice that are unrelated to the contrast in autotomy. One way

51

of coming closer to identifying candidate autotomy genes is to look only at genes harboured in

the Pain1 autotomy QTL and select those that contrast between the strains.

In Pain1 alone, there were 20 probes encoding 16 genes that differentially contrasted between A

and B intact mice in the DRGs (Table 4). In the spinal cord, a total of 25 probes encoding 22

genes were constitutively and significantly regulated between the two mouse strains (Table 5).

Each of these was a possible candidate gene responsible for the production and/or response to

injury discharge immediately after the nerve injury, and thereby possibly explaining the

contrasting auotomy behaviour in these strains. In section 3.2.9 we provide the SNP analysis for

candidate genes in Pain1 and discuss further the candidacy of potential genes that may be

associated with injury discharge.

Table 3: Probes and genes with contrasting expression levels between naïve A vs. B mice are

presented. Spinal cord (SC) and DRG probes throughout the genome show a significant contrast

in the expression levels in naïve/intact A and B mice. These probes include genes likely

associated with the production of, and/or the response to injury discharge, and are therefore

susceptibility genes to developing autotomy behaviour in A mice or its prevention in B mice.

P<0.05 FDR<0.05 FDR<0.01 FDR<0.001

5116 1296 760 439 Probes

4600 1160 680 395 Genes

5164 973 594 321 Probes

4650 880 535 290 Genes

DRG

SC

52

FDR<0.05 FDR<0.01 FDR<0.001

P Fold Diff.

78962213-78962154 1700088E04Rik Unknown − 9.09E-11 2.06 1 √ − √ √ √

76548954-76548895 C030006K11Rik Unknown − 9.06E-06 1.59 1 √ − √ √ −

77946181-77946122 Cacng2 calcium channel, voltage-dependent, gamma subunit 2 − 0.0076602 -2.68 0 √ √ − − −

78602622-78602563 Card10 caspase recruitment domain family, member 10 (Card10)105844

6.08E-08 2.21 1 √ √ √ √ √

78678076-78678135 Cdc42ep1 CDC42 effector protein (Rho GTPase binding) 1 − 1.48E-07 -2.17 0 − √ √ √ √

78177993-78178052 Csf2rb1

colony stimulating factor 2 receptor, beta, low-affinity

(granulocyte-macrophage) − 0.0360918 1.69 1 √ √ − − −

78110010-78109951 Csf2rb2

colony stimulating factor 2 receptor, beta 2, low-affinity

(granulocyte macrophage) − 0.00922538 2.04 1 √ − − − −

76361994-76361935

EG666504 =

Gm8140 Predicted Gene − 0.0313923 -1.86 0 − − − − −

75425309-75425368 Gpihbp1 GPI-anchored HDL-binding protein 1 26730 0.00152915 1.63 1 − √ − − −

74872596-74872537 Ly6c lymphocyte antigen 6 complex, locus C 10741 0.00450767 1.77 1 √ √ − − −

74810352-74810293 Ly6i lymphocyte antigen 6 complex, locus I (Ly6i) 20498 0.00222233 1.81 1 √ √ − − −

74624413-74624354 Ly6k lymphocyte antigen 6 complex, locus K (Ly6k) 29627 0.0440461 -1.82 0 − − − − −

78770811-78770870 Nol12 nucleolar protein 12 − 5.52E-07 -2.07 0 √ √ √ √ −

76062077-76062136 Plec1 Plectin − 0.000656955 -2.29 0 − √ √ − −

78448785-78448844 Pscd4 pleckstrin homology, Sec7 and coiled/coil domains 4 − 0.000323216 1.48 1 − − √ − −

76715441-76715500 Zfp7 KRAB-zinc finger protein 65 − 0.0265229 -1.55 0 √ − − − −

LiteratureGenomic Coordinates

Chr. 15 (from-to, Mb)Gene Symbol Gene Product

SNP

Variation

Statistics Contrast Type

(A>B=1) (B>A=0)Accession NM

Table 4: Pain1 genes in lumbar DRGs showing a significant contrast in the expression level

between intact A and B mice at P<0.05 and a fold change of more than 1.5 or less than -1.5 (listed

in alphabetical order). Cited in the literature are genes that are connotated with key words, for a

complete list of connotations see Methods Section 2.4.6; SNP Varation denotes genes that have

polymorphic regions that contrast between A and B mice.

53

3.2.3.2 Expression of DRG genes in denervated A and B mice

The following section introduces the genome-wide array analysis to seek for potential ‘autotomy

genes’ in A mice following hind paw denervation. Equivalent analyses were performed in

parallel for DRG and spinal cord tissues. To distinguish and select for the most optimal

candidate ‘autotomy genes’ in A mice, we compared the gene expression levels of A high

autotomy mice (MHS-A) with gene expression levels of low autotomy mouse groups in the

FDR<0.05 FDR<0.01 FDR<0.001

P Fold Diff.

74705824-74705765 2010109I03Rik Unknown 25929 0.05415 -1.60 0 √ − − − −

77411931-77411872

9130218O11Rik=

Apol10b apolipoprotein L 10B − 0.01565 -2.99 0 √ − − − −

75947885-75947826 BC024139 cDNA sequence BC024139 198172 0.02372 1.69 1 − − − − −

75049228-75049287 BC025446 cDNA sequence BC025446 146058 0.02124 -1.58 0 − − − − −

77822064-77822005 Cacng2

calcium channel, voltage-dependent, gamma

subunit 2 − 0.01056 -1.56 0 √ √ − − −

78602863-78602804 Card10 caspase recruitment domain family, member 10 130 0.00001 2.69 1 √ √ √ √ √

78678076-78678135 Cdc42ep1 CDC42 effector protein (Rho GTPase binding) 1 − 0.00000 -3.07 0 − √ √ √ √

73398751-73398810 Dennd3 DENN/MADD domain containing 3 − 0.02855 -1.63 0 √ − − − −

74752329-74752388 ENSMUST00000096398 Predicted Gene − 0.01923 -1.56 0 − − − − −

78498028-78497969 Lrrc62 = Elfn2

leucine rich repeat and fibronectin type III,

extracellular 2 − 0.01863 1.45 1 − − − − −

74589435-74589376 Ly6d lymphocyte antigen 6 complex, locus D ( 10742 0.04082 -2.01 0 √ − − − −

75392855-75392555 Ly6h lymphocyte antigen 6 complex, locus H 11837 0.02714 1.56 1 − √ − − −

75574346-75574287 Mafa

v-maf musculoaponeurotic fibrosarcoma oncogene

family, protein A (a − 0.04180 1.54 1 − √ − − −

78770811-78770870 Nol12 nucleolar protein 12 − 0.00068 -1.76 0 √ √ √ − −

79083778-79083837 Pick1 protein interacting with C kinase 1 − 0.00050 -2.43 0 √ √ √ − −

76016579-76016520 Plec1 plectin 1 , transcript variant 6 201389 0.01409 2.26 1 − √ − − −

78018531-78018472 Pvalb parvalbumin 13645 0.00843 2.24 1 √ √ − − −

76911278-76911219 Rbm9 = RBfox2 RNA binding protein, fox-1 homolog (C. elegans) 2 − 0.00185 3.13 1 √ √ − − −

76531533-76531294 Recql4 RecQ protein-like 4 58214 0.00823 -1.84 0 √ √ − − −

76345755-76345696 Scrt1 scratch homolog 1, zinc finger protein (Drosophila) − 0.02167 1.89 1 √ − − − −

73404944-73404885 Slc45a4 solute carrier family 45, member 4 − 0.04403 1.71 1 √ − − − −

74553969-74553910 Slurp1 Ly6/Plaur domain containing 1 − 0.00685 -2.19 0 √ √ − − −

SNP

VariationLiterature

Genomic Coordinates

Chr. 15 (from-to, Mb)Gene Symbol Gene Product

Statistics Contrast Type

(A>B=1) (B>A=0)Accession NM

Table 5: Pain1 genes in lumbar spinal cord showing a significant contrast in the expression level

between naïve/intact A and B mice at P<0.05 and a fold change of more than 1.5 or less than -1.5

(listed in alphabetical order). Cited in the literature are genes that are connotated with key words,

for a complete list of connotations see Methods Section 2.4.6; SNP Varation denotes genes that

have polymorphic regions that contrast between A and B mice.

54

following manner. MHS-A mice were compared to sham-operated A mice (Group comparison

1), MHS-A mice were compared to NLS-B mice (Group comparison 2), MHS-A mice were

compared to NLS-A mice (group comparison 3). Each of the 3 group comparisons from the

DRG dataset resulted in a list of thousands of genes throughout the genome that differed at a

significance level of P<0.05 and at a 1.5<fold difference<-1.5 (Figure 4). The 3 group

comparisons and their outcome are detailed below.

Comparison (1): Mice of the ADH group underwent a denervation procedure involving

anesthesia, wound formation, major nerves injured, followed by wound healing, degeneration of

the cut nerves, and expression of MHS of the toes. The AS mice had the same procedures but

they lacked the denervation procedure, no major nerves were injured and no degeneration of the

cut nerves was followed. Despite the identity in the genomes of the mice of these two groups,

the difference in procedures and behavioural outcomes manifested in gene expression level

differences in 2194 probes encoding 1964 genes throughout the genome. This large number of

genes correlates to genes associated directly with denervation + autotomy behaviour. Reducing

the significance level to an FDR<0.05 reduced the numbers to 335 probes encoding 300 genes; at

FDR<0.01 there was an additional reduction in the number of probes to 162 and 145 genes,

respectively, and at an FDR<0.001 the numbers dropped further to 58 and 50 genes, respectively

(see Table 6). One should keep in mind that shortlisting the gene lists by lowering FDR

thresholds concurrently increases the type 2 error, thereby eliminating genes, which may be

relevant to the pain outcome.

Comparison (2): ADH and BD mice had undergone exactly the same surgical procedures

involving anesthesia, wound formation, major nerves injured, followed by wound healing, and

degeneration of the cut nerves. The only two differences between these groups were the

differing expression levels of autotomy of the toes and strain-specific differences in DRG

neuronal, satellite cells and other cell types that are unrelated to the autotomy behaviour. The

genomes of DRG cells in these groups differed in 5034 probes that mark 4679 genes. These are

compared to 5116 probes (that mark 4600 genes) in intact A vs. B mice. Reducing the

significance level to an FDR<0.05 lowered the numbers to 977 probes encoding 880 genes; at

FDR<0.01 an additional reduction in the number of probes brought it to 584 and 525 genes,

55

respectively, and at an FDR<0.001 the numbers dropped further to 342 probes and 300 genes,

respectively (see Table 6).

Comparison (3): ADH and ADL mice have an identical genome and underwent exactly the same

surgical procedure, same nerves that degenerated post-axotomy and the same wound that healed

post-operatively, and only differed on the autotomy levels. This difference in autotomy levels

was associated with 3375 probes throughout the genome that mark 3037 DRG genes. Some

mice responded by autotomy and some did not. We do not know the reason why the latter did

not but this unknown environmental parameter drove these mice to lack the drive for autotomy

behaviour and they are listed in Table 6. These genes may be regarded as genes whose

expression level was differentially affected by the environment in a GXE manner. Setting the

significance level to FDR<0.05 reduced the numbers to 1336 probes encoding 1200 genes; at

FDR<0.01 the number of probes got down to 74 and 65 probes, respectively, and at FDR<0.001

the numbers dropped further to 7 probes and genes, respectively (see Table 6).

Table 6: Probes and genes that are regulated with autotomy behaviour in A and B mice. Spinal

cord (SC) and DRG probes throughout the genome showing significant gene expression levels in

MHS-A, NLS-A and B mice.

P<0.05 FDR<0.05 FDR<0.01 FDR<0.001

2194 335 162 58 Probes

1964 300 145 50 Genes

798 0 0 0 Probes

756 0 0 0 Genes

3375 1336 74 7 Probes

3037 1200 65 7 Genes

677 0 0 0 Probes

666 0 0 0 Genes

5037 977 584 342 Probes

4679 880 525 300 Genes

2465 783 465 261 Probes

2215 700 420 235 Genes

SC

DRG

SC

A h

igh

vs.

A

low

A h

igh

vs.

B

low

DRG

SC

A h

igh

vs.

A

sham

DRG

56

To shorten the above lists even further we used a Venn analysis in order to isolate autotomy-

specific genes. This analysis applies filters that screen the dataset according to a set of criteria,

leaving out genes that do not fit a specific profile, thereby isolating autotomy-associated genes.

The profile sought genes shared between all 3 group comparisons: ADH vs. AS, ADH vs. BD

and ADH vs. ADL. Also, these genes came up as significantly different at a P value<0.05 and a

1.5<fold change<-1.5 in each of the three group comparisons (Figure 4). When correcting for

multiple comparisons, we were unable to get differentially expressed genes in the spinal cord,

and only a few that regulated in the DRGs. Therefore, in order to use the Venn analysis strategy

for selecting candidate genes for autotomy, we gave each gene a fair chance, not to miss any

false negatives, and used a cut off of P value<0.05 and a 1.5<fold change<-1.5. The resulting list

included genes in lumbar DRGs of A mice whose regulation is not related to the response to

surgery per se (i.e., the anesthesia, wounds in skin, fascia and muscles), but related to the

response to nerve degeneration and regeneration that drive denervated A mice, but not B mice, to

express autotomy, and whose regulation is sensitive to environmental variables that cause some

A mice to express autotomy (ADH) while protecting other A mice (ADL) from neuroglial

mechanisms originating in the DRGs that drive for autotomy. The resulting Venn diagram in

Figure 4 shows a subset of 284 probes, encoding 226 unique genes throughout the DRG genome,

which fitted these filters. These genes are listed in Appendix I by name, protein product they

encode, genomic coordinates, fold difference, direction of effect, and P value. Some of these

genes showed the same direction, i.e. up-regulation or down-regulation in all 3 group

comparisons. Other genes showed a mixture of up-regulation in some comparisons and down-

regulation in others.

57

Figure 4: Numbers of DRG genes throughout the genome whose expression level is regulated

by the effects of: (1) denervation in A mice expressing high autotomy vs. sham operated A mice,

(2) denervation in different strains expressing contrasting autotomy levels, and (3) the effect of

different environmental controls. The number of genes that resulted from each individual group

comparison is shown in the circles. The 226 genes that are common to all three comparisons had

significantly different expression levels (at P<0.05 and at a -1.5<fold difference>1.5). These

genes are therefore considered as “autotomy associated” genes.

3.2.3.3 Spinal cord genes whose expression is associated with

autotomy levels

As in the analysis of the DRG gene expression levels dataset, Tables 6 and Appendix II provide

the number of probes and genes, and the respective list of genes that significantly differentiated

in the spinal cord at P<0.05 and 1.5<fold change <-1.5 between ADH vs. AS, ADH vs. BD and

ADH vs. ADL. Figure 5 shows that 798 probes marking 756 genes contrasted between ADH and

AS mice, 2465 probes marking 2215 genes contrasted between ADH and BD mice, and 677

probes marking 666 genes contrasted between ADH and ADL mice. The Venn analysis shown

in Figure 5 also indicates that 76 probes marking 73 genes across the whole genome

significantly contrasted between the ADH mice and mice of all other NLS groups (AS, BD, and

ADL).

226 shared genes for autotomy in DRGs

A Highvs.

A Low

A Highvs.

B Low

A Highvs.

A Sham

3037

4679

1964

58

Figure 5: Number of spinal cord genes throughout the genome whose expression level is

regulated by the effects of: (1) denervation in A mice expressing high autotomy vs. sham

operated A mice, (2) denervation in different strains expressing contrasting autotomy levels, and

(3) the effect of different environmental controls. The numbers of genes that resulted from each

individual group comparison are shown in the circles. The 73 genes that are common to all three

comparisons had significantly different expression levels (at P<0.05 and at a -1.5<fold

difference>1.5). These genes are therefore considered as “autotomy associated” genes.

3.2.4 Network analysis

3.2.4.1 Network analysis for DRG genes in D14PO denervated A

and B mice

In the next step to select for candidate ‘autotomy genes’, we looked at gene networks that

resulted from our gene lists, to isolate potential candidates that may be involved in pain

mechanisms. Using the GeneMANIA website, the 226 candidate genes in the DRG resulting

from the Venn analysis were further analyzed for network interactions, to determine with which

biological processes and cellular pathways they are engaged. Table 7 lists genes from the 226

genes’ list, and their proteins, which are known from the literature to functionally interact with

other proteins in biological processes. This mechanistic network analysis includes co-

73 shared genes for autotomy in spinal cord

A Highvs.

A Low

A Highvs.

B Low

A Highvs.

A Sham

666

2215

756

59

expression, co-localization, and physical interaction, whereby genes are co-expressed in the same

tissue, proteins are co-localized in the same cell organelle, or proteins are physically bound to

one another as a complex in a physical interaction, respectively. As shown in the Table 7, some

of the 226 genes interact with other candidate autotomy genes in this list (in bold), whereas many

other genes only interact with genes not on this list (non-bold). Out of these 226 DRG candidate

autotomy genes, some genes interacted within the following biological pathways: response to

metal ions and/or inorganic substance (Bcl2, Prnp, Gdi1, Ncam1, Cnga3, Homer1, Mef2c, Ect2,

Mapk8, Ccl19 and Ppargc1b), B cell proliferation (Ada, Bcl2, Il13ra1, Irs2, Mef2c), chemokine

receptor binding (Ccl17, Ccl19, Cxcl10, Stat1, Stat3), histone deacetylase binding (bx5, Mapk8,

Mef2c, Nr2c1, Nrip1), lymphocyte activation (Gata3, Cd47, Mef2c, Ada, Bcl2, Il4ra, Il13ra1,

Irs2), cellular component movement and/or cell migration (Bcl2, Ccl19, Cxcl10, Igf1r, Irs2,

Mapk8, Podxl, Fer, Gata3), and excitotoxicity, apoptosis, and neuroprotection (Bcl2).

60

Table 7: Candidate autotomy genes in the DRGs and biological processes in which they

operate. In bold are genes from the 226 autotomy candidate genes. In non-bold are other genes

that interacted in a network with other genes. In a Network genes that are either co-expressed,

co-localized or in physical interaction with another gene/s (i.e. 2 or more proteins that are found

bound to each other in a complex); individual= genes that were not interacting with other gene/s.

Underlined is Stat3 that was also studied by us in this project and reported herein (Chapter 5).

Homer1 Chr 13 94406010-94442522 homer homolog 1 (Drosophila) 26556

Mef2c Chr 13 84141092-84141151 myocyte enhancer factor 2C 25282

Bcl2 Chr 1 108538339-108538280 B-cell leukemia/lymphoma 2 9741

Cnga3 Chr 1 37206871-37206930 cyclic nucleotide gated channel alpha 3 9918

Ccl19 Chr 4 42776430-42776371 (C-C motif) ligand 19 11888

Gdi1 Chr X 74305012- 74311867 guanosine diphosphate (GDP) dissociation inhibitor 1 14567

Ncam1 Chr 9 49257408-49257349 neural cell adhesion molecule 1 17967

Prnp Chr 2 131909928- 131938431 prion protein 11170

Ect2 Chr 3 27321129-27318861 ect2 oncogene 7900

Mapk8 Chr 14 32207646-32207587 mitogen activated protein kinase 8 26419

Ppargc1b Chr 18 61421963-61421904peroxisome proliferative activated receptor, gamma,

coactivator 1 beta170826

Ada Chr 2 163726584- 163750177 adenosine deaminase 7398

Bcl2 Chr 1 108538339-108538280 B-cell leukemia/lymphoma 2 9741

Il13ra1 Chr X 32601604-32601663 interleukin 13 receptor, alpha 1 133990

Irs2 Chr 8 10986961- 11008430 insulin receptor substrate 2 1081212

Mef2c Chr 13 84141092-84141151 myocyte enhancer factor 2C 25282

Ccl17 Chr 8 97700999-97701058 chemokine (C-C motif) ligand 17 (Ccl17), mRNA 11332

Ccl19 Chr 4 42776430-42776371 (C-C motif) ligand 19 11888

Cxcl10 Chr 5 93422014-93421955 chemokine (C-X-C motif) ligand 10 21274

Stat1 Chr 1 52066524-52066583 signal transducer and activator of transcription 1 20846

Stat3 Chr 11 100886810-100939511 signal transducer and activator of transcription 3 20848

bx5 Chr 15 103191546..103239816 chromobox homolog 5 (Drosophila HP1a) _

Mapk8 Chr 14 32207646-32207587 mitogen activated protein kinase 8 26419

Mef2c Chr 13 84141092-84141151 myocyte enhancer factor 2C 17260

Nr2c1 Chr 10 93580081-93580140 nuclear receptor subfamily 2, group C, member 1 22025

Nrip1 Chr 16 76234512-76234453 nuclear receptor interacting protein 1 268903

Gata3 Chr 2 9857078- 9878600 GATA binding protein 3 14462

Cd47 Chr 16 49831819-49831878Mus musculus adult male hippocampus cDNA, RIKEN

full-length enriched library, cl16423

Gata3 Chr 2 9857078- 9878600 GATA binding protein 3 14462

Mef2c Chr 13 84141092-84141151 myocyte enhancer factor 2C 25282

Ada Chr 2 163726584- 163750177 adenosine deaminase 7398

Bcl2 Chr 1 108538339-108538280 B-cell leukemia/lymphoma 2 9741

Il4ra Chr 7 125552282- 125579474 interleukin 4 receptor, alpha 16190

Il13ra1 Chr X 32601604-32601663 interleukin 13 receptor, alpha 1 133990

Ind

ivi

du

al

Irs2 Chr 8 10986961- 11008430 insulin receptor substrate 2 1081212

Bcl2 Chr 1 108538339-108538280 B-cell leukemia/lymphoma 2 9741

Ccl19 Chr 4 42776430-42776371 (C-C motif) ligand 19 11888

Cxcl10 Chr 5 93422014-93421955 chemokine (C-X-C motif) ligand 10 21274

Igf1r Chr 7 68105625-68105684 insulin-like growth factor I receptor 10513

Irs2 Chr 8 10986961- 11008430 insulin receptor substrate 2 1081212

Mapk8 Chr 14 32207646-32207587 mitogen activated protein kinase 8 26419

Podxl Chr 6 31450627-31450568 Mus musculus podocalyxin-like 13723

Fer Chr 17 63896018- 64139496 er (fms/fps related) protein kinase 103921

Gata3 Chr 2 9857078- 9878600 GATA binding protein 3 14462

cell

ula

r co

mp

on

en

t

mo

vem

en

t an

d/o

r ce

ll

mig

rati

on

In a

Ne

two

rkIn

div

idu

al

che

mo

kin

e

rece

pto

r

bin

din

g

In a

Ne

two

rk

his

ton

e

de

ace

tyla

se

bin

din

g

In a

Ne

two

rk

lym

po

ho

cyte

act

ivat

ion

In a

Ne

two

rk

resp

on

se t

o in

org

anic

su

bst

ance

/ m

eta

l

ion

s In a

Ne

two

rkIn

div

idu

al

B c

ell

pro

life

rati

on

Ind

ivid

ual

DRG VENN genes Gene SymbolGenomic coordinates (Chr, from-

to Mb)Gene Product

Accession

NM

61

3.2.4.2 Network analysis of genes expressed in the spinal cord of

denervated A and B mice

An additional network analysis was performed, in a similar manner to the dataset of the DRG

autotomy genes, for the autotomy associated genes in the spinal cord. Table 8 lists genes in

biological processes found using a GeneMania analysis for some of the 73 candidate genes for

autotomy in the spinal cord, yielding the most significant contrasts in the three comparisons. Out

of these 73 spinal cord candidate autotomy genes, some genes interacted within the following

biological pathways: phagocytosis/endocytosis (Sirpb1a, Hck, Fcgr1 Slc11a1, Fcer1g, Vav1,

Coro1a), myeloid leukocyte/dendrite activation (Gimap5, Lilrb3, Pilrb1, Slc11a1, Fcer1g, Sfi1,

Tyrobp), adaptive immune response (Lilrb3, Fcgr1, Icam1, Slc11a1, Fcer1g, Samsn1, Gimap5),

homeostasis of number of cells (Lilrb3, Heph, Sox6, Fcer1g, Sfpi1, Coro1a, Gimap), external

side of plasma membrane (Vwf, Icam1, Fcgr1, Cd244, Emr1, Fcer1g, Ccr5), immunological

synapse (Cd53, Coro1a, Icam1), immunoglobulin binding (Fcgr1, Fcer1g, Vwf), myeloid cell

differentiation (Gimap5, Pilrb1, Lilrb3, Sox6, Cebpa, Heph, Sfpi1), antigen processing and

presentation (Fcgr1, Slc11a1, Fcer1g, Icam1), endocytosis and phagocytosis (Sirpb1a, Fcgr1,

Hck, Slc11a1, Fcer1g, Vav1, Coro1a), and inflammatory response (Fcgr1, Fcer1g, Ccr5, Kl).

62

Table 8: Candidate autotomy genes in the spinal cord and biological processes in which they

operate. In bold are genes from the 73 autotomy candidate genes. In non-bold are other genes

that interacted in a network with other genes. In a Network genes that are either co-expressed,

co-localized or in physical interaction with another gene/s (i.e. 2 or more proteins that are found

bound to each other in a complex); individual genes that were not interacting with other gene/s.

Sirpb1a Chr 3 015348529-015348470 signal-regulatory protein beta 1A 1002898

Hck Chr 2 152840517-152842444 hemopoietic cell kinase 10407

Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Slc11a1 Chr 1 74375203-74386051 solute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1

13612

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Vav1 Chr 17 57279100-57329236 vav 1 oncogene 22324Coro1a Chr 7 126699774-126704754 coronin, actin binding protein 1A 12721Gimap5 Chr 6 48676977-48677036 GTPase, IMAP family member 5 175035

Lilrb3 Chr 7 3315702-3315643 leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM domains),

member 3

18733

Pilrb1 Chr 5 138082142-138082083 paired immunoglobin-like type 2 receptor

beta 1 170741

Slc11a1 Chr 1 74375203-74386051 solute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1

13612

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Sfpi1 Chr 2 91096797-91115756 SFFV proviral integration 1 20734Tyrobp Chr 7 30413788-30417582 TYRO protein tyrosine kinase binding protein 11662

Lilrb3 Chr 7 3315702-3315643 leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM domains),

member 3

18733

Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Icam1 Chr 9 20779144-20779203 intercellular adhesion molecule 1 10493

Slc11a1 Chr 1 74375203-74386051 solute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1

13612

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

samsn1 Chr 16 75858794- 75909266 SAM domain, SH3 domain and nuclear

localization signals, 167742

Ind

ivi

du

al Gimap5 Chr 6 48676977-48677036 GTPase, IMAP family member 5 175035

Lilrb3 Chr 7 3315702-3315643 leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM domains),

member 3

18733

Heph Chr X 92733954-92734013 hephaestin , transcript variant 2 181273

Sox6 Chr 7 115332904-115332845 SRY-box containing gene 6 , transcript

variant 120679

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Sfpi1 Chr 2 91096797-91115756 SFFV proviral integration 1 20734Coro1a Chr 7 126699774-126704754 coronin, actin binding protein 1A 12721

Ind

iv

idu

al Gimap5 Chr 6 48676977-48677036 GTPase, IMAP family member 5 175035

ad

ap

tiv

e i

mm

un

e r

esp

on

seh

om

eo

sta

sis

of

nu

mb

er

of

cell

s

In a

Ne

two

rk

Genomic coordinates (Chr, from-to

Mb)Gene Product

Accession

NM

ph

ag

ocy

tosi

s /

en

do

cyto

sis

In a

Ne

two

rk

Gene SymbolSC VENN genes

In a

Ne

two

rk

my

elo

id l

eu

ko

cyte

/de

nd

rite

act

iva

tio

n

In a

Ne

two

rk

63

Table 8: Continued

Vwf Chr 6 125652152-125652211 Von Willebrand factor homolog 11708

Icam1 Chr 9 20779144-20779203 intercellular adhesion molecule 1 10493

Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Cd244 Chr 1 173419775-173419834 CD244 natural kil ler cell receptor 2B4 18729

Emr1 hr 17 57358686-57483529 EGF-like module containing, mucin-like,

hormone receptor-like sequence 1 13733

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Ccr5 Chr 9 124121543-124127183 chemokine (C-C motif) receptor 5 12774Cd53 Chr 3 106758861-106790149 CD53 antigen 7651

Coro1a Chr 7 126699774-126704754 coronin, actin binding protein 1A 12721Icam1 Chr 9 20779144-20779203 intercellular adhesion molecule 1 10493

Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

In di vi du al Vwf Chr 6 125652152-125652211 Von Willebrand factor homolog 11708

Gimap5 Chr 6 48676977-48677036 GTPase, IMAP family member 5 175035

Pilrb1 Chr 5 138082142-138082083 paired immunoglobin-like type 2 receptor

beta 1 170741

Lilrb3 Chr 7 3315702-3315643 leukocyte immunoglobulin-like receptor,

subfamily B (with TM and ITIM domains),

member 3

18733

Sox6 Chr 7 115332904-115332845 SRY-box containing gene 6 , transcript

variant 120679

Cebpa Chr 7 34829102-34829161 Mus musculus CCAAT/enhancer binding

protein , alpha

Heph Chr X 92733954-92734013 hephaestin , transcript variant 2 181273

Sfpi1 Chr 2 91096797-91115756 SFFV proviral integration 1 20734Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Slc11a1 Chr 1 74375203-74386051 solute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1

13612

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

In di vi du al Icam1 Chr 9 20779144-20779203 intercellular adhesion molecule 1 10493

Sirpb1a Chr 3 015348529-015348470 signal-regulatory protein beta 1A 1002898

Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Hck Chr 2 152840517-152842444 hemopoietic cell kinase 10407

Slc11a1 Chr 1 74375203-74386051 solute carrier family 11 (proton-coupled

divalent metal ion transporters), member 1

13612

Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Vav1 Chr 17 57279100-57329236 vav 1 oncogene 22324Coro1a Chr 7 126699774-126704754 coronin, actin binding protein 1A 12721Fcgr1 Chr 3 096369943-096369880 Fc receptor, IgG, high affinity I 14129Fcer1g Chr 1 171229572-171234349 Fc receptor, IgE, high affinity I, gamma

polypeptide

10185

Ccr5 Chr 9 124121543-124127183 chemokine (C-C motif) receptor 5 12774

Kl Chr 5 151255290-151255349 secreted isoform of Klotho protein 16591

SC VENN genes Gene SymbolGenomic coordinates (Chr, from-to

Mb)Gene Product

Accession

NM

In a

Ne

two

rk

ext

ern

al s

ide

of

pla

sma

me

mb

ran

e

In a

Ne

two

rk

en

do

cyto

sis

and

ph

ago

cyto

sis

Infl

amm

ato

ry

resp

on

se

In a

Ne

two

rkIn

a N

etw

ork

imm

un

o-

glo

bu

lin

bin

din

g

In a

Ne

two

r

k

In a

Ne

two

rk

mye

loid

ce

ll d

iffe

ren

tiat

ion

In a

Ne

two

rk

imm

un

o

logi

cal

syn

apse

anti

gen

pro

cess

ing

and

pre

sen

tati

on

64

3.2.5 Pain1 DRG genes in denervated A and B mice

In the next step of the search for candidate ‘autotomy genes’ having a major effect on this pain

behaviour, we screened the list of candidate DRG autotomy genes to isolate those harboured

exclusively in Pain1. As shown in Table 8, seven genes were significantly regulated at P<0.05

in all 3 group comparisons (Csf2rb1, Cyhr1, Hemt1, Oplah, Ptk2, Rabl4, Rbm9). However, only

Hemt1 gene, encoding for hematopoietic cell transcript 1, fitted the criteria of the Venn analysis,

with fold differences ranging from -3.0 to +5.8. Hemt1 was up regulated in the ADH group 5.8

fold more compared to the AS group (P<0.00003), and 3.0 fold more compared to the ADL

group (NLS mice; P<0.01), but down-regulated by -2.0 fold compared to the BD group (P<0.03).

Hemt1 was not found to interact with other genes in the network analysis, nor was it found in

databases (http://www.ncbi.nlm.nih.gov/gene/?term=hemt1) of cellular pathways or biological

pathways. However, while Hemt1 is part of the genes mapped by the Allen Brain Atlas,

indicating that its cDNA probes did hybridize to brain sections of the B mouse used by this atlas,

there are no hybridization maps to spinal cord sections (http://mouse.brain-

map.org/gene/show/14978) and therefore, further indications about where in the spinal cord of

intact B mice it is expressed are not available from this resource.

65

Table 9: Pain1 genes in sham-operated and denervated A mice and denervated B mice that

reacted to the treatments by changed expression levels in the DRG. The 3 group comparisons

(ADH vs. AS; ADH vs. BD; and ADH vs. ADL) list candidate genes at P<0.05. In red are genes

that differed between each two compared groups at a significance level of P<0.05 in all 3

comparisons, regardless of the fold difference; in black are fold differences that did not reach the

cut off -1.5<fold difference>1.5. Those genes that were significant at P<0.05 in all 3 group

comparisons are Csf2rb1, Cyhr1, Hemt1, Oplah, Ptk2, Rabl4, Rbm9. Only Hemt1 gene made the

cut off of P<0.05 at a -1.5<fold difference>1.5 in all 3 group comparisons. Underlined is the

gene we report in this dissertation study.

P Fold Diff. P Fold Diff. P Fold Diff.

74433385-74433326 1700016M24Rik =

Mroh4

maestro heat-like repeat

family member 4

0.04103 -1.8

78962213-78962154 1700088E04Rik Unknown 138581 0.00673 -1.2 0.00000 1.7

0.05296 -1.5

75808056-75807997 2410075B13Rik =

Ccdc166

coiled-coil domain containing

166

75474451-75474510 2810039B14Rik Unknown 0.04725 -1.2

73054667-73054608 8030476L19Rik Unknown 0.03068 -1.2 0.04770 -1.2

75828441-75828382 AA409316 =

Fam83h

family with sequence

similarity 83, member H

134087 0.01302 -1.7

77659374-77659315 AK037579 Unknown 0.00980 1.3

78964090-78964149 AK147274 Unknown 0.01449 -2.5

78964090-78964149 AK147274 Unknown 0.00249 1.6 0.00463 1.6

76884283-76884342 Apol6 apolipoprotein L 6 0.04203 -2.1

74496481-74496422 Arc activity regulated cytoskeletal-

associated protein

0.01711 1.3 0.00515 -1.4

74416405-74416464 Bai1 brain-specific angiogenesis

inhibitor 1

174991 0.01264 -1.7

77234114-77234055 BC020489 = Apol9a apolipoprotein L 9a 0.00347 -2.5

76548954-76548895 C030006K11Rik Unknown 176828 0.00003 1.6

76549038-76548979 C030006K11Rik Unknown 176828 0.00047 1.5

77819931-77819872 Cacng2 calcium channel, voltage-

dependent, gamma subunit 2

0.04829 -1.6 0.01956 -1.8

78602622-78602563 Card10 caspase recruitment domain

family, member 10

130 0.00000 2.4

78602863-78602804 Card10 caspase recruitment domain

family, member 10

130 0.00000 2.3

78678076-78678135 Cdc42ep1 CDC42 effector protein (Rho

GTPase binding) 1

0.00000 -2.7

76728465-76728524 Commd5 COMM domain containing 5 25536 0.00667 -1.3 0.00130 -1.5

78175465-78175524 Csf2rb1 colony stimulating factor 2

receptor, beta 1, low-affinity

(granulocyte macrophage)

0.01223 2.0 0.01745 1.9 0.04768 1.3

78177993-78178052 Csf2rb1 colony stimulating factor 2

receptor, beta 1, low-affinity

(granulocyte macrophage)

0.04485 1.6

76172763-76172822 Cyc1 cytochrome c-1 25567 0.00193 -1.7

76473085-76473026 Cyhr1 cysteine and histidine rich 1 0.00002 -1.3 0.00992 -1.4 0.00037 -1.5

76485515-76485456 Cyhr1 cysteine and histidine rich 1 0.00048 -1.4 0.02838 -1.1 0.03946 -1.1

76471038-76470979 Cyhr1 cysteine and histidine rich 3 0.05218 -1.3 0.00981 -1.7

6255912-76256422 D330001F17Rik =

Mroh1 =

ENSMUST0000009

2595

maestro heat-like repeat

family member 1

0.03852 1.9

ADH-ADLAccession NM

Genomic Coordinates

Chr. 15 (from-to, Mb)Gene Symbol Gene Product

ADH-AS ADH-BD

66

Table 9: Continued (2)

P Fold Diff. P Fold Diff. P Fold Diff.

76330532-76330316 Dgat1 diacylglycerol O-

acyltransferase 1

10046 0.00311 1.4 0.00316 1.5

76330099-76329953 Dgat1 diacylglycerol O-

acyltransferase 1

10046 0.02450 1.3 0.01214 1.4

75722464-75722405 Eef1d eukaryotic translation

elongation factor 1 delta

(guanine nucleotid

0.00019 -1.5 0.00984 1.3

77792875-77792816 Eif3s7 eukaryotic translation

initiation factor 3, subunit D

0.02130 2.4

76266416-76266475 ENSMUST0000009

2595 = Mroh1 =

D330001F17Rik

maestro heat-like repeat

family member 1

0.04691 -1.8

76363529-76363209 Fbxl6 F-box and leucine-rich repeat

protein 6

13909 0.01310 -1.5

77780514-77780455 Foxred2 FAD-dependent

oxidoreductase domain

containing 2

0.03926 -2.7

78864613-78864672 Gcat glycine C-acetyltransferase (2-

amino-3-ketobutyrate-

coenzyme A ligase)

0.01022 -1.3 0.02096 -1.3

74641133-74641074 Gml Glycosyl-phosphatidylinositol

(GPI)-anchored molecule like

protein

0.00124 3.4

75425309-75425368 Gpihbp1 GPI-anchored HDL-binding

protein 1

26730 0.00005 2.0

76368517-76368576 Gpr172b G protein-coupled receptor

172B

29643 0.02519 -1.3

74648553-74648612 Hemt1 hematopoietic cell transcript 1 0.00003 5.8 0.02911 -2.0 0.00915 3.0

76327923-76327982 Hsf1 heat shock factor 1 8296 0.00910 -1.4 0.02673 -1.3

76328001-76328060 Hsf1 heat shock factor 2 8296 0.04863 -1.2

76495330-76495399 Kifc2 kinesin family member C2 0.00004 -1.8 0.00314 -1.5

78307993-78307934 Il2rb interleukin 2 receptor, beta

chain

8368 0.01853 -2.5

78757391-78757450 Lgals1 lectin, galactose binding,

soluble 1

8495 0.00874 -1.7

78678199-78678140 Lgals2 lectin, galactose binding,

soluble 2

25622 0.05136 -1.3

77454209-77454268 LOC193676 Predicted gene 0.02147 2.4

76543586-76543645 Lrrc14 leucine rich repeat containing

14

0.03123 1.5

76542790-76542731 Lrrc24 leucine rich repeat containing

24

198119 0.01138 -1.5 0.00373 -1.6

74872596-74872537 Ly6c lymphocyte antigen 6

complex, locus C

10741 0.00366 1.9

74810352-74810293 Ly6i lymphocyte antigen 6

complex, locus I

20498 0.00108 2.0

75392855-75392555 Ly6h lymphocyte antigen 6

complex, locus H

11837 0.00556 -1.8

74559568-74559509 Lypd2 Ly6/Plaur domain containing 2 26671 0.01568 -1.8

74575318-74575259 Lynx1 Ly6/neurotoxin 1 11838 0.02059 1.4

78583437-78583378 Mfng O-fucosylpeptide 3-beta-N-

acetylglucosaminyltransferase

8595 0.01625 -1.5

76530688-76530747 Mfsd3 major facilitator superfamily

domain containing 3

0.00136 -1.4 0.00015 -1.6

78943124-78943183 Micall1 microtubule associated

monooxygenase, calponin and

LIM domain containing -like 1

0.03267 -1.4

ADH-BD ADH-ADLGenomic Coordinates

Chr. 15 (from-to, Mb)Gene Symbol Accession NMGene Product

ADH-AS

67

Table 9: Continued (3)

P Fold Diff. P Fold Diff. P Fold Diff.

78770811-78770870 Nol12 nucleolar protein 11 133800 0.00000 -2.1 0.00335 1.5

78767955-78768014 Nol12 nucleolar protein 12 133800 0.01301 1.2

76850644-76850585 NP063612 unidentified reading frame 0.00759 -1.8

76124074-76124133 Oplah 5-oxoprolinase (ATP-

hydrolysing)

153122 0.00322 -1.4 0.03733 -1.3 0.03093 -1.3

76124269-76124210 Oplah 5-oxoprolinase (ATP-

hydrolysing)

153122 0.05059 1.3

78746524-78746583 Pdxp pyridoxal (pyridoxine, vitamin

B6) phosphatase

0.04924 -1.3 0.03681 -1.4

79076615-79076674 Pick1 protein interacting with C

kinase 1

0.00035 -1.2 0.01626 -2.0

76016579-76016520 Plec1 plectin 1 201389 0.03081 -2.0

78971861-78973309 Polr2f polymerase (RNA) II (DNA

directed) polypeptide F

0.03283 -1.4

78968684-78971902 Polr2f polymerase (RNA) II (DNA

directed) polypeptide F

0.03421 -1.4

78448785-78448844 Pscd4 = Cyth4 Mus musculus pleckstrin

homology, Sec7 and

coiled/coil domains 4 =

cytohesin4

0.00023 1.5 0.01208 1.3

78448892-78448951 Pscd4 = Cyth4 Mus musculus pleckstrin

homology, Sec7 and

coiled/coil domains 4 =

cytohesin 4

0.00104 1.5 0.04259 1.4

73032517-73032458 Ptk2 PTK2 protein tyrosine kinase 2 7982 0.02145 1.2 0.03614 -1.2 0.01909 1.3

73043295-73043236 Ptk2 PTK2 protein tyrosine kinase 2 7982 0.04570 1.4

78018531-78018472 Pvalb parvalbumin (Pvalb) 13645 0.00656 2.3

77986794-77986735 Rabl4 RAB, member of RAS

oncogene family-like 4

25931 0.00018 -1.4 0.00056 -1.4 0.03014 -1.2

76909655-76909596 Rbm9 RNA binding motif protein 9 0.01360 1.2 0.03089 1.7 0.00053 1.4

76345755-76345696 Scrt1 scratch homolog 1, zinc finger

protein (Drosophila)

0.01628 -2.1 0.00285 -2.7

76286533-76286592 Scx scleraxis 198885 0.00000 -2.0 0.00026 -1.5

76174354-76174295 Sharpin SHANK-associated RH domain

interacting protein

0.00563 -1.2

73404944-73404885 Slc45a4 solute carrier family 45,

member 4

0.01241 -1.7 0.05082 -1.5

74553969-74553910 Slurp1 secreted Ly6/Plaur domain

containing 1

20519 0.00577 1.6

78982389-78982330 Sox10 SRY-box containing gene 10 0.04871 1.4

78367604-78367545 Sstr3 somatostatin receptor 3 9218 0.02139 -1.7

75484985-75484457 Top1mt DNA topoisomerase 1,

mitochondrial

28404 0.05324 -1.2

75752876-75752619 Tsta3 tissue specific transplantation

antigen P35B

312 0.04354 -1.3

76449556-76449497 Vps28 vacuolar protein sorting 28

(yeast)

25842 0.00204 -1.3

76449439-76449380 Vps28 vacuolar protein sorting 28

(yeast)

25842 0.02439 -1.2 0.00933 -1.3

76678942-76678883 Zfp251 zinc finger protein 251 0.00172 -1.7

5452356-75452415 Zfp41 zinc finger protein 41 117 0.01194 -1.2

76737906-76737847 Zfp647 zinc finger protein 647 172817 0.00347 1.3 0.01499 1.3

76715441-76715500 Zfp7 zinc finger protein 7 0.01845 -1.6

Genomic Coordinates

Chr. 15 (from-to, Mb)Gene Symbol Accession NMGene Product

ADH-AS ADH-BD ADH-ADL

68

3.2.6 Pain1 genes associated with autotomy in spinal cord

Table 10 shows that within Pain1, the only genes that came up as significantly contrasting

between ADH and all three other groups (Venn shared genes) were Csf2rb1 and Csf2rb2.

Csf2rb1 encodes the beta subunit 1 of the common receptor for the following three cytokines:

granulocyte-macrophage colony stimulating factor (GM-CSF), and the interleukin-3 (IL3) and -5

(IL5). Its other aliases are: Bc, Il3r, AIC2B, Il3rb, Il5rb, CDw131, Il3rb1, Csfgmrb, AI848964

and MGC130522. Csf2rb2, on the other hand, encodes the beta subunit 2 of the common

receptor for the following three cytokines: granulocyte-macrophage colony stimulating factor

(GM-CSF), and the interleukin-3 (IL3) and -5 (IL5), and is located juxtaposed and upstream of

Csf2rb1 on mouse chromosome 15 within Pain1. Its other aliases are AIC2A, BetaIl3, Bil3,

Csfgmrb, Il3r, Il3rb and Il3rb2. It is likely a gene duplicate of Csf2rb1. Csf2rb1 was up-

regulated in ADH mice expressing MHS, compared to A mice that had the sham operation and

did not express autotomy (1.8 fold; P<0.0001), and to denervated B mice (BD) who expressed

NLS (1.7 fold; P<0.00003), and also to the denervated NLS-A mice despite undergoing the same

denervation and carrying the same A genome (1.9 fold; P<0.00009).

69

Table 10: Pain1 genes in the individual 3 group comparisons that yielded in the spinal cord

(ADH vs. AS; ADH vs. BD; ADH vs. ADL), P<0.05. In red are genes that yielded P<0.05 and

made the the cut off -1.5<fold difference>1.5. Those were Csf2rb1 and Csf2rb2.

P Fold Diff. P Fold Diff. P Fold Diff.

76884283-76884342 Apol6 Apolipoprotein L 6 0.04768 1.5

74496481-74496422 Arc activity regulated cytoskeletal-associated 0.02168 -1.3 0.02209 -1.3

78678076-78678135 Cdc42ep1 CDC42 effector protein (Rho GTPase 0.00000 -2.8

76777840-76777899 1110038F14Rik Unknown 54099 0.02957 -1.2

78962213-78962154 1700088E04Rik Unknown 138581 0.00182 1.4

77352227-77352168 2210421G13Rik Unknown 175391 0.04079 7.5

77352479-77352420 2210421G13Rik Unknown 175391 0.04762 4.2

77317715-77317774 9030421J09Rik Unknown 177744 0.02945 1.7 0.03415 1.7

74496481-74496422 Arc activity regulated cytoskeletal-associated 0.00007 -1.6

78602863-78602804 Card10 caspase recruitment domain family, 130 0.00002 2.4

78602622-78602563 Card10 caspase recruitment domain family 130 0.00006 2.1

78677249-78677308 Cdc42ep1 CDC42 effector protein (Rho GTPase 0.02491 -1.5

76728465-76728524 Commd5 COMM domain containing 5 25536 0.03226 1.3

78177993-78178052 Csf2rb1

colony stimulating factor 2 receptor, beta

1, low-affinity (granulocyte macrophage) 0.00013 1.8 0.00005 1.8 0.00009 1.9

78110010-78109951 Csf2rb2

colony stimulating factor 2 receptor, beta

2, low-affinity (granulocyte macrophage) 0.03975 1.7 0.01284 1.9 0.04049 1.7

76157502-76157561 Exosc4 exosome component 4 175399 0.01966 -1.2

78721389-78721448 Gga1

golgi associated, gamma adaptin ear

containing, ARF binding protein 0.04180 1.3

76327923-76327982 Hsf1 heat shock factor 1 8296 0.03284 -1.4

78678199-78678140 Lgals2 lectin, galactose-binding, soluble 2 25622 0.01818 -1.4

77454209-77454268 LOC193676 Predicted gene 0.01654 2.5

74822437-74822378 Ly6a

Mus musculus lymphocyte antigen 6

complex, locus A 10738 0.00429 1.9

74935520-74935462 Ly6c mRNA for Ly-6C variant 0.00016 2.0

74872596-74872537 Ly6c lymphocyte antigen 6 complex, locus C 10741 0.00017 2.2

74810352-74810293 Ly6i lymphocyte antigen 6 complex, locus I 20498 0.00029 2.1

78089728-78089787 Ncf4 neutrophil cytosolic factor 4 8677 0.03332 1.8

76453642-76453583 Nfkbil2

nuclear factor of kappa light polypeptide

gene enhancer in B-cells 0.03995 1.3

78770811-78770870 Nol12 nucleolar protein 12 0.00189 -1.6

78767955-78768014 Nol12 nucleolar protein 12 133800 0.02868 1.2

79083778-79083837 Pick1 protein interacting with C kinase 1 0.03420 -1.6

78448785-78448844 Pscd4

pleckstrin homology, Sec7 and coiled/coil

domains 4 0.04177 1.3 0.04387 1.3

73032517-73032458 Ptk2 PTK2 protein tyrosine kinase 2 7982 0.02290 -1.3

76911278-76911219 Rbm9 RNA binding motif protein 9 0.00441 2.6

78739245-78739304 Sh3bp1 SH3-domain binding protein 1 9164 0.00784 1.7

73407821-73407762 Slc45a4 solute carrier family 45, member 4 0.00353 1.2

75484985-75484457 Top1mt DNA topoisomerase 1, mitochondrial 28404 0.04252 -1.2

75452356-75452415 Zfp41 zinc finger protein 41 0.05080 -1.2

Genomic

Coordinates Chr. 15

ADH-AS ADH-BD ADH-ADLAccession

NMGene ProductGene Symbol

70

3.2.7 In Situ Hybridization (ISH) labelling of Csf2rb2 in the spinal

cord and brain of the mouse

The resulted Pain1 ‘autotomy genes’ from our spinal cord data analysis, Csf2rb1 and Csf2rb2,

were further investigated by us using in silico methodology. The Allen Institute has produced

the Allen Brain Atlas (ABA; Allen Brain Atlas) by using in situ hybridization (ISH) methods to

map nuclei of many expressed genes throughout the brain and spinal cord of a naïve young adult

C57BL6/J male mouse. Using this resource, investigators can screen photomicrographs of

sagittal or coronal views of brain sections and transverse views of spinal cord sections labelled

with the gene under study. In addition, investigators can view matching Nissl stained sections

and anatomical reference views that schematically annotate the same sections as those under

investigation, pinpointing anatomical structures harbouring the gene under study. Amongst the

genes studied by the ABA is Csf2rb2, a gene that is also located in Pain1, which was also

detected by us using the whole genome gene expression analysis of the DRG and spinal cord, but

at a marginal significance compared to Csf2rb1. Csf2rb2 juxtaposes in Pain1 the gene Csf2rb1

(Figure 6), the gene we studied in this dissertation work. Csf2rb2 has a sequence and orientation

that suggest that it is a result of gene duplication.

Figure 6: Physical genetic map of Csf2rb1 and Csf2rb2. These two genes are located

juxtaposing each other in Pain1.

Csf2rb2 Csf2rb1 = Csf2rb

71

While the ABA did not map Csf2rb1, it is of value to analyze the cell types that express Csf2rb2

and their location in the brain and spinal cord vis-à-vis those of Csf2rb1. We carefully studied

these maps and provide a summary below. The following are photomicrographs of sections from

the ABA for Csf2rb2 showing intense labelling of somata of cells in the dentate gyrus

polymorph layer and a careful observation also shows many more smaller cells in the granule

layers (Figure 7A, C). Labelled cells were also found in the peri-aqueductal grey (PAG; cells of

which engage in endogenous modulation of pain (Ho et al., 2013), the hypothalamic

supramamillary and arcuate nuclei (data not shown), the midbrain ventral tegmental area (VTA)

and on the pial surface of the brain, and in the grey matter surrounding the 3rd

ventricle, seen as a

black thin rim in Figure 7C. The spinal cord is also labelled, mainly with neurons in the white

and grey matter, including in specific dorsal horn regions that take part in processing nociceptive

inputs (Layer I, substantia gelatinosa, deeper Layers, Layer X, central canal, but also the ventral

horn neurons; Figure 8). Since these labelled sections are from B mice, it is possible that A mice

constitutively express more of this gene in dorsal horn and central canal regions that process

nociceptive inputs. Moreover, in the brain of a 56 week old B mouse, the Atlas shows highly

specific and very intense labelling of Csf2rb2 expressing neuronal nuclei in select regions,

mainly the Polymorph Layer of the Hippocampal Formation Dentate Gyrus, the

Supramammilary Nucleus, Arcuate nucleus of the hypothalamus, Subthalamic nucleus, and

72

Periaqueductal Gray Matter (Figure 9).

A B

C

73

Figure 7: Photomicrographs of Csf2rb2 ISH in select regions of mouse brain

(http://mouse.brain-map.org/experiment/show/73992937). (A, C) Coronal sections from the

ABA for Csf2rb2 in the mouse brain showing strong and specific labelling of cells in the dentate

gyrus (red ovals), peri-aqueductal grey (PAG, blue oval), the midbrain ventral tegmental area

(VTA), the pial surface of the brain (green oval), and in the grey matter surrounding the 3rd

ventricle (blue oval).

Figure 8: Photomicrographs from the Allen Brain Atlas (http://mousespinal.brain-

map.org/imageseries/show.html?id=100038817) showing a strong labelling of in situ

hybridization of Csf2rb2 in the lumbar spinal cord of a 56 weeks old intact B male mouse.

Transverse sections of the spinal cord show widely distributed labelling of Csf2rb2 expression in

neurons throughout the gray matter, more in the upper dorsal horn (1). There is also labelling of

glia cells under the pial surface of the dorsal columns (2) and around the central canal (3).

1

2

3

74

1

1

2

1

1

A B

C D

1

2

2

2

4

2

2

4

2

1

2

SMN

5

3

3

3

2

E

B

A

F G

Figure 9: Photomicrographs from the Allen Brain Atlas (http://mouse.brain-

map.org/experiment/show/73992937) showing a strong labelling of in situ hybridized reaction product to

Csf2rb2 in nuclei of certain cells clustering in specific brain nuclei and regions of an intact 56 weeks

male old B mouse (A, C, D, E). Sagittal (A, C) and coronal (D) sections of the mouse brain showing

selective areas having Csf2rb2 expression: (1) dentate gyrus of the hippocampal formation; shown in

light green in the reference atlas (B). Cells marked by (2) are labelled in the Peri-aqueductal Gray (PAG),

an area that is involved in descending modulation of pain (E-I). Strong labelling was also noted in the

Supramamilary Nucleus (SMN), marked as (3) in panels E-G, and other hypothalamic nuclei, including

the Arcuate Nucleus (ARH) marked as (7) in panels (H-J). What appears as glia cells in linings of

ventricles and the pia mater is marked by (4) and (5) in panels E-G. (B, F, G, J) show reference maps.

75

6

J

H

B

A

I

B

A

7

7

7

76

3.2.8 Csf2rb2 gene networks

The ISH data from the Allen Atlas of Csf2rb2, together with the strong indications from our own

gene expression data, pointing at Csf2rb1 and Csf2rb2 as possible candidate genes for autotomy

behaviours has prompted us to have a closer look at network interactions specifically with these

genes. Figure 10 illustrates the gene networks in which Csf2rb1 is involved. As can be seen,

Csf2rb1 is co-expressed with Csf2rb2, Csf2ra and Jak2, among other genes. Jak2 is one of the

kinases that phosphorylates CSF2RB1 and is discussed in greater detail in Chapter 5 & 6 in

mechanisms of the Jak/Stat pathway. Csf2rb1 is co-localized with and is found in a physical

interaction with Csf2rb2, among other genes. Csf2rb1 shares similar protein domains found in

Csf2rb2 and Csf2ra, as shown in Figure 10.

Figure 10: Gene networks of Csf2rb1. Presented in grey circles are genes found by GeneMania

to correlate with Csf2rb1 in one or more networks. Every line presents the correlation between

two genes, and the color specifies the type of correlation: purple designates co-expressed genes,

genes that are expressed together in the same tissue or system; physical interactions is marked by

pink lines, gene products are found physically bound to one another in the same complex, thus

serve in the same cellular pathway; co-localization (in blue lines), gene products that are

localized together within the same tissue or in the same cellular location; and shared protein

domains (in green lines), gene products that have the same protein domains. The thickness of the

lines symbolizes the weight of the correlation, the thicker the line the more correlated are 2 gene

or gene products.

77

Co-expression Co-localization

Physical interactionsShared protein domains

78

3.2.9 SNP analysis in candidate autotomy genes – Pain1 genes in

intact A and B mice

In addition to different mRNA levels in candidate genes that may produce differential levels of

autotomy behaviour, other genetic mechanisms may produce the same effect on chronic pain

behaviour, conferred by differences in the genetic sequence that control the contrasting

phenotypes in two genetic backgrounds, such as A vs. B mouse strains. One type of such genetic

alteration is mismatching single nucleotides between the two strains. These alterations are called

single nucleotide polymorphisms (SNPs), which can be found in exons that relay the genetic

message, in intronic regions that are not translated, or in the untranslated regions normally found

upstream of the gene where transcription factors bind and begin gene transcription. SNPs can

affect mRNA stability, production of different splice variants, gene-gene interactions, and the

production of a dysfunctional/truncated protein. In the present study, we looked at SNPs in

candidate genes, preferentially in coding regions, that exist between autotomy prone mouse

strains and autotomy resistant strains, such as A vs. B. This analysis served as another criterion

for prioritizing a candidate autotomy gene for future study, because genes lacking SNPs that

differ between A and B mice can be excluded from the shortlisted candidates for being

considered autotomy genes.

Using the Jax Labs Mouse Genome Informatics (MGI) website

(http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=snpQF) we compared the

sequence of a select number of mouse strains for which we had previous knowledge regarding

their typical level of autotomy post-hindpaw denervation, including some that are known to

express moderate-high levels of autotomy (such as A mice), some that express no or low

autotomy levels (including B mice) and some that express moderate autotomy levels. Table 11

and Appendix III list all candidate autotomy genes in Pain1, differentially expressed in the

DRGs, spinal cord, or both, and their SNPs according to following types.

(1) SNPs in coding regions: Several synonymous and non-synonymous SNPs were found in

these genes when comparing A to B mice, as well as comparing other high autotomy strains

(A/HeJ, BALB/cByJ, BALB/cJ), low autotomy strains (AKR/J, C57BL10/J), and moderate

autotomy strains (129X1/SvJ, 129S1/SvImJ, and DBA/2J).

79

(i) Non-synonymous SNPs, located in exons and thereby encode for a change in the amino acid

sequence of the gene product, were found in Csf2rb2 (2 such SNPs), and one SNP in each of the

genes Csf2rb1, Ly6i, Zfp7 Pick1 and Recql4. In Recql, the same SNP was also present in other

high (C3H/HeJ and BALB/cByJ) vs. low (AKR/J) and vs. moderate (DBA/2J, 129S1/SvImJ, and

129X1/SvJ) autotomy strains.

(ii) Four synonymous SNPs were found in exons. These are “silent” mutations, which do not

alter the amino acid sequence of the protein. Four such SNPs were noted in Csf2rb2 and 1 SNP

in each of the genes Csf2rb1, Ly6i, Zfp7, Pick1 and the currently not annotated gene

2010109I03Rik. In 2010109I03Rik the same SNP was also present in other high autotomy

strains (C3H/HeJ and BALB/cByJ) vs. low autotomy strains (AKR/J) and vs. moderate autotomy

strains (DBA/2J). As well, another SNP in this gene contrasted between the same strains;

however the allele in B mice is unknown.

(2) SNPs in untranslated regulatory regions (UTRs): UTRs also included many polymorphisms

in nucleotides mismatching between A and B mice. Seven genes contained SNPs within these

regions in Pain1: Nol12, differentially regulated in both DRGs and spinal cord of A vs. B mice,

had 2 SNPs; Csf2rb2, Csf2rb1, 1700088E04Rik, and Ly6i, differentially regulated in DRGs of A

vs. B mice, had 7, 4, 1, and 1 SNPs, respectively; 2010109I03Rik and Rbm9, that were

differentially regulated in the spinal cords of A vs. B mice, had 3 and 2 SNPs, respectively,

differing between A and B mice in the mRNA UTR. In 2010109I03Rik the same two SNPs were

also present in other high autotomy strains (C3H/HeJ and BALB/cByJ) vs. low autotomy strains

(AKR/J) and vs. moderate autotomy strains (DBA/2J, 129S1/SvImJ, and 129X1/SvJ).

(3) Intronic SNPs: Most genes had SNP variations in introns, and most variations in general were

intronic, as expected. Of the 34 candidate genes in Pain1, 20 genes had SNPs within introns.

Cacng2 (Nissenbaum et al., 2010) and Card 10, which were differentially regulated in both

DRGs and spinal cords of A vs. B mice, had 90 SNPs and one SNP within introns, respectively,

contrasting between the two strains, 9 SNPs contrasted between other high autotomy strains

(C3H/HeJ and BALB/cByJ) and low autotomy strain (AKR/J), and 6 SNPs contrasted between

high autotomy strains (C3H/HeJ and BALB/cByJ) and moderate autotomy strains (DBA/2J,

129S1/SvImJ, and 129X1/SvJ). In each of these SNPs, one allele was dedicated to all high

autotomy mouse strains and the other allele was present in low and/or the moderate autotomy

80

groups mentioned above. Rbm9, which was differentially regulated in the spinal cord of A vs. B

mice, had 94 SNPs within introns, which differed in these two strains, 23 SNPs differed between

other high autotomy strains (C3H/HeJ and BALB/cByJ) vs. low autotomy strain (AKR/J), and 10

SNPs contrasted between high autotomy strains (C3H/HeJ and BALB/cByJ) vs. moderate

autotomy strains (DBA/2J, 129S1/SvImJ, and 129X1/SvJ). Some of the other genes that

contained SNPs within introns that contrasted between A and B mice included: Csf2rb2,

Csf2rb1, Zfp7, 170008E04Rik, Ly6i, Ly6c and C030006K11Rik, which were differentially

expressed in the naïve DRGs, having 24, 13, 12, 7, 5, 2 and 1 SNPs, respectively; Pick1 Pvalb,

Apol10b, Recql4, and 2010109I03Rik that was differentially expressed in the naïve spinal cord,

had 23, 11, 5, 1, and 1 SNPs within introns, respectively.

The SNP analysis herein has advanced our knowledge on the genotypic contrast in several

candidate genes in Pain1 between A and B mice, and between other contrasting autotomy

strains. These differences in genotype within certain candidate genes may explain the

differential gene expression levels constitutively, as well as following hindpaw denervation, in

the contrasting autotomy strains. Indeed, we point out several candidates that were differentially

expressed in A vs. B mice before the denervation in intact mice, and following the denervation

with the appearance of autotomy. These 12 genes, Card10, Nol12, 1700088E04Rik,

CO30006K11Rik, Csf2rb1, Csf2rb2, Ly6c, Ly6i, Zfp7, Pick1, Pvalb, and Rbm9, were

differentially regulated with autotomy in at least one group comparison (ADH vs. AS, BD and

ADL; P<0.05;), and polymorphisms in coding, non-coding, and untranslated regulatory regions

were detected as discussed above. Of the four polymorphic type regions, the most significant

SNP type is in coding regions, whereby the mutation causes a change in the amino acid of the

protein and can potentially change its function. We thus prioritized genes with non-synonymous

SNPs at the highest level. SNPs in coding regions that were ‘silent’ and did not change the

amino acid were considered next, whereby the coding sequence in mRNA does not change the

protein function or its secondary structure. Third to be considered were genes whose SNPs were

located in UTRs, indicating whether mRNA levels may change for example, due to malfunctions

in the promoter binding site. Intronic SNPs were considered last because they were insignificant

unless they were found juxtaposing an exon-intron boundary, which might have affected the

splice site and could have created a splice variant. It is noteworthy that only three genes,

Csf2rb2, Csf2rb1, and Ly6i conveyed polymorphisms in all 4 regions discussed above, whereby

81

Csf2rb2 and Csf2rb1 were highly polymorphic within intronic and UTR regions in A vs. B mice,

some of which may explain the significant elevated mRNA levels in MHS- A mice compared to

NLS- B mice. Polymorphisms in Csf2rb2 and Csf2rb1 found in coding regions may or may not

support the notion that a truncated or non-functional protein receptor is produced in either the B

or the A strain, but not both. In conclusion to the SNP analysis Csf2rb2 and Csf2rb1 remain the

best candidate genes for autotomy in Pain1.

Table 11: SNPs in Pain1 candidate autotomy genes regulated constitutively between A and B

mice and between other MHS (A/HeJ, C3H/HeJ, BALB/cByJ, BALB/cJ) vs. NLS (AKR/J,

C57BL/10J) strains; MOD=Moderate autotomy strains: 129X1/SvJ, 129S1/SvI

Total SNPs A vs. B High vs. Low High vs. ModLow vs. ModTotal SNPs A vs. B High vs. Low High vs. ModLow vs. Mod Total SNPs A vs. B High vs. Low High vs. ModLow vs. ModTotal SNPs A vs. B High vs. Low High vs. ModLow vs. Mod

Card10 2 − − − − 0 − − − − 0 − − − − 1 1 − − −

Cdc42ep1 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Cacng2 0 − − − − 0 − − − − 0 − − − − 109 90 9 6 0

Nol12 7 2 − − − 1 − − − − 1 − − − − 44 − − − 5

Plec1 0 − − − − 0 − − − − 0 − − − − 0 − − − −

1700088E04Rik 1 1 − − − 0 − − − − 0 − − − − 13 7 − − −

C030006K11Rik 1 − − − − 0 − − − − 0 − − − − 3 1 2 − −

Csf2rb1 6 4 − − − 1 1 − − − 5 1 − − − 47 13 − − −

Csf2rb2 15 7 − − − 9 4 − − − 11 2 − − − 126 24 − − 1

EG666504 = Gm8104 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Gpihbp1 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Ly6c 1 − − − − 0 − − − − 1 − − − − 28 2 4 − −

Ly6i 10 1 1 − − 1 1 − − − 2 1 − − − 37 5 5 − −

Ly6k 0 − − − − 0 − − − − 1 − − − − 2 − − − −

Pscd4 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Zfp7 0 − − − − 1 1 − − − 1 1 − − − 18 12 4 1 −

2010109I03Rik 9 3 − 2 − 2 1 1 − − 0 − − − − 3 1 − − −

9130218O11Rik=

Apol10b 2 − − − − 3 − − − − 3 − − − − 47 5 3 1 1

BC024139 0 − − − − 0 − − − − 0 − − − − 0 − − − −

BC025446 0 − − − − 0 − − − − 0 − − − − 39 − − − −

Dennd3 7 − − − − 13 − − − − 7 − − − − 511 − − 9 −

ENSMUST00000096398 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Lrrc62 = Elfn2 1 − − − − 0 − − − − 0 − − − − 8 − − − −

Ly6d 2 − − − − 0 − − − − 0 − − − − 9 − − 2 −

Ly6h 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Mafa 0 − − − − 0 − − − − 0 − − − − 0 − − − −

Pick1 0 − − − − 1 1 − − − 2 1 − − − 83 23 − 5 −

Pvalb 0 − − − − 0 − − − − 0 − − − − 24 11 − − −

Rbm9 = RBfox2 2 2 − − − 0 − − − − 0 − − − − 145 94 23 10 −

Recql4 0 − − − − 0 − − − − 1 − 1 − − 3 1 1 − −

Scrt1 1 − − − 1 0 − − − − 0 − − − − 0 − − − −

Slc45a4 17 − − − − 9 − − − − 1 − − − − 207 − − 4 4

Slurp1 0 − − − − 0 − − − − 0 − − − − 3 − − 1 −

Coding Synonymous Coding Nonsynonymous IntronGene

mRNA UTR

DR

Gs &

Sp

ina

l

Co

rd

DR

Gs

Sp

ina

l co

rd

82

3.2.10 Other candidate genes related to autotomy

Another analytical algorithm for prioritizing the 34 Pain1 candidate genes for further in depth

work, resulting from the comparisons of the expression data in the DRGs and spinal cord of

intact and denervated A vs. B mice, was a literature search performed for each gene to examine

whether it has been studied within the context of pain or another related neural or neurological

function. A Pubmed search was performed for each of the candidate genes in Pain1 with the

following 14 searching terms: ‘brain’, ‘nerve’, ‘CNS’, ‘spinal cord’, ‘nerve injury’,

‘inflammation’ and ‘inflammatory response’,‘neurodegeneration’, ‘analgesia’, ‘pain’,

‘nociception’, ‘neuropathic’, ‘hyperalgesia’ and ‘allodynia’.

Table 12 lists genes reported in articles associated with one or more of these 14 searching terms,

including the product they encode, accession number, and chromosomal location (Mb). Genes

appearing in reports that included 1 of the 14 searching terms (‘brain’) were Ly6i, Ly6h and

Scrt1. Genes reported with 2 searching terms were Nol12, and Pvalb (‘brain’ and ‘nerve’) and

Gpihbp1 (‘brain’ and ‘analgesia’). Genes reported with 3 terms were Plec1 (‘brain’, ‘nerve’ and

‘inflammatory response’) and Recql4 (‘brain’, ‘CNS’ and ’neurodegeneration’). The gene Card

10 was reported with 4 terms (‘brain’, ‘nerve’, ‘inflammatory response’, and ‘analgesia’). Genes

reported with 5 terms were Ly6c and Rbm9 (‘brain’, ‘nerve’, ‘CNS’, ‘nerve injury’,

‘inflammation’and ‘inflammatory response’). The gene Mafa was reported with 6 terms (‘brain’,

‘nerve’, ‘spinal cord’, ‘nerve injury’, ‘inflammation’, ‘inflammatory response’ and ‘analgesia’).

The gene Csf2rb1 was reported with 7 terms (‘brain’, ‘nerve’, ‘CNS’, ‘spinal cord’, ‘nerve

injury’, ‘inflammatory response’ and ‘neurodegeneration’). The gene Cacng2 was reported with

8 terms (‘brain’, ‘nerve’, ‘CNS’, ‘spinal cord’, ‘nerve injury’, ‘inflammatory response’,

‘neurodegeneration’ and ‘pain’; some of these were actually contributed by our publication of it:

Nissenbaum et al., 2010). Finally, Pick1 was associated with 10 search terms (brain’, ‘nerve’,

‘CNS’, ‘spinal cord’, ‘nerve injury’, ‘inflammation’, ‘neurodegeneration’ ‘analgesia’,

‘nociception’, ‘neuropathic’ and ‘hyperalgesia’). Thus, based on this literature search, any one

of the above-listed genes could be a candidate gene for autotomy, moreover, it is important to

keep in mind that a new gene never implicated before with this trait could be missed with this

analysis.

83

Table 12: Candidate autotomy genes in Pain1 and their known relevance to pain. In red are

genes that are reported in articles associated with one or more of the following14 searching

terms: ‘brain’, ‘nerve’, ‘CNS’, ‘spinal cord’, ‘nerve injury’, ‘inflammation’ and ‘inflammatory

response’,‘neurodegeneration’, ‘analgesia’, ‘pain’, ‘nociception’, ‘neuropathic’, ‘hyperalgesia’

and ‘allodynia’.

3.2.11 Selecting the best candidate gene in Pain1

The gene expression profiling results from this study identified a number of candidate genes for

autotomy before and after the nerve injury. Autotomy genes were found throughout the genome

in both DRGs and spinal cords. In Pain1 there were a few candidates that differentially

regulated between A vs. B mice before the nerve injury, which could explain autotomy behaviour

Gene Brain Nerve CNSSpinal

cord

Nerve

injury

Inflammation,

Inflammatory

response

Neurodegeneration Analgesia

Pain,

Nociception,

Neuropathic

Hyperalgesia,

Allodynia

Cacng2 √ √ √ √ √ √ √ x √ x

Card10 √ √ x x x √ x √ x x

Plec1 √ √ x x x √ x x x x

Nol12 √ √ x x x x x x x x

Cdc42ep1 √ x x x x x x x x x

Csf2rb1 √ √ √ √ √ √ √ x x x

Ly6c √ √ √ x √ √ x x x x

Gpihbp1 √ x x x x x x √ x x

Ly6i √ x x x x x x x x x

Csf2rb2 x x x x x x x x x x

1700088E04Rik x x x x x x x x x x

C030006K11Rik x x x x x x x x x x

EG666504 = Gm8104 x x x x x x x x x x

Ly6k x x x x x x x x x x

Pscd4 x x x x x x x x x x

Zfp7 x x x x x x x x x x

Pick1 √ √ √ √ √ √ √ √ √ √

Mafa √ √ x √ √ √ x √ x x

Rbm9 = RBfox2 √ √ √ x √ √ x x x x

Slurp1 x √ √ √ √ x x x √ x

Recql4 √ x √ x x x √ x x x

Pvalb √ √ x x x x x x x x

Ly6h √ x x x x x x x x x

Scrt1 √ x x x x x x x x x

2010109I03Rik x x x x x x x x x x

9130218O11Rik=

Apol10b x x x x x x x x x x

BC024139 x x x x x x x x x x

BC025446 x x x x x x x x x x

Dennd3 x x x x x x x x x x

ENSMUST00000096398 x x x x x x x x x x

Lrrc62 = Elfn2 x x x x x x x x x x

Ly6d x x x x x x x x x x

Slc45a4 x x x x x x x x x x

DR

Gs

& S

pin

al

Co

rdD

RG

sS

pin

al

cord

84

in A mice devoid of B mice. However, only 3 candidate Pain1 genes were differentially

regulated with autotomy behaviour following the nerve injury, Hemt1 in the DRGs and Csf2rb1

and Csf2rb2 in the spinal cord. Hemt1 was up-regulated with autotomy in MHS-A mice

compared to sham A mice and to denervated NLS-A mice, but down-regulated in denervated

MHS-A mice compared to denervated NLS-B mice. Moreover, Hemt1 lacked polymorphisms

mismatching between A and B mice. Furthermore, Hemt1 was not cited in the literature in any

category that may associate it with pain. Taken together, Hemt1 was not considered by us as a

candidate gene for autotomy and was not selected for further gene follow-up analyses.

Thus, the only remaining candidate autotomy genes in Pain1 were Csf2rb2 and Csf2rb1 (mainly

the latter) based on the evidence detailed below:

(i) Csf2rb1 was significantly up-regulated with autotomy in the spinal cord of denervated MHS-

A mice vs. sham operated A mice, and vs. denervated NLS-A mice as well as vs. denervated

NLS-B mice.

(ii) Csf2rb1 was associated in a network with other genes, which indicate that it participates in a

few cellular processes known to be relevant to nociception and chronic pain.

(iii) Csf2rb1 contains polymorphic nucleotides contrasting between A and B mice, which could

be causative in explaining the contrast in autotomy. Moreover, it contained SNPs in four

different genetic regions, including coding regions, some of which are non-synonymous -

altering the protein product.

(iv) Csf2rb1 was cited in the literature in relation with many keywords associating it with pain

(see Discussion).

Despite the fact that Csf2rb2 was significantly up-regulated with autotomy in the spinal cord of

denervated MHS-A mice vs. the other low autotomy mouse groups indicated above, the P values

were not as robust as for Csf2rb1. Csf2rb2 was not significantly regulated in DRGs of

denervated MHS-A mice vs. the other low autotomy mouse groups. Csf2rb2 was associated with

networks with other genes and contains SNPs contrasting between A and B mice; however, this

gene is not cited in the literature in relation to pain.

85

On this basis, Csf2rb1 was selected as the best candidate gene for autotomy, and the subject of

the gene follow-up assays described below in Chapter 4. While working on these experiments

on this gene, another gene in Pain1, Cacng2, has been studied as a candidate gene for autotomy

collaboratively by us and another research group at the Hebrew University in Jerusalem

(Nissenbaum et al., 2010). Some experimental work contributed by both groups resulted in a

publication to which we co-author, and this work is discussed in detail in the Discussion.

3.3 Discussion

3.3.1 Csf2rb1 as a candidate autotomy gene in Pain1

The present study used whole genome expression profiling to identify Csf2rb1 (a gene encoding

for the common receptor beta of GM-CSF, IL-3 and IL-5) as another candidate autotomy gene in

Pain1, in addition to Cacng2 (Nissenbaum et al., 2010). Chapter 4 describes additional

experiments we undertook to further support its candidacy. Csf2rb1 was expressed in

significantly higher levels in DRGs of naïve A mice compared to naïve B mice, and in the spinal

cord of denervated A mice expressing MHS of autotomy compared to denervated A mice with

NLS of autotomy . Csf2rb1 is part of a heterodimer receptor complex, the Colony Stimulator

Factor Receptor (CSFR) that is composed of 2 α and 2 β subunits. Csf2rb1 encodes the β

receptor subunit which is common for a number of cytokines, and is analogous to other cytokine

receptor complexes (such as the IL-6, IL-4, IL-13, and IL-2 receptors), each of which uses a

shared signalling subunit, suggesting an evolutionary conserved structural and functional

arrangement whereby a single polypeptide receptor chain can recognize more than one cytokine

to mediate multiple biological activities (Hercus et al., 2009). Indeed, Csf2rb1 encodes the beta

chain of a cytokine receptor which is shared among three ligands, GM-CSF, IL-3 and IL-5. It is

conserved in humans, chimpanzees, Rhesus monkeys, dogs, cows, rats, mice and chicken. Its

protein product is involved in multiple biological mechanisms, including cell survival,

proliferation, differentiation and apoptosis or its repression (Hercus et al., 2009; Kao et al., 2008;

Lee et al., 1999b; Lopez et al., 2010). Upon ligand binding, with any ligand of the three: GM-

CSF, IL-3, or IL-5, and receptor dimerization and activation via Janus kinase-2 (Jak-2) (Lopez et

al., 2010), intracellular signalling is followed, including the Jak/Signal transducer and activator

86

of transcription (Jak/Stat) pathway, Ras/Mitogen-activated protein kinase (Ras-MAPK) pathway,

and the phosphatidylinositol-3 (PI-3) kinase pathway (Hercus et al., 2009).

Many chronic inflammatory diseases are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine

receptor signalling, highlighting their importance in disease (Lopez et al., 2010). Thus, Csf2rb1

holds several roles in important cellular mechanisms that have been conserved through

evolution, which may suggest that this gene product may play the same role in neuropathic pain

mechanisms in many other mammalian and avian species. If corroborated by additional

experiments, this conservation would attest to the importance this gene product plays in pain

mechanisms. While not much is known about the functions of Csf2rb1 in the nervous system,

more is known about one of its ligands, GM-CSF, in relation to neurological diseases, response

to nerve injury, and in peripheral pain mechanisms. In this dissertation we describe the possible

involvement of CSF2RB1 in the CNS, and propose its association with pain.

3.3.2 Cacng2 in Pain1 as a candidate autotomy gene

In a recent publication we proposed that Cacng2, a gene in Pain1, is also a candidate autotomy

controlling gene (Nissenbaum et al., 2010). To prioritize the genes in Pain1, Nissenbaum et al.

applied four criteria to the genes in this QTL: (1) segregation between known SNPs in genes in

the confidence length of the QTL with the autotomy phenotype, i.e., having one SNP in all MHS

autotomy strains following nerve injury while having the other SNP in NLS autotomy strains. (2)

reported functional relevance of the candidate gene to neuropathic pain, or other pain syndromes

or traits (e.g., acute and/or inflammatory pain) as found by browsing PubMed using a set of

relevant searching terms, (3) significant up- or down-regulation of the gene expression levels

following hindpaw denervation, in a direction compatible with the gene’s putative function, and

(4) differential expression in several MHS vs. NLS autotomy strains.

The mouse strains studied by Nissenbaum et al. were different from those we used in the present

study in the following ways. The only MHS strain used for gene expression by Nissenbaum et

al. was C3H/HeJ. The A mouse strain used in the present study was not included in the gene

analysis (Criterion-4), but was only used for the SNP variation analysis

(http://www.informatics.jax.org). That study did not identify SNPs that: (i) alter protein

sequence in Cacng2, and (ii) segregate between mouse strains with known NLS and MHS,

87

suggesting an alteration of gene expression levels only between the studied mouse strains. In

contrast to Cacng2, there were 6 SNP variations in the non-intronic regions of Csf2rb1, 4 in the

UTR, and 2 in the coding region, 1 of which was found non-synonymous, and alters the protein

sequence, that differ in MHS strains (A/J, Balb/cByJ, 129X1/SvJ, 129S1/SvInJ) vs. NLS strains

(C57BL/6J and DBA) (Mogil et al., 1999a). The Nissenbaum et al. paper included the following

gene validation experiments for Cacng2’s candidacy as an autotomy gene: (i)

Immunohistochemical demonstration of positively labelled CACNG2 somata in the DRGs of

naïve mice of the CBA/J strain, showing that the somata of primary afferents indeed express this

protein. However, the article did not include differential labelling of such somata in denervated

MHS mice vs. sham-operated or naïve mice. The mere showing that CACNG2 is expressed in

the DRGs of naïve mice is not enough, when presented alone, to support its candidature as a

gene associated with autotomy, (ii) Phenotyping of autotomy levels following hindpaw

denervation was carried out in mice of the F2 generation of the Stargazer CACNG2

hypomorphic mutant mice. However, no significance was found in the autotomy levels when the

denervated mutant mice were housed communally. It was only when studying the denervated

AND single-caged that the differential display of autotomy was shown in the denervated mutants

vs. wild type mice. The latter manipulation is known to be a stressor. Single housing is not

known to exert any other effect. The article showed that the effect that CACNG2 in the DRGs

has on autotomy is mediated by increasing the expressed levels of the protein. (iii) Using

electrophysiological recording methods they showed that spontaneous ectopic discharge in DRG

neurons of Stargazers denervated mice is higher than sham operated mice. But as CACNG2

operates in the periphery, it is not clear how does the stress of single housing affect the firing rate

of the injured primary afferents in the periphery. Moreover, regardless of the mechanism, the

mere fact that the effect of CACNG2 on autotomy can only be demonstrated when there is an

added condition (i.e., stress) indicates that CACNG2 alone has a very weak effect on autotomy.

And finally: (iv) The authors demonstrated a significant segregation of Cacng2 SNPs in women

post-mastectomy with neuropathic pain vs. operated women who were pain-free.

While our contribution to that study was in genotyping women post-mastectomy, a similar

genotyping of the same patients for SNPs in Csf2rb1 also showed a significant segregation by

neuropathic pain levels (data not shown). Thus, taken together, our results suggested that

Csf2rb1 may be another candidate gene for autotomy in Pain1 and prompted us to carry out to

88

undertake additional follow-up experiments including immunohistochemistry (see Chapter 4) to

further support its candidacy.

3.4 Conclusions

Experiments we carried out in the study reported in Chapter 3 identified Csf2rb1 as the gene in

Pain1 that significantly up-regulated in the spinal cord with pain behaviour induced by

denervation of the hindpaw in A mice compared to B mice and sham operated A mice. Its

mRNA levels were constitutively higher in the DRGs of naïve A vs. B mice and significantly

elevated in the ipsilateral spinal cord following hindpaw denervation in A mice vs. B mice.

Moreover, SNPs in several exons of Csf2rb1 were polymorphic between A and B mice.

Furthermore, Csf2rb1 has been documented to participate in some peripheral pain pathways,

such as Jak/Stat3, PI3K, MAPK and nuclear factor kappa B (NFκB) signalling. As these studies

did not test its role in the CNS, it is possible that should those articles tested it in the CNS its role

there could have been demonstrated as well. In addition, one of its ligands (i.e., GM-CSF) is a

crucial contributor to microglial activation in the CNS as well as contributes to membrane

excitability of peripheral sensory neurons (Duport and Garthwaite, 2005; Giulian and Ingeman,

1988; Hutter et al., 2010; Lin and Levison, 2009; Maresz et al., 2005; Mukaino et al., 2010;

Nakajima et al., 2006; Quan et al., 2009; Schweizerhof et al., 2009; Sheikh et al., 2009; Suzuki et

al., 2008; Volmar et al., 2008) - two important cellular mechanisms in pain. Results reported in

the next chapter additionally validate, in part, the candidacy of Csf2rb1 as an autotomy gene.

89

Chapter 4

Study II: Expression of CSF2RB1 by spinal central canal ependymal cells/radial glia/tanycytes correlates with autotomy levels in mice

4.1 Introduction

The Neuroma model (Wall et al., 1979) of spontaneous neuropathic pain mimics in rodents pain

experienced by humans following limb amputation, plexus avulsion, and major denervations.

The model manifests in progressive self-mutilation (‘autotomy’) of the denervated hindpaw, and

when studied under the same environmental conditions, the degree of autotomy is highly variable

across inbred rodent stains, indicating that this trait is controlled genetically (Devor et al., 2007;

Mogil et al., 1999a). Yet, mice of the same inbred strain, that are studied under fixed

experimental conditions, also show some variability in autotomy levels, indicating that some

environmental parameters have not been fully controlled and that they play some role via gene-

by-environment interactions.

The A and B strains are inbred (see Chapter 1 Section 1.4.2), and when tested under the same

environmental conditions they show contrasting levels of autotomy, where A express MHS and

B express NLS following the same hindpaw denervation procedure (Chapter 3). These strains,

together with the 23 recombinant inbred lines of the AXB-BXA set that were derived from

crossing mice of the A and B inbred strains, have been used by us previously to map a

quantitative trait locus (QTL) on mouse chromosome 15 that has a major effect on controlling

the variable levels of autotomy. We termed this QTL Pain1 (Seltzer et al., 2001). The presence

of this QTL was replicated in a study that used two strains that are also known to contrast on this

trait but are different strains from those used by Seltzer et al. 2001: C3H/HeJ and C58/J (Devor

et al., 2005b). This replication study indicated that Pain1 may contain autotomy genes in

additional contrasting strains, different from A versus B mice, that can be generalized to

operating in this function in mice in general. We reported that Cacng2, a gene harboured at the

Pain1 QTL, is a candidate gene for autotomy and neuropathic pain in women post-mastectomy.

This gene encodes the calcium channel subunit g2 (Nissenbaum et al., 2010).

90

In our preceding study, using whole genome expression analysis of DRGs and spinal cord in

naïve, sham-operated and denervated mice of the A and B strains, we identified another gene

located in Pain1 associated with autotomy behaviour. This gene, Csf2rb1, encodes the common

beta receptor of the cytokines GM-CSF, IL-3 and IL-5. In the present study, our aim was to

associate the candidacy of Csf2rb1 as another gene for autotomy in Pain1, and identify the type

of cells expressing it as ependymal cells/radial glia/tanycytes located in certain locations in the

spinal cord and brain.

4.2 Results

4.2.1 Autotomy behaviour in denervated A and B mice

To determine whether expressed levels of the Csf2rb1 gene correlate with the levels of pain

behaviour, autotomy was scored in denervated A and B mice following sciatic and saphenous

neurectomy, and these levels were compared with those of sham operated and naïve mice of the

same strains. Figure 11 displays the autotomy behaviour in these groups. As expected from

previous experiments by others and us, the figure shows that naïve and sham-operated A and B

mice remained free of autotomy throughout the 14 day behavioural follow-up, while denervated

A mice expressed considerably more autotomy behaviour compared to denervated B mice. Also

expected was the finding that denervated A mice expressed on day 14 PO final autotomy scores

that varied highly from animal to animal ranging from an AFS=0-11. As done in Chapter 3,

denervated A mice were divided into two subgroups, one comprising mice that expressed no/low

scores (NLS) on day 14 PO (i.e., having AFSs=0-1; ‘low-autotomy’), and the other subgroup of

mice who expressed moderate-high scores (MHS) on day 14 PO (i.e., had AFSs=2-11; ‘high

autotomy’). Figure 11b shows the average course of autotomy behaviour in these two subgroups

of denervated A mice. The final autotomy score on day14 PO significantly differed between

MHS and NLS phenotypes at P<0.0001 and was used as the final pain outcome measure for the

following correlative assessment of Csf2rb1 gene expression levels.

A second phenotype that contrasted between denervated A and B mice was the autotomy onset

day, i.e., the day in which mice began to self-mutilate and their autotomy score was ≥1. MHS-A

91

mice began to self-mutilate much earlier (day 4.84 PO ±0.77), compared with NLS- A mice (day

13.0 PO ±0.85, P<0.0001) or NLS-B mice (day 12.5 PO ±0.99, P<0.0001) (Figure 12b).

The third pain outcome of autotomy behaviour that was used to assess the expression level of

Csf2rb1 was the level of self-mutilation expressed by denervated mice on the onset day of

autotomy, a phenotype we named Autotomy Onset Score (‘AOS’). Figure 13a shows that the

average AOS in MHS- A mice was significantly higher (4.08±1.05) compared to the NLS-A

mice (0.14±0.14; P<0.0001) and NLS-B mice (0.41±0.09; P<0.0001), and this low score retained

low (scores of 0-1) throughout the experiment. MHS-A mice expressed a wider range of self-

mutilation scores on the day of onset. Therefore, it was important to determine whether Csf2rb1

gene expression levels also drove A mice to express a higher autotomy score at the behaviour’s

onset or only at the end of the behavioural follow up on day 14.

92

Figure 11: Post-operative course of average autotomy scores in denervated, sham and naïve A

and B mice. Following sciatic and saphenous neurectomy, mice were followed up daily and

their autotomy behaviour was scored up to day 14. (A) Average autotomy scores per day, of

denervated A mice (AFSs on day 14PO ranged from 0-11; N=42) vs. denervated B mice (AFSs

on day 14PO ranged from 0-1; N=32), A-sham (AFS on day 14PO was 0 for all mice; N=12), B-

sham (AFS on day 14PO was 0 for all mice; N=12), A-naïve (AFS on day 14PO was of 0 for all

mice; N=8;) and B-naïve mice (AFS on day 14PO was of 0 for all mice; N=8;) (B) Average

autotomy scores of MHS-A mice (AFSs on day 14PO were 2-11; N=25) vs. NLS-A subgroup

(AFSs on day 14 were 0-1; N=17), A-sham (AFS on day 14PO of all mice was 0; N=12), and A-

naïve mice (AFS of all mice on day 14PO was 0; N=8). For A and B, Two-way ANOVA for

0

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ave

rage

au

toto

my

sco

re

Post operative day

Autotomy in A and B mice

A-denervated

B-denervated

A-sham

B-sham

A-naive

B-naive

0

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ave

rage

au

toto

my

sco

re

Post operative day

Autotomy in A mice

A-high

A-low

A-sham

A-naive

B

A

*

****

*** **** ********

************

************

*

**

* ** *****

** ** ****************

93

repeated measures was performed followed by Tukey’s post hoc test; *P<0.05, **P<0.01,

***P<0.001, ****P<0.0001

Figure 12: Average autotomy onset day in denervated A and B mice. Following sciatic and

saphenous neurectomy, mice were followed up daily and their autotomy behaviour was scored

up to day 14. (A) Average autotomy onset day is presented for mice of the MHS-A subgroup

(N=25) vs. NLS-A mice (N=20) and NLS-B mice (N=32), sham-operated A and B mice (N=12

and N=12, respectively), and naïve A and B mice (N=8 and N=8, respectively). A one-way

ANOVA with Tukey’s post hoc showed that denervated MHS-A mice had a significantly shorter

autotomy onset day than all other NLS groups. (B) The number of mice which developed

autotomy on each day post-operation is represented in the columns. The frequency of autotomy

scores at onset day is shown for the MHS-A, NLS-A and NLS-B autotomy subgroups, as well as

for sham-operated and naïve A and B subgroups.

Figure 13: Autotomy scores at onset day in denervated A and B mice. Following sciatic and

saphenous neurectomy, mice were followed up daily and their autotomy behaviour was scored at

onset. (A) Average scores of autotomy at onset day in denervated A and B mice are presented

for MHS-A subgroup (N=25) vs. NLS-A (N=20) and NLS-B mice (0.28±0.08, N=32), sham

operated A and B mice (N=12 and N=12, respectively), and naïve A and B mice (N=8 and N=8,

respectively). A one-way ANOVA with Tukey’s post hoc showed that denervated A-high mice

had significantly higher autotomy onset scores than all other low autotomy groups. (B) The

0

2

4

6

8

10

12

14

16

A mice B mice

Au

toto

my

on

set

day

Autotomy onset day in A and B mice

denervated-high

denervated-low

sham

naive

0

5

10

15

20

25

30

35

40

45

50

1 2 4 5 6 7 8 9

10

11

12

13

A m

ice

14

B m

ice

14

Nu

mb

er

of

mic

e

Autotomy onset day

Number of mice per Autotomy Onset Day

denervated-high

denervated-low A

denervated-low B

sham

naive

p<0.0001

A B

0

0.5

1

1.5

2

2.5

3

3.5

4

A mice B mice

Au

toto

my

on

set

sco

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Autotomy onset score in A and B mice

denervated-high

denervated-low

sham

naive

p<0.0001

0

5

10

15

20

25

30

35

40

45

50

A m

ice

0

B m

ice

0

A m

ice

1

B m

ice

1 2 3 4 5 7

11

Nu

mb

er

of

mic

e

Autotomy onset score

Number of mice per Autotomy Onset Score

denervated-high

denervated-low

sham

naive

BA

94

frequency of certain autotomy scores is shown in the denervated MHS-A, NLS-A and NLS-B

subgroups, as well as the sham-operated and naïve subgroups.

4.2.2 Csf2rb1 pattern of expression correlates with autotomy

behaviour in denervated A mice

Csf2rb1 gene expression levels contrasted in spinal cords of denervated MHS-A vs. NLS-A

mice, as expected from previous studies (Figure 14A). The significantly contrasting Csf2rb1

expression levels were noted between autotomizing and non-autotomizing mice, whether

denervated, sham operated or naïve. In denervated MHS-A mice, Csf2rb1 was expressed 2-fold

higher compared to the denervated NLS-A mice, and to sham-operated A mice (2.47±0.33 vs.

1.20±0.09; P<0.002, and 1.07±0.06; P<0.01, respectively). Moreover, Csf2rb1 was expressed 2-

fold higher in the MHS-A mice compared to the NLS-B mice (2.47±0.33 vs. 1.19±0.05;

P<0.004). No significant difference was observed between its expression levels in intact A and

B mice. Thus, constitutive gene expression levels of Csf2rb1 are comparable in the spinal cords

of these mice and therefore, the behavioural contrast post-denervation cannot be attributed to

differing constitutive levels of expression that would manifest at the time of nerve injury or a few

hours thereafter. Furthermore, no significant difference in gene expression levels was observed

between sham-operated A and B mice. Thus, the behavioural contrast post-denervation between

these strains cannot be attributed to differing levels of expression that are caused by anesthesia

and cutting skin and exposure of the nerves alone. Moreover, no significant difference was

observed in Csf2rb1 expression levels between the denervated NLS-A and NLS-B mice. This

result is compatible with the explanation that despite strain differences, same levels of Csf2rb1

gene expression are associated with the same autotomy levels. In fact, these expression levels

were generally comparable in all six groups that expressed NLS. Figure 14B shows the

correlation between Csf2rb1 gene expression and the autotomy levels in denervated A and B

mice on day 14 PO (R2 = 0.31; P<0.004).

Csf2rb1 gene expression levels were also correlated with the onset day of autotomy in A and B

mice (Figure 15A). MHS-A mice that had an early onset of autotomy that ranged from PO days

1-7 , had significantly higher Csf2rb1 mRNA levels in the ipsilateral spinal cord compared with

NLS-A and NLS-B mice that had a significantly later onset, ranging from PO days 8-14

(2.43±0.38 vs. 1.19±0.10; P<0.01 and 1.19±0.05; P<0.01, respectively). Within MHS-A mice,

95

no significant differences in Csf2rb1 spinal cord mRNA levels were observed between early vs.

late onset days of autotomy. This result suggests that expression levels of this gene are not

related to the onset of auotomy and its trigger but to the maintenance phase of chronic pain that

is at its peak at the time the animals were sacrificed, correlating significantly with the contrast at

the AFS levels rather than the onset days of the behaviour. Despite this conclusion, Csf2rb1

gene expression levels significantly correlated with the level of autotomy at its onset, both in A

and B mice (Figure 15B). Csf2rb1 mRNA levels of mice expressing at onset MHS (i.e., scores

≥2) did not significantly differ from those of mice expressing NLS at onset (i.e., autotomy onset

score of 1). However, high levels of autotomy at onset in MHS-A mice were correlated

significantly with higher Csf2rb1 mRNA levels on day 14 PO of A and B mice that did not

develop autotomy at all (2.32±0.51 vs. 1.27±0.07; P<0.05 and 1.19±0.05; P<0.05, respectively).

In conclusion Csf2rb1 was regulated significantly with autotomy behaviour, especially with the

autotomy final score on day 14PO in A and B mice.

96

Figure 14: Correlation of Csf2rb1 gene expression levels with autotomy behaviour on PO day

14 in denervated A and B mice. (A) Average Csf2rb1 mRNA levels (normalized relatively to

the expression levels of Hprt, the reference gene, see Appendix IV) of naïve (N=4, and N=4,

respectively), sham-operated (N=4 and N=4, respectively), denervated A and B mice with NLS

(N=8, and N=6, respectively), and denervated MHS-A mice (N=11). Csf2rb1 expression was 2-

fold higher in MHS-A mice compared to NLS mice (one-way ANOVA followed by Tukey’s

post hoc test). (B) Correlation analysis of Csf2rb1 mRNA levels (normalized by the levels of

Hprt) of individual denervated A and B mice and their autotomy levels on PO day 14.

0

0.5

1

1.5

2

2.5

3

A strain B strain

mR

NA

level of

Csf2

rb1

rela

tive t

o H

prt

Csf2rb1 gene expression in A and B mice

Naive

Sham

Low Autotomy

High Autotomy

P<0.01P<0.05 P<0.01P<0.01

A

B

R = 0.3137

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12

mR

NA

leve

l of

Csf

2rb

1re

lati

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o H

prt

Final autotomy score

Correlation of Csf2rb1 mRNA and autotomy levels in A and B mice

P = 0.003593

97

Figure 15: Csf2rb1 gene expression levels correlation with the onset day of autotomy and

autotomy onset scores in denervated A and B mice. (A) Average Csf2rb1 mRNA levels (relative

to Hprt) are represented for Low autotomy/Late onset B and A mice, which developed an

autotomy score of 1 on days 8-14 PO (N=6 and N=7), MHS-A/Late onset mice, which developed

autotomy scores of 2-11 on days 8-14 PO (N=5), and MHS-A/Early onset mice, which

developed autotomy scores of 2-11 on day 1-7 PO (N=8). Csf2rb1 expression was 2-fold higher

in MHS-A/Early onset mice, compared to Low autotomy/Late onset mice (one-way ANOVA

followed by LSD’s post hoc test). (B) Average Csf2rb1 mRNA levels (relative to Hprt) are

shown for denervated A and B mice, which did not develop autotomy and remained with an

autotomy score of 0 up to day 14 PO (N=6 and N=6), denervated A mice, which developed high

or low autotomy and their score at autotomy onset day was 1 (N=6), and A-MHS mice, which

developed high autotomy and their score at onset was ≥2 (N=8). Csf2rb1 expression was 2-fold

higher in denervated A mice that developed high autotomy scores of ≥2 at onset, compared to

denervated A and B mice with no autotomy until PO day 14, hence their score at onset day of 15

PO was 0 (one-way ANOVA followed by LSD’s post hoc test).

4.2.3 Autotomy behaviour in denervated C3H/HeJ and AKR/J

mice

If Csf2rb1 expression regulates the autotomy behaviour in mice in general and not just the

contrast between denervated A and B strains, then other autotomy contrasting strains should also

show the corresponding contrast in expression levels of this gene in the spinal cord. Therefore,

two additional strains that are known from previous work to contrast in autotomy behaviour

following hindpaw denervation were used next, to determine whether Csf2rb1 correlates with

autotomy in these strains as well: C3H/HeJ mice, which express MHS, and AKR/J, which

express NLS following the same sciatic and saphenous neurectomy. Table 11 (Chapter 3) shows

that these two strains carry many SNPs on candidate Pain1 autotomy genes we considered in the

previous Chapter. For this reason, we compared Csf2rb1 gene expression levels in these strains

0

0.5

1

1.5

2

2.5

3

Low autotomy/Lateonset

High autotomy/Lateonset

High autotomy/Earlyonset

mR

NA

leve

l of

Csf

2rb

1re

lati

ve t

o H

prt

Csf2rb1 gene expression is correlated with onset day of autotomy in A and B mice

B

A

0

0.5

1

1.5

2

2.5

3

Onset score 0 Onset score 1 Onset score >2mR

NA

leve

l of

Csf

2rb

1re

lati

ve t

o H

prt

Csf2rb1 gene expression is correlated with autotomyonset score in A and B mice

B

A

B

p<0.01

A

p<0.01p<0.05

p<0.05

_

98

correlating these levels with autotomy behaviour. Mice of the two strains were denervated, or

sham-operated, or left intact, and were followed up for autotomy behaviour. As most C3H/HeJ

mice that were denervated did not develop autotomy in the first 14 days post-operation, they

were followed up for 21 days PO. Upon reaching a final autotomy score of 11 or on post-

operative day 21 mice were perfused and their spinal cords removed for gene expression

analysis. As in A mice (on PO day 14), on day 21 autotomy scores of denervated C3H/HeJ mice

varied from 0-11, and like A mice they were divided into two separate subgroups: NLS (having

AFS=0-1) and MHS (having scores AFS=2-11) subgroups. Autotomy scores in denervated

AKR/J mice were as expected from the literature (AFS=0). Figure 16 shows the average

autotomy behaviour in the MHS-C3H/HeJ mice compared to NLS-AKR/J mice (16A), and NLS-

C3H/HeJ mice (16B).

Autotomy onset day of denervated C3H/HeJ and AKR/J mice is shown in Figure 17A. As

shown above for A and B mice, denervated MHS-C3H/HeJ mice had an earlier onset day

(8.3±1.26, ranging from 2-15) than NLS-C3H/HeJ mice (16.22±2.04, ranging from day 1-21;

P<0.01) and NLS -AKR mice (22.0±0.00; P<0.001) that did not express autotomy. The number

of mice that expressed autotomy on each day PO is shown in Figure 17B.

The score of autotomy at onset day also varied highly in C3H/HeJ and AKR/J mice (Figure

18A). This score was significantly higher in C3H/HeJ mice with high autotomy scores on PO

day 21 (2.9±0.66, ranging from 1-7) than C3H/HeJ and AKR/J mice with no or low levels of

autotomy on PO day 21 (0.29±0.13 P<0.0001, and 0±0; P<0.0001, respectively). The number of

mice that expressed autotomy with onset scores 0-7 is represented in the columns (Figure 18B).

99

Figure 16: Post-operative course of average autotomy scores in C3H/HeJ and AKR/J mice.

Following sciatic and saphenous neurectomy, mice were followed up daily and their autotomy

behaviour was scored up to day 21. (A) Average autotomy scores per day of denervated

C3H/HeJ mice (AFSs on PO day 21 ranged from 0-11; N=28) vs. denervated AKR/J mice (AFSs

0

0.5

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1.5

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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Post operative day

Autotomy in C3H/HeJ and AKR/J mice

C3H/HeJ denervated

AKR/J denervated

C3H/HeJ sham

AKR/J sham

C3H/HeJ naive

AKR/J naive

0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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Post operative day

Autotomy in C3H/HeJ mice

C3H/HeJ high

C3H/HeJ low

C3H/HeJ sham

C3H/HeJ naive

A

B

C

0

2

4

6

8

10

12

0 5 10 15 20

Fin

al a

uto

tom

y sc

ore

Mouse number

Autotomy in C3H and AKR mice on PO day 21

C3H/HeJ high

C3H/J moderate

C3H/HeJ low

AKR/J low

* *****

****

**

** ** **** ** ** ** ** ** **

********

**

**** **

** ** ** ** ** ** **

100

of all mice on PO day 21 were 0; N=5), sham-operated C3H/HeJ and AKR/J mice, and naïve

C3H/HeJ and AKR/J mice (AFSs were 0 for all sham-operated and naïve mice of both strains on

PO day 21; N=6 mice per group). (B) Average autotomy scores per day of MHS-C3H/HeJ mice

(AFSs on PO day 21 ranged from 2-11; N=10) vs. NLS-C3H/HeJ mice (AFSs ranged on PO day

21 from 0-1; N=18), C3H/HeJ sham (AFSs on day 21 PO were 0; N=6), and C3H/HeJ naïve

mice (AFSs of 0 on day 21 PO; N=6). Two-way ANOVA for repeated measures was performed

followed by Tukey’s post hoc test; *P<0.05, **P<0.01, ****P<0.0001 (C) Final autotomy scores

of individual mice on PO day 21.

Figure 17: Autotomy average onset day in C3H/HeJ and AKR/J mice was followed up daily to

PO day 21. (A) Average autotomy onset days of denervated MHS-C3H/HeJ, NLS-C3H/HeJ and

NLS-AKR/J mice, and sham operated and naïve mice of both strains. A one-way ANOVA

followed by Tukey’s post hoc test was performed. MHS-C3H/HeJ mice had a significantly

lower autotomy onset day than the denervated NLS-C3H/HeJ, the C3H/HeJ sham-operated and

naive groups and the denervated, sham and naïve AKR/J mice. (B) The number of mice of these

groups that expressed certain autotomy scores at autotomy onset day.

Figure 18: Score of autotomy at onset day of denervated C3H/HeJ and AKR/J mice. (A)

Average autotomy score at onset day of denervated MHS-C3H/HeJ mice, denervated NLS-

C3H/HeJ and NLS-AKR/J mice, sham-operated and naïve mice of both strains. A one-way

ANOVA followed by Tukey’s post hoc test was performed. MHS-C3H/HeJ mice had

significantly higher autotomy scores at onset than the denervated mice with NLS, sham-operated

0

5

10

15

20

25

C3H/HeJ AKR/J

Au

toto

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on

set

day

Autotomy onset day in C3H/HeJ and AKR/J mice

denervated-high

denervated-low

sham

naive

A

0

5

10

15

20

25

Nu

mb

er

of

mic

e

Autotomy onset day

Number of mice per Autotomy Onset Day

denervated-high

denervated-low

sham

naive

BP<0.001

P<0.01

P<0.0001

0

0.5

1

1.5

2

2.5

3

3.5

4

C3H/HeJ AKR/J

Au

toto

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on

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sco

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Autotomy onset score in C3H/HeJ and AKR/J mice

denervated-high

denervated-low

sham

naiveP<0.0001

0

5

10

15

20

25

Nu

mb

er

of

mic

e

Autotomy onset score

Number of mice per Autotomy Onset Score

denervated-high

denervated-low

sham

naive

A B

101

and naive groups (P<0.0001 in all 5 comparisons). (B) The number of mice that expressed

autotomy scores 0-7 at the onset day of autotomy.

4.2.4 Csf2rb1 expression levels in C3H/HeJ vs. AKR/J mice

Csf2rb1 expression levels in denervated NLS-C3H/HeJ mice was significantly higher than in

NLS-AKR/J mice (1.37±0.07 vs. 1.05±0.07; P<0.01) (Figure 19). These data indicate that in

these strains Csf2rb1 expression levels were not differentially regulated with high autotomy

behaviour. No significant differences in Csf2rb1 gene expression levels in the spinal cord were

found between intact C3H/HeJ and AKR/J mice. Thus, the constitutive gene expression levels of

Csf2rb1 are comparable in the spinal cords of these strains. This suggests that the CSF2RB1

protein levels present at the time of nerve injury are not relevant for the contrasting levels of

autotomy seen PO on day 21. Furthermore, no significant differences were observed between

the sham-operated mice of the two strains. Csf2rb1 expression levels did not contrast

significantly between MHS- C3H/HeJ mice, NLS-C3H/HeJ and NLS-AKR/J mice (Figure 19).

There was no significant correlation between the autotomy onset day and expression levels of

Csf2rb1 in denervated C3H/HeJ and AKR/J mice (Figure 20A). Only AKR/J mice, which were

all classified as late onset, as none of these mice expressed autotomy up to PO day 21, had a

significantly lower mRNA levels of Csf2rb1 (1.05±0.07) compared to the late-onset C3H/HeJ

mice (P<0.05), and to the early onset C3H/HeJ mice (P<0.02). These data indicate that the

differences in Csf2rb1 expression are unrelated to the levels of autotomy but to inherent strain

differences between C3H/HeJ and AKR/J mice.

Lastly, AKR/J mice that all had an onset score of 0 up to PO day 21 expressed a significantly

lower level of Csf2rb expression (1.05±0.07) compared to the two C3H/HeJ subgroups, (P<0.03

and P<0.04, for the onset score of 0, and onset score ≥1, respectively) (Figure 20B). Csf2rb1

gene expression levels, however, were not significantly different in the two subgroups of

denervated C3H/HeJ mice, regardless of the degree of self-mutilation at onset. This was the

third indication that autotomy variables in the C3H/HeJ and AKR/J strains are not regulated by

Csf2rb1 expression levels, although there are strain-related differences.

102

Figure 19: Csf2rb1 gene expression levels in the spinal cord of C3H/HeJ and AKR/J mice.

Average Csf2rb1 mRNA levels relative to Hprt reference gene (see Appendix IV) are shown for

naïve C3H/HeJ and AKR/J mice (N=6 and N=5, respectively), sham-operated mice of these

strains (N=6 and N=3, respectively), denervated mice that expressed NLS (N=5 and N=5,

respectively), and denervated mice of the C3H/HeJ strain expressing MHS (N=4). These values

indicate that Csf2rb1 expression levels did not correlate significantly with autotomy scores.

Expression of Csf2rb1 was significantly higher in NLS-C3H/HeJ mice compared to NLS-AKR/J

mice (One-way ANOVA followed by LSD post hoc test).

Figure 20: Csf2rb1 gene expression levels in denervated C3H/HeJ and AKR mice sub-grouped

by the autotomy onset day (early onset: 0-7 days PO and late onset: 8-21 days PO, and by its

score (score=0 and scores≥1). (A) Csf2rb1 mRNA levels of AKR/J and C3H/HeJ mice

(calculated relatively to those of Hprt, the reference gene), which expressed autotomy with a late

onset (N=7 and N=5) and C3H/HeJ mice with an early onset (N=6). Thus, Csf2rb1 expression

levels were significantly elevated in C3H/HeJ mice compared to AKR/J mice, regardless of their

onset day. (B) Csf2rb1 mRNA levels (relative to those of the Hprt reference gene) of denervated

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

C3H/HeJ AKR/J

mR

NA

leve

l of

Csf

2rb

rela

tive

to

Hp

rt

Csf2rb1 gene expression in C3H and AKR mice

Naive

Sham

Low Autotomy

High Autotomy

p<0.01

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Late onset Early onset

mR

NA

leve

l of

Csf

2rb

re

lati

ve t

o H

prt

Csf2rb1 and Autotomy onset day

AKR/J

C3H/HeJ

BA

P<0.05P<0.05P<0.05

P<0.05

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Onset score 0 Onset score =>1

mR

NA

leve

l of

Csf

2rb

rela

tive

to

Hp

rt

Csf2rb1 and Autotomy onset score

AKR/J

C3H/HeJ

103

AKR/J and C3H/HeJ mice, which did not express autotomy at all up to PO day 21 (N=5 and

N=5, respectively), and in denervated C3H/HeJ mice, which expressed at onset MHS (N=8).

Thus, Csf2rb1 expression levels were higher in denervated C3H/HeJ mice regardless of their

autotomy onset score. One-way ANOVA followed by LSD post hoc test was performed.

4.2.5 CSF2RB1 protein is differentially expressed in spinal cords

of A and B mice

The results we got in A, B, C3H/HeJ and AKR/J mice, denervated, sham-operated and naïve, are

compatible with the following two scenarios that affect our rationale for the following

experiments: (i) Csf2rb1 is a gene whose upregulated expression post-denervation is needed for

producing MHS of autotomy in the spinal cord of some denervated A mice, but not of

denervated C3H/HeJ mice. In her dissertation, Tina Elahipanah from our lab (Elahipanah, 2010)

showed that the drive for autotomy in MHS mice is controlled not only by a Pain1 gene(s) but

also by (an)other gene(s) on mouse chromosome 14 whose identity is unknown. This would

suggest that there are transcription factors that are upstream these genes and whose function is to

activate expression of Csf2rb1 in the spinal cord and/or those other genes, depending on the

environment and strain. (ii) Proactive, genetically controlled prevention of expression of Csf2rb1

in the spinal cord protects some denervated mice from developing pain and expressing autotomy.

The results we got so far indicate that there is a significant correlation between levels of

autotomy and differences in Csf2rb1 levels of expression in the DRGs of intact A vs. B mice,

and changes in the spinal cord post-denervation in some A but not B mice. But mRNA levels

are not final proof that this also manifest in the protein it encodes, one needs to show that it is

indeed differentially expressed in the spinal cord of denervated A and B mice. Moreover, it is

important to find out which cell types express this gene, and where in the spinal cord are they

located. To address these questions, immunohistochemical staining was carried out by way of

labelling CSF2RB1 with antibodies against the receptor. After preliminary experiments we were

successful with antibodies against CSF2RB1. The results of experiments that used this as a

marker are shown below. Transverse sections of perfusion fixed spinal cords of naïve, sham

operated, and denervated A and B mice were processed and compared.

104

As in the gene expression studies, based on their expressed levels of autotomy, denervated A

mice were subdivided into MHS and NLS subgroups (as done in previous chapters of this

dissertation), while denervated B mice were all expressors of NLS. Visibly detectable levels of

the reaction product were located in select parts of the spinal cord: (i) in small glia cells in the

white matter including the dorsal (mainly the medio-dorsal part) and ventral columns, and to a

lesser extent around the spinal cord under the pial surface, (ii) fine reaction product was found

throughout the grey matter, including around blood vessels, and (iii) mainly in cells around the

central canal. Of these locations, the most prominent difference between A and B mice was seen

in the CSF2RB1+ cells located around the central canal. These bipolar cells had a spindle-

shaped, very thin soma, extending a single long and thick process that was strongly labelled with

the reaction product, which could be easily followed for many hundreds of μm until terminating

dorsally in the dorsal septum, ventrally in the ventral septum, and laterally in lamina X of the

spinal cord (Figure 21A-I). To compare the number of such cells in mice of the various tested

groups, we counted the number of such processes extending longer than 20 μm from the outer

edge of the central canal into the dorsal, ventral and lateral directions, per section 50 μm thick

(Figure 23). No differences were found in the total number of processes between naïve A and

sham-operated A mice, and between naïve B and sham-operated B mice (Appendix V). As there

were no significant differences in any of these comparisons the numbers for naïve and sham

operated mice were combined into one control group for each strain. As well, no differences

were observed in the numbers of processes per section in denervated MHS- mice and NLS- A

mice (Appendix V). Therefore, the values for denervated animals were combined into one

denervated group per strain. The numbers of CSF2RB1+ processes extending to the dorsal and

ventral midsagittal septa were not significantly different. In non-denervated naïve and sham

operated animals there were significantly more processes per section extending dorsally and

ventrally in A mice than B mice (dorsally extending processes: 4.74±0.64 vs. 0.75±0.70

(P<0.0001); ventrally extending processes: 4.75±0.63 vs. 0.85±0.79 (P<0.0001); respectively).

The number of such processes increased significantly after denervation in A mice compared to

its control mice (dorsally extending processes: 6.47±0.19 vs. 4.74±0.64 (P<0.018); ventrally

extending processes: 7.12±0.42 vs. 4.75±0.63 (P<0.003). This increase was also observed after

denervation in B mice (dorsally extending processes: 6.33±0.63 vs. 0.75±0.70, P<0.0001;

ventrally extending processes: 6.33±0.38 vs. 0.85±0.79, P<0.0001; respectively). Comparable

105

number of cells extending dorsally and ventrally were observed in A and B mice after

denervation, as is seen in Figures 23A and 23B.

The average numbers of CSF2RB1+ processes per section that extended laterally were

significantly higher in A mice compared to B mice both in naïve, sham operated and after

denervation (control mice: 12.49±2.28 vs. 1.42±0.99 (P<0.0001); denervated mice: 18.89±1.14

vs.7.58±1.33 (P<0.002) respectively; Figure 23C). In both strains, denervated mice showed a

significant increase in the number of lateral extensions compared to their respective controls (A

mice: 18.89±1.14 vs. 12.49±2.28, P<0.008; B mice: 7.58±1.33 vs. 1.42±0.99, P<0.085,

respectively).

The overall number of processes per section extending dorsally, ventrally and laterally was

significantly higher in A mice compared to B mice, both prior to, and after denervation: control

mice: 20.27±3.09 vs. 2.71±2.13, respectively (P<0.0001); denervated mice: 30.33±1.31 vs.

18.13±1.43, respectively (P<0.0001), Figure 23D). In summary, CSF2RB1-labelled processes in

the spinal cord of control mice were significantly more abundant in A mice compared to B mice,

but increased in numbers after denervation.

Figure 24 shows the number per section of CSF2RB1-labelled, laterally extending processes of

individual control (naïve and sham) and denervated A and B mice. It can be seen that all B mice

and a small number of A mice had 10 lateral processes per section or fewer. Most A mice had

10 or more of these processes per section that extend laterally deeper into the grey matter of the

spinal cord.

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Figure 21: Immunohistochemical analysis of CSF2RB1-expressing cell types in the spinal cord.

CSF2RB1 is present in white matter (green arrows, A), dorsal horn (white arrows, A, F, H) and

dorsal column (yellow arrows, A, F, G, H), around some blood vessels (red arrows, A, F), and

abundantly surrounding the central canal with strongly labelled ependymal cells/radial

glia/tanycyte cells (A-E) in MHS-A mice (A, B, D-H) and NLS-B mouse (C). Elongated bipolar

CSF2RB1+ cells ≥20 μm have their cell body lining the central canal extending a single process

in a centrifugal manner dorsally or ventrally into the dorsal and ventral midsagittal septa and

laterally into Lamina X (A). Considerably longer and thicker CSF2RB1+ processes are present

in A mice (B) compared to B mice (C). In these ependymal cells/radial glia/tanycytes CSF2RB1

did not co-localize with NeuN that labels neurons (B), GFAP that labels astrocytes (C, blue),

MAP-2 that also labels neurons (D), NG2 that labels oligodendrocyte precursor glia cells (E,

blue). In glia present in the dorsal column CSF2RB1 did not co-localize with NeuN (F, G, blue)

or GFAP (H). (I) Schematic diagram of spinal cord sectioning, showing the staining of the

central canal (CC) region and the cell extensions into dorsal and ventral columns; DH=Dorsal

horn; VH=Ventral Horn; scale bar - 160 µm (A-B), 39 µm (C-H); magnification - 5x (A), 20x

(B-H).

A

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Figure 22: CSF2RB1 expression levels in the spinal dorsal horn. (A) Cross section of a high-

autotomy A mouse expressing CSF2RB1 (red) in white and grey matter, and neuronal nuclei

(NeuN, green) in the white matter of the spinal cord. Magnification, 5x; scale bar, 310 μm. (B)

Higher magnification (20x) of the dorsal horn showing expression of CSF2RB1 in laminae I and

II, some of which co-localized with neuronal nuclei, marked by NeuN (C & F). (D) Spinal

dorsal horn labelling CSF2RB1 (red) and neuronal nuclei (blue) in a denervated B mouse

expressing low autotomy. Some of the CSF2RB1 profiles co-localized with neuronal nuclei

(purple). (F) A replication of the Figure in B, showing only CSF2RB1 expression in white, with

dense dots appearing in laminae I and II. Scale bar, 39 μm

A B C

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Figure 23: The number of long CSF2RB1+ processes in the spinal cord of A and B mice. Cells

that extend labelled processes to a distance ≥20 μm from the outer edge of the central canal into

dorsal, ventral and lateral aspects of the spinal cord were counted in A and B mice, before and

after denervation. Average process numbers is presented separately for dorsal (A), ventral (B),

lateral (C), and the sum of all extensions (D; N≥4 mice per group, ≥6 sections per mouse).

Control designates pooled data of naïve and sham operated mice that were not found to be

significantly different from each other. Neurectomized= all denervated mice, MHS and NLS in

A strain, NLS in B strain. This data was pooled per strain regardless of the autotomy levels

expressed by these groups since the number of processes did not correlate autotomy levels. One-

way ANOVA was carried out followed by LSD post hoc test.

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Figure 24: The number of long CSF2RB1+ processes extending laterally in A and B mice. (A)

Average number of CSF2RB1+ processes extending ≥20 μm from the outer edge of the central

canal into dorsal, ventral and lateral aspects of the spinal cord, in naïve and sham-operated

(control) A and B mice. (B) Average number of CSF2RB1+ lateral processes in denervated A

and B mice. Average numbers are presented as lateral extensions (≥ 6 sections per mouse).

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4.2.6 Evidence that CSF2RB1 is localized in ependymal

cells/radial glia/tanycytes of the central canal

Mice were subjected to hindpaw denervation, sham operation or were left intact, and their

perfusion-fixed spinal cords were removed for immunohistological staining with CSF2RB1 and

various other antibodies that are differentially expressed in particular cell types within the spinal

cord. This was done to determine what type(s) of cells express CSF2RB1 in the spinal cord.

The results of these immunohistological experiments are shown in Figures 22A-G. They

indicate that CSF2RB1+ cells did not co-localize with OX-42 (not shown) and GFAP, markers

for microglia and astrocytes, respectively (Figure 21C, H). IL-3Rβ labelled in red co-localized

with NeuN in some of the neuronal nuclei that were labelled by green or blue fluorescence,

showing that green and red merged into yellow in the upper dorsal horn layers (Figure 22B-C, F)

and red and blue merged into purple (Figure 22D). However, this observation may be an artefact

due to the fact that a z-stack image that is shown in Figure 22F collectively gathers tens of

images, some of which include neuronal nuclei and others that do not. CSF2RB1 did not co-

express with dendrites and neurites of neurons that were labelled blue with the MAP-2 antibody

(Figure 21D). CSF2RB1 also did not co-express with NG2 that labels a precursor cell of

oligodendroglia (Figure 21E). However, CSF2RB1 co-localized with Vimentin, a marker of

ependymal cells/radial glia/tanycytes. Figure 25 shows that the same ependymal cells/radial

glia/tanycytes around the central canal in both A and B mice were labelled by CSF2RB1 and

Vimentin. CSF2RB1 co-localized with Vimentin significantly more in A mice in this region,

compared to B mice, as shown by the scatter plots in Figures 25D, H, and by measure of the

average Pearson’s Correlation Coefficients ±SEM (0.69±0.06 vs. 0.14±0.05, P<0.0002,

respectively; Figure 25I). In A and B mice, denervated, sham-operated and naïve mice all

showed co-localizations of CSF2RB1 with Vimentin. Figure 25J shows the average Pearson’s

Correlation Coefficients for the individual mice, demonstrating higher correlations in A mice

compared to B mice. These results indicate that A mice express significantly more CSF2RB1-

expressing ependymal cells/radial glia/tanycytes compared to B mice.

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Figure 25: Co-localization of CSF2RB1 and Vimentin in ependymal cells/radial glia/tanycytes

surrounding the central canal in control and denervated A and B mice. Around the central canal

CSF2RB1 (A, E) co-localized with Vimentin (B, F) significantly more in A mice (MHS),

compared to B mice (naïve) (C vs. G), as shown by the scatter plots in D (for A mice) vs. H (for

B mice). Each scatter plot indicates the amount of labelled CSF2RB1 (red; x axis) and labelled

Vimentin (green; y axis). (I) Co-localization is also shown by measure of the average Pearson’s

Correlation Coefficients ±SEM (panel I, N≥4 mice, 5-9 sections per mouse, two-tailed t-test).

Co-localization of individual A and B mice is presented at panel J, showing that many more

ependymal cells/radial glia/tanycytes in A mice had co-localizations of CSF2RB1 and Vimentin

(r ranging from 0.5-0.8) than in B mice (r ranging from 0-0.3). Scale bar - 46 µm; magnification

- 20x.

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4.2.7 CSF2RB1 in the brain is associated with high autotomy

levels in denervated A mice

In Chapter 3 (Section 3.2.7) of this thesis report we showed that Csf2rb2 is expressed in

numerous regions of the mouse spinal cord and brain, demonstrated by in situ hybridization in

the Allen Brain Atlas. This analysis suggested that Csf2rb1, the gene we studied in this

dissertation, might also be expressed in brain regions known to process pain input, and whether

its expression in those structures correlates with autotomy behaviour in denervated A and B

mice. To this end, brain sections (50μm thick) of perfusion-fixed naïve, sham and denervated

MHS-A and NLS-A mice, and naïve, sham and denervated NLS-B mice, were immunolabelled

with IL-3Rβ (i.e., CSF2RB1). As with the Csf2rb2 gene, CSF2RB1+ cells were abundantly seen

in a select number of regions in the brain, very similar to those harbouring the Csf2rb2 gene.

CSF2RB1 was located in small cells in the dentate gyrus granule layer and in the polymorph

layer (Figure 26A-F). Labelled cells were also found in the hypothalamic arcuate nuclei, lining

the 4th ventricle (Figure 28A-D), and the pial surface of the brain, areas exposed to cerebrospinal

fluid (Figure 28E). The most profound difference between A vs. B mice was present in the

dentate gyrus and the arcuate nucleus. In the granule layer, there were many more CSF2RB1+

cells with processes crossing in a perpendicular orientation through the layer in MHS-A mice,

compared to denervated NLS-A and sham operated A mice or naïve, sham and denervated NLS-

B mice (Figure 26).

Naïve and sham-operated mice did not differ significantly in the number of processes crossing

the dentate gyrus granule cell layer (Appendix VI). Therefore, data from naïve and sham-

operated mice were pooled separately for A and B strains into strain-specific control subgroups

as shown in the summarizing histograms in Figure 26E.

The histograms also show that MHS-A mice had 3-folds more CSF2RB1+ cells with processes

traversing the granule layer, per unit length of 200 μm, compared to denervated NLS-A, NLS-B,

and control A and B mice [MHS-A: 6.56±3.79 vs. NLS- A: 4.35±2.30 (P<0.03); NLS-B:

2.35±0.69 (P<0.008); control A: 5.10±2.71 (P<0.01); control B: 6.31±2.07 (P<0.03)]. A

positive correlation between CSF2RB1+ processes in the granule layer and autotomy behaviour

on day 14 PO of A and B mice is shown in Figure 27 (R=0.82, P<0.0003).

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The polymorph layer of the dentate gyrus in denervated A mice contained significantly more

CSF2RB1+ somata than control A mice and the same was observed for B mice (Figure 26F).

But in denervated A mice there was no significant difference between MHS and NLS mice in the

number of CSF2RB1 labelled cells per ROI of 2500 μm2 for a 50 μm thick section, (13.82±2.21

vs. 13.93±0.43, respectively, P<0.96). Thus, these cells do not seem to a play a role in

correlating autotomy levels by gene-by-environment interactions that protects some denervated

A mice from expressing autotomy.

Control A and B mice comprised pooled data of CSF2RB1+ cells in the polymorph layer of

naïve and sham-operated animals, since no significant differences were observed between these

two groups in A or B mice (Appendix VII). No significant differences were found between the

number of such cells in control A and B mice. However, denervation caused a significant

increase in the number of CSF2RB1 expressing cells in the polymorph layer of the dentae gyrus

compared to control A and B mice: 13.88±1.01 and 5.89±1.86 cells per 2500 μm2 of a 50 μm

think section, respectively, (P<0.01), and 16.47±2.54 and 8.10±0.98 cells per 2500 μm2 of a 50

μm think section, respectively (P<0.01). But no difference was found between denervated A and

B mice. Thus, this finding is most likely associated with changes due to the denervation, rather

than changes of strain specificity between A and B mice.

Figure 28 shows the co-localization of very few CSF2RB1+ cells in the polymorph cell layer

with Vimentin, the same marker that labelled ependymal cells/radial glia/tanycytes in the central

canal. CSF2RB1 did not co-localize neurons, labelled by NeuN, or with GFAP or OX-42 that

label astroglia and microglia, respectively.

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Figure 26: Photomicrographs of hippocampal dentate gyrus sections labelled

immunohistologically for CSF2RB1 (red), expressed in granule and polymorph layers. These

cells and processes were present in sham-operated A and B mice (A, C), but were significantly

increased in denervated mice of both strains (B, D). (E) The average number of processes

traversing through the granule layer per unit length of 200 μm was significantly higher in MHS-

A mice, (B), compared to control A mice, and denervated NLS- A mice (N≥3 mice per group, ≥

5 sections per mouse). (F) CSF2RB1+ cells in the polymorph layer of the dentate gyrus of A

and B mice. The average number of cells per region of interest of 2500 μm2 per section is shown

for control and denervated A and B mice (N≥3 mice per group, ≥ 5 sections per mouse). Control

(i.e., naïve and sham-operated mice); Denervated (data of MHS and NLS was combined for the

A strain, and of mice with NLS for the B strain. One-way ANOVA followed by the LSD post

hoc test was performed. Scale bar- 46 µm, magnification - 20x.

A mouse B mouse E

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Figure 27: Correlation of the number of CSF2RB1+ long processes per 200 μm of the dentate

gyrus with autotomy behaviour. The number of CSF2RB1+ processes traversing the granule

layer of the dentate gyrus per 200 μm/section is shown as a function of the Final Autotomy

Scores (FASs) on PO day 14 of denervated A and B mice. The number of processes ranged from

4-10 per 200 μm/50 μm thick section per mouse, red circles designate mice expressing MHS in

A mice; blue circles denote A-NLS mice and black diamonds mark B-NLS mice.

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Figure 28: CSF2RB1 expression in the dentate gyrus. CSF2RB1 (red) is expressed in pyramidal

and granule layers of the dentate gyrus. It did not co-localize with (A) NeuN (neurons; green),

(B) OX42 (microglia; green), or (C) GFAP (astrocytes; green) expressed in the same areas of A

and B mice. CSF2RB1 co-localized with very few Vimentin+ cells (indicated by white arrows).

(D) Vimentin (shown in green) labels ependymal cells/radial glia/tanycytes but not astroglia or

microglia. Notable CSF2RB1 somata are observed in both A denervated mice with autotomy

(A-B), and B denervated mice with no autotomy (C-D). Magnification - 20x; scale bar - 46 μm.

CSF2RB1NeuN

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CSF2RB1Ox42

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4.2.8 CSF2RB1 in ependymal cells/ radial glia/tanycytes in other

brain regions

CSF2RB1 was present in additional regions in the brain, specifically around the 3rd ventricle

(V3 in Figure 29), in the peri-ventricular hypothalamic nucleus (PVI in Figure 29), in the

hypothalamic arcuate nucleus (ARH in Figure 29), in the pial surface of the brain in some

regions (e.g., Figure 29H1-3). Figure 29 shows considerable differences in CSF2RB1-

expressing cells in the hypothalamic PVI nucleus of A mice compared to B mice. These cells

have a soma located near the ventricle, sending a short process to the lining of the ventricle and a

very long process extending perpendicularly to the surface of the ventricle, deep into the peri-

ventricular hypothalamic zone. These processes are robustly visible in the A mouse, but are

almost devoid in the B mouse (Figure 29C, D). CSF2RB1 was not expressed by neurons, as

shown by the green NeuN fluorescence in Figure 29A, B, which did not co-localize with the red

fluorescence of the CSF2RB1+ cells. CSF2RB1+ cells also did not co-localize with GFAP or

OX-42 that label astroglia and microglia, respectively (data not shown). However, CSF2RB1+

cells co-localized with Vimentin, as shown in Figure 29H at the pial surface in the ventral aspect

of the brain, and in cells lining the ventricles (Figure 29E1-3).

Figure 29: CSF2RB1 immunoreactive cells in select brain regions. CSF2RB1+ cells were

present in the peri-ventricular hypothalamic nucleus surrounding the 3rd ventricle of sham A

mice, extending very long processes into the hypothalamus (A-C), whereas in sham B mice (D-

E) these CSF2RB1+ cells did not extend such processes into the hypothalamus. Figures A-C

show that CSF2RB1+ cells (red fluorescence) did not co-localize with NeuN (green

fluorescence) in the PVi (B), but did co-localize with Vimentin (E1-3). (F) Nissl stained brain

section of an intact B mouse taken from the Allen Brain Atlas (http://www.brain-map.org)

showing neuronal clusters in the same rostrocaudal location of the sections shown in (A-D). (G)

An anatomical reference brain atlas annotating the brain nuclei located in the same sections as in

A-F. (H1-3) Co-localization of CSF2RB1 with Vimentin is shown at the pial surface in the

ventral aspect of the brain in a B sham mouse. PVi = peri-ventricular nucleus pars internal; V3 =

third ventricle; ARH = arcuate nucleus of the hypothalamus. Scale bar - 160 μm (A), 46 μm (B-

D), 39 μm (E1-E3, H1-H3); Magnification - 5x (A), 20x, (B-H).

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4.3 Discussion

The present study validated the results of the expression profiling data we presented in Chapter

3, where spinal cord expression levels of Csf2rb1, a gene located in Pain1, was shown to be

significantly associated with autotomy behaviour following peripheral nerve injury. Here, using

RT-PCR and immunohistochemical analyses, we validated, in part, these findings by showing

that the post-operative changes in mRNA levels indeed translate into changes in expressed

protein product of Csf2rb1 in the spinal cord and brain of nerve injured mice, and that these

expressed levels correspond to the autotomy behaviour. Thus, the major findings of the present

results garnered in Chapter 4 can be summarized as follows: (i) Csf2rb1mRNA expression levels

were up-regulated in MHS-A mice, but not in NLS-A or NLS-B mice, and this up-regulation

significantly correlated the autotomy scores, (ii) In MHS-C3H/HeJ mice vs. NLS-C3H/He/J and

NLS-AKR/J mice, we were unable to find differences in the expressed levels of Csf2rb1 mRNA

levels, (iii) CSF2RB1 was demonstrated in ependymal cells/radial glia/tanycytes of lamina X of

the spinal central canal, and (iv) CSF2RB1 was also expressed in select regions of the brain,

including the hypothalamus, hippocampus, peri-ventricular nuclei and pia, correlating

significantly the autotomy behaviour with ependymal cells/radial glia/tanycytes cell processes in

the dentate gyrus. The following sections discuss these results in detail.

4.3.1 Csf2rb1 is correlated with pain levels in denervated A mice

Gene expression assays for Csf2rb1 in A vs. B mice validated the findings in Chapter 3 on the

expression profiling study, whereby Csf2rb1 was up-regulated in the ipsilateral spinal cord of

denervated MHS-A mice. Data we describe here indicate that Csf2rb1 mRNA levels

significantly correlated with the autotomy scores in A mice. As well, denervated A mice that

scored autotomy levels of 2 or higher at onset had significantly higher number of Csf2rb1

transcripts compared to denervated A mice that did not express autotomy at all. However, within

the MHS-A mice, no significant difference in Csf2rb1 mRNA levels was observed in mice that

developed autotomy early (on day 1-7 PO) vs. mice that developed autotomy later (on day 8-14

PO), indicating that Csf2rb1 is associated with pain maintenance in these animals, represented by

their final scores on day 14 PO rather than with pain initiation, represented by their autotomy

scores at onset of the behaviour.

120

In addition to the contrast in Csf2rb1 gene expression levels in A vs. B mice, mRNA levels of

this gene also contrasted within the A strain between MHS and NLS mice, indicating that

regulation of this gene is also affected by the environment in a GXE interaction. However, this

could not be demonstrated for denervated C3H/HeJ vs. AKR/J mice, that while contrasted in

their autotomy behaviour, ipsilateral spinal cord Csf2rb1 gene expression levels did not differ

significantly, indicating that Csf2rb1 does not play the same role in autotomy between these two

strains as it does in A and B mice. It may be that in these strains Csf2rb2, rather than Csf2rb1,

differentially regulates autotomy behaviour. More research is needed to answer this question. In

conclusion to the above results, it seems that Csf2rb1 up-regulation is mechanistically involved

in producing the drive for autotomy in some denervated A mice. Alternatively, it is possible that

the genome interacts with the environment to protect some denervated A mice, and all

denervated B mice from autotomy by preventing up-regulation of Csf2rb1 in the spinal cord.

4.3.2 CSF2RB1 is expressed in ependymal cells/radial

glia/tanycytes of the spinal central canal

We have localized CSF2RB1 to ependymal cells/radial glia/tanycytes of the spinal central canal,

since it was co-expressed with Vimentin, but not with neuronal or other glial markers.

Moreover, we showed that the CSF2RB1 positive cells are characterized by a very typical

morphology of ependymal cells/radial glia/tanycytes of the spinal cord, as they extend long

processes deep into the spinal cord grey matter and to the midsagittal septa of the white matter

dorsally and ventrally. Since we showed that there was a significantly higher number of such

processes in naïve A mice compared to naïve B mice, could suggest that CSF2RB1+ ependymal

cells/radial glia/tanycytes of the spinal cord participate in the spinal response to injury discharge

at the time of nerve injury. As reviewed in Chapter 1, injury discharge is one of the main

triggers that initiate the development of neuropathic pain after nerve injury. However, Csf2rb1

gene expression data contradict its possible role in initiating neuropathic pain, as discussed above

(Section 4.3.1), since no differences in Csf2rb1 mRNA levels were detected between naïve A vs.

B mice. We propose that the morphology of these CSF2RB1+ ependymal cells/radial

glia/tanycytes in the central canal region may be important in mechanisms of initiating pain at

the time of nerve injury, and that the number of CSF2RB1 receptors that these cells express may

be important in pain maintenance mechanisms. The morphology of these cells characterized by

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long processes that are expressed in A mice and are absent in B mice may be important perhaps

for interaction with other cell types, such as neurons or glia. In a similar way that glial cells

cross talk with neurons in the dorsal horn and activate them to induce hyperexcitablity,

CSF2RB1+ ependymal cells/radial glia/tanycytes may cross talk with other cell types and

contribute to the central sensitization in the spinal cord. One way could be by releasing

inflammatory mediators that can respond to neurons thereby exciting them. Another way is by

having the same inflammatory mediators activate microglia and astrocytes. We also suggest that

in addition to the potential impact of physical appearance of CSF2RB1+ ependymal cells/radial

glia/tanycytes may have on pain mechanisms, the number of transcripts that encode the CSF2

receptors on these cells may be significant in pain maintenance, since Csf2rb1 mRNA levels

were associated with autotomy behaviour in A mice.

A significant increase of CSF2RB1+ ependymal cells/radial glia/tanycytes of the spinal central

canal was also observed after the denervation in B mice. The increase in the number of

CSF2RB1+ processes both in A and B strains may suggest that these cells proliferate in response

to nerve injury and not necessarily related to pain per se. Indeed, studies indicate that ependymal

cell proliferation increases dramatically following spinal cord injury, and is necessary for the

production of neurons, oligodendrocytes, and more abundantly astorcytes that migrate to the site

of injury where they serve critical role in glial scar formation (Meletis et al., 2008). But to the

best of our knowledge we are the first to show that these cells respond to peripheral nerve injury.

In this respect, ependymal cells/radial glia/tanycytes in the central canal are known to serve as

neural stem cells, and are very important following nerve injury (Guo et al., 2011; Mothe and

Tator, 2005). This phenomenon is well known in spinal cord and trigeminal microglia and

astrocytes, which change their appearance from the normal state, proliferate and become

‘activated’ in response to nerve injury, particularly within areas of pain perception, such as the

spinal and trigeminal dorsal horn. These microglia and astrocytes communicate with nociceptive

neurons through inflammatory agents and cytokines released by inflammatory cells, which

promote neuronal excitation and disinhibition processes associated with chronic pain (Jo et al.,

2009; Kim et al., 2009a; Liu et al., 2012b; Matsui et al., 2010; Wen et al., 2011). Likewise, it

may be that ependymal cells/radial glia/tanycytes of the spinal central canal act in a similar way

to astrocytes and microglia following nerve injury, in that they may release inflammatory

mediators (either GM-CSF, IL-3, IL-5, or others, which may promote the release of the former

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agents from other cell types) to act on their receptor, CSF2RB1. It is important to note that

because CSF2RB1+ cells did not co-localize with microglia and astrocytes or with neurons in the

central canal region, they may be a new type of cell responding to nerve injury in a function that

is currently unknown. Given that we detected CSF2RB1+ ependymal cells/radial glia/tanycyte

increase after denervation in both A and B mice, we propose that the number of CSF2RB1+

processes and the extent in which they invade deep into the spinal cord significantly more in A

than B mice, could be major determinants that affect autotomy behaviour.

Future research is needed to test whether differences exist in the number of lateral processes

between the ipsi- and the contra-lateral sides of the spinal cord following unilateral nerve injury,

especially in denervated A mice. Such a difference was not obvious to the eye of the investigator

just by observing the two sides of the dorsal horn, as is robustly ipsilateral for other glial cells

such as microglia and astrocytes. We already know that with the proliferation and activation of

microglia and astrocytes following nerve injury, the increase in the number of these cells at the

side of the injury are associated with pain mechanisms (Calvo and Bennett, 2012; Tsuda et al.,

2013). Emirandetti et al. have shown that A mice produce robustly more astrocytes compared to

B mice one week following sciatic nerve transection, especially around axotomized motoneurons

(Emirandetti et al., 2006). The study also showed that significant differences existed between

ipsi- and contra-lateral sides within the same animal. Although motoneurons are located in the

ventral horn where pain inputs are not processed, the increased production of astrocytes in the

ipsilateral spinal cord may contribute to overall changes in the dorsal horn associated with pain

perception. Similarly, the present study showed an increase in the number of ependymal

cells/radial glia/tanycytes in spinal cord of A mice, which may also drive them to pain

mechanisms involving these cells. The significant fold change in CSF2RB1+ ependymal

cells/radial glia/tanycytes number that we detected around the central canal in B mice after

denervation may not be sufficient for activating pain mechanisms involving these cells, since

these mice do not develop autotomy. Further studies are needed to confer this possibility.

It is noteworthy that we also located CSF2RB1+ cells in the dorsal columns, mostly near the

midline and a few in white matter surrounding the grey matter of the spinal cord. These cells did

not react with Vimentin, suggesting that they may be another subtype of cell, other than

ependymal cells/radial glia/tanycytes. These non-Vimentin, CSF2RB1+ cells, in white matter

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also did not co-localize with NeuN, OX-42, and GFAP, suggesting that they are not neurons (not

expected in white matter), microglia, or astrocytes. It may be that these cells are another type of

specialized glia that is reactive with Nestin, that like Vimentin is a marker for neural stem cells

(Busch et al., 2010; Mothe and Tator, 2005; White et al., 2010).

4.3.3 CSF2RB1 cells in the hippocampal dentate gyrus

Csf2rb2 expression, documented by the ABA for naïve B mice, was found in the hippocampus

and other brain structures that process pain inputs. This has led us to look closer at CSF2RB1

protein expression in these regions, especially in the hippocampus. Indeed, we found CSF2RB1

expression in almost identical areas of the brain, including the peri-ventricular region of the

hypothalamus, the pial surface, and the hippocampal dentate gyrus, where Csf2rb2 expression

was found. But while the cells expressing the latter protein were neurons, the vast majority of

the expression of CSF2RB1 in the brain was not in neurons, and also not in microglia or

astroglia. It is interesting that these two genes, juxtaposing on Pain1, comprising what may very

likely be the result of gene duplication, are present in the same structures in the central nervous

system, but on different cell types: neurons (expressing CSF2RB2 and known to respond to

peripheral nerve injury and be a major part of the chronic pain pathway) and ependymal

cells/radial glia/tanycytes (expressing CSF2RB1, shown in this dissertation to take part in

chronic pain pathways), suggesting the possibility that as they both respond to the same 3 ligands

(GM-CSF, IL-3 and IL-5), they may be part of the same mechanism. It is possible that they are

recruited to produce chronic pain at two different time periods: neurons may respond earlier, at

the triggering phase of pain chronicity, while ependymal cells/radial glia/tanycytes may respond

later, taking a role during the chronic pain maintenance phase. Like the cells expressing

CSF2RB1 in the spinal cord, those in the brain were characterized by a bipolar thin soma

extending a single long process perpendicularly through the granule layer of the dentate gyrus.

The number of such cellular processes crossing through that layer correlated with high autotomy

scores, and appeared in much greater numbers in these animals compared to control and

denervated A and B mice that did not express autotomy. The polymorph and granule cell layers

in control A and B mice were dotted with many CSF2RB1+ somata, proliferating in numbers

both in A and B mice post-injury, regardless of autotomy. The mere fact that a peripheral nerve

injury can trigger proliferation of certain glia cells in the brain (i.e., the hippocampus) is

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fascinating. More research is needed to unravel the importance of this mechanism, the signals

triggering it, and the relevance to pain.

Evidence for pain processing in the hippocampus is discussed in detail in Chapter 6.

4.3.4 CSF2RB1 cells in the hypothalamic peri-ventricular and

arcuate nuclei

In the grey matter near the 3rd ventricle of the brain we saw some remarkable differences in the

morphology of CSF2RB1 labelled cells between sham operated mice of different strains.

CSF2RB1+ cells in A mice were characterized by long processes extending perpendicularly to

the granule layer, traversing throughout that layer, whereas the cells in B mice lacked those

processes and the reaction product was limited to the soma of cells expressing it. As in the

central canal of the spinal cord, these hypothalamic CSF2RB1+ cells co-reacted with Vimentin.

Like the dentate gyrus, the peri-ventricular zone harbours many neural stem cells that respond to

brain injury by neoneurogenesis, including ependymal cells. Ependymal cells/radial

glia/tanycytes typically form a single layer of ciliated neuroepithelial cells that line the lumen of

the ventricles, aqueducts and spinal central canal. Our results show clearly that, as in the spinal

central canal, the hypothalamic nuclei expressing CSF2RB1+ cells comprise a much thicker

layer in both A and B mice, with contrasting morphologies between the strains. In response to

nerve injury these cells proliferate, increasing in numbers 2.5 times. This fits the process that

characterizes astroglia and microglia after peripheral nerve injury (Beggs and Salter, 2007;

Echeverry et al., 2008). Whether these morphological differences between A and B mice have a

functional role associated with their autotomy levels, is not known as of yet. It has been shown

that ependymal cells/radial glia/tanycytes lining the lateral ventricles are quiescent under normal

conditions and serve as a reservoir that is recruited by injury (Carlén et al., 2009). Following

stroke, these cells are activated to produce neuroblasts and glial cells (Carlén et al., 2009). We

propose that these cells may have a similar role following peripheral nerve injury in the

autotomy model, but we have no evidence to support this hypothesis. It is also not clear, why

neoneurogenesis and proliferation of glia cells should be instrumental in producing chronic pain.

We could not co-localize CSF2RB1 positive cells in this region with GFAP expressing

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astrocytes nor to OX-42 expressing microglia, yet other types of glial cells may emerge from

ependymal lineage in this region.

4.3.5 Autotomy behaviour in denervated C3H/HeJ mice

We aimed to extend our findings of Csf2rb1 gene expression levels correlating with MHS in A

mice to another autotomy susceptible strain, C3H/HeJ mice, which generate MHS following the

same hindpaw denervation. The expression of Csf2rb1 in denervated vs. sham-operated and

naïve C3H/HeJ mice did not correlate with autotomy behaviour. Mice of this strain often

showed variable levels of autotomy behaviour following hindpaw denervation like A mice. As

noted, autotomy behaviour was followed in C3H/HeJ mice for up to day 21 PO (unlike A mice

which were followed up to day 14 PO) to ensure that at least 50% of the denervated mice

developed autotomy. However, by day 14 PO, for an unknown reason most C3H/HeJ mice

failed to develop autotomy and in fact this outcome was the case even when the follow up period

was extended to day 21 PO. In contrast, in denervated A mice up to 50% developed autotomy by

day 14 PO or earlier. This phenotypic difference in autotomy behaviour between these strains

may be explained by differences in their genetic background and/or differences that may develop

as their genes interact with environmental factors in a ‘GXE interactions’ fashion.

4.4 Conclusions

The present study validated, in part, the candidacy of Csf2rb1 as a gene in Pain1 whose

expression levels are linearly and significantly correlated with scores of autotomy behaviour in

some denervated A mice. Expression profiling results for Csf2rb1 in the spinal cord ipsilateral to

the injury (shown in Chapter 3) were replicated here using quantitative PCR methodology. We

also identified that CSF2RB1 is expressed in ependymal cells/radial glia/tanycytes in the spinal

cord central canal. This identification is based on the co-localization of CSF2RB1 with the

neural stem cell marker Vimentin. In addition, we identified ependymal cells/radial

glia/tanycytes expressing CSF2RB1 in various brain regions including the dentate gyrus, peri-

ventricular and arcuate nuclei of the hypothalamus, and in the pial surface of the brain. We also

found that the number CSF2RB1-expressing cells in the dentate gyrus extend processes through

the granule cell layer in MHS-A mice , but significantly less abundant or even completely absent

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in denervated NLS-A and B mice and in sham mice of these strains. Although CSF2RB1 co-

localized with Vimentin in the pial surface of the brain we could not locate Vimentin in the

CSF2RB1+ cells of the dentate gyrus. It is possible that in the dentate gyrus these cells co-

localize with another neural stem cell marker named Nestin (Busch et al., 2010; Kirsch et al.,

2008; Mothe and Tator, 2005; White et al., 2010). Future studies are needed to test whether

Nestin co-localizes with CSF2RB1 or not. As well, functional studies are needed to identify the

mechanism by which Csf2rb1 gene product may control the development and/or maintenance of

neuropathic pain in these brain areas and/or the spinal cord areas mentioned in this report.

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Chapter 5

Study III: Tlr4 and pStat3 as candidate genes controlling

autotomy behaviour in mice

5.1 Introduction

In the last set of experiments of this thesis we studied 2 additional candidate genes for autotomy

(Tlr4 and Stat3), which relate to the work on Csf2rb1 (see Chapters 3-4) and further seek to

explain the role of Csf2rb1 in neuropathic pain. The following paragraphs introduce the current

knowledge reported on those 2 additional genes as well as elaborate on the rationale for inclusion

of the following experiments in this dissertation.

5.1.1 Tlr4

Toll-like receptors (TLRs) comprise a family of proteins that are assembled in the outer cell

membrane of neurons, glia, macrophages, Schwann cells and pericytes in the nervous system,

and following peripheral nerve injury they serve as activators of Schwann cells in the PNS and

glia in the spinal cord (Hutchinson et al., 2008; Kim et al., 2009a; Piccinini and Midwood, 2010;

Tanga et al., 2005). TLR4 is one of 13 identified mammalian TLRs (Bowie and O’Neill, 2000),

a recognition receptor that responds to diverse invading pathogens and/or endogenous ligands.

Upon ligand binding TLRs undergo dimerization, activating intracellular signalling cascades that

regulate gene transcription via transcription factors, such as NFκB and AP-1, thereby generating

numerous proinflammatory cytokines, including IL-1β, IL-6 and TNFα (Kaisho and Akira, 2006;

Kawai and Akira, 2007; Okun et al., 2011). Endogenous signals are considered the source of

TLR activation in neuropathic pain conditions (Hutchinson et al., 2008; Kim et al., 2009a; Tanga

et al., 2005), particularly by activating glial cells in the spinal cord (Piccinini and Midwood,

2010; Scholz and Woolf, 2007). Those signals are referred to as damage- (or the risk of damage,

i.e., danger-) associated molecular patterns (DAMPs), and examples include fibronectin, β-

defensins, high mobility group box-1 (HMGB1), and heat shock proteins (Asea et al., 2002;

Costigan et al., 1998; Ohashi et al., 2000; Piccinini and Midwood, 2010; Vabulas et al., 2002).

DAMPs are believed to originate from long-term Wallerian degeneration of nociceptive fibres

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that can last for years after the original trigger (Kim et al., 2009a; Vargas and Barres, 2007).

When bound to TLRs, DAMPs also regulate degeneration of the distal axonal segments that got

disconnected from the parent axon and the soma, a process that according to some investigators

initiates neuropathic pain via additional peripheral and central mechanisms (Kim et al., 2009a;

Piccinini and Midwood, 2010).

Scheme 1 (reprinted with permission from Kim et al., 2009a) illustrates a model of TLRs

involved in nerve injury and neuropathic pain. Following nerve injury, TLRs on peripheral

Schwann cells respond to DAMPs and regulate Wallerian degeneration and nerve regeneration.

Subsequently, DAMPs in the spinal dorsal horn activate microglial TLRs thereby releasing

inflammatory mediators and other factors on site, such as IL-1β, IL-6, TNFα, PGE2, NO and

BDNF (Jo et al., 2009; Kim et al., 2009a; Liu et al., 2012b). Some of these factors sensitize

neurons and others activate pain pathways indirectly, resulting in pain hypersensitivity (Matsui et

al., 2010; Wen et al., 2011). Specifically, these microglial mediators strongly regulate synaptic

transmission by enhancing excitatory and suppressing inhibitory synaptic transmission in spinal

cord neurons (Coull et al., 2005; Kawasaki et al., 2008). TLR4 microglial activation increases

p38 MAPK signalling in the spinal cord after SNL injury, which enhances release of TNFα, IL-

1β, and BDNF (Liu et al., 2012b). Astrocytes in the spinal cord and brain also express TLR4 on

the cell membrane (not shown in Scheme 1) and also contribute to the maintenance of

neuropathic pain conditions (Gao et al., 2009; Gao and Ji, 2010; Tsuda et al., 2011; Wei et al.,

2008). Gilmore and Skinner (1979) were the first to report that peripheral nerve injury (PNI)

induces astrocyte activation (Gilmore and Skinner, 1979), which is involved in the maintenance

of pain hypersensitivity (Raghavendra et al., 2003a). As well, TLR4 expression can be induced

in astrocytes in response to inflammation (Takeda and Akira, 2004). Among the many mediators

released from activated astrocytes to promote pain following PNI are ATP, NO, IL-6, PGE2, IL-

1β, CCL2 (MCP-1), CXCL-1 and CXCL-10 (reviewed in Liu et al., 2012b; McMahon et al.,

2005), many of which are induced by TLR4. Nerve injury and inflammation activate NFκB and

MAPK-mediated signalling pathways ERK and JNK in spinal astrocytes via TLR4, leading to

the synthesis and release of inflammatory mediators (Liu et al., 2012b). In the PNS, TLR4 is

expressed in TRPV1-expressing trigeminal neurons (Wadachi and Hargreaves, 2006), and

increases TRPV1 activity by LPS-induced intracellular Ca2+

release and inward currents in these

cells (Diogenes et al., 2011). In addition, TLR4 co-localizes with CGRP in primary sensory

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neurons, and LPS enhances the TRPV-1 dependent release of CGRP (Ferraz et al., 2011), which

modifies the excitability of sensory neurons. Since LPS is a ligand for TLR4, these studies

indicate that TLR4 is involved in neuronal excitability also in the PNS.

Scheme 1: A model for TLRs in peripheral nerve injury and neuropathic pain (Kim et al.,

2009a).

Immediately following nerve injury, inflammatory and immune cells are recruited to the

nerve injury site including blood-derived immune cells, nerve-resident macrophages, and

Schwann cells, and these become activated upon binding endogenous TLR agonists

including cell membrane components, plasma proteins, heat shock proteins, IL-1β, and

TNFα (Hameed et al., 2010), to release cytokines, chemokines and neurotrophic factors

that facilitate nerve degeneration and regeneration processes (as shown in the bottom

inset of Scheme 1). These growth factors, as well as other inflammatory mediators, may

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directly sensitize neurons and contribute to the development of neuropathic pain (Coull et

al., 2005; Kawasaki et al., 2008). Additionally, binding of endogenous agonists of TLRs

on resting microglia in the spinal dorsal horn activate these cells and release

inflammatory mediators that promote neuropathic pain (top rectangle). These agonists,

including IL-1β and TNFα, possibly originate from necrotic cells in the spinal cord after

spinal injury or by release from the microglia themselves. Proinflammatory cytokine

production results from the NFκB/MAPK pathways initiated by TLR4 (Jo et al., 2009).

TLRs are expressed in the PNS in Schwann cells (Tang et al., 2007) and in trigeminal

sensory neurons (Wadachi and Hargreaves, 2006).

5.1.2 Tlr4 up-regulation in pain behaviour

The following numerous reports have indicated an association between up-regulation of

Tlr4 and pain behaviour. In the intraplantar Complete Freund’s Adjuvant (CFA)-induced

peripheral inflammation model, Tlr4 was up-regulated within 4 h in various CNS regions

including the lumbar spinal cord, brain stem and forebrain, and remained elevated for

more than 14 days (Raghavendra et al., 2004), in parallel with dissipation of the allodynia

Tlr4 expression was similarly up-regulated after L5 nerve transection in the lumbar spinal

cord within 4 h, and continued to increase until day 14 post-injury, also correlating with

the time course of allodynia (DeLeo et al., 2004; Tanga et al., 2004). Furthermore, TLR4

contributed to neuropathic pain behaviour in the SNL L5 spinal nerve ligation and CCI

models and to long established neuropathic pain (2-4 months), not just to pain that

follows shortly after nerve damage (up to 14 days) (Lewis et al., 2012). Both Tlr4 gene

and its protein expression levels were significantly elevated on days 3 and 7 post-CCI in

rats compared to their sham controls, as were IL-1β and TNFα (proinflammatory

cytokines contributing to neuropathic pain states) (Kuang et al., 2012). Following a CCI

injury in rats, both Tlr4 gene expression levels and proinflammatory cytokines IL-1 and

TNFα were elevated significantly on days 1, 3, 7, 10 and 14 PO (Wu et al., 2010). In the

SNL model in B mice, Tlr4 mRNA levels were elevated significantly 14 days post-spinal

nerve injury on the ipsilateral dorsal horn, compared with the contralateral dorsal horn

(Kuboyama et al., 2011). Finally, in a rat model of bone cancer pain, both Tlr4 gene and

protein expression levels were significantly increased on days 6, 12 and 18 post-induction

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of the pain model, compared to sham and intact rats (Lan et al., 2010). These studies,

while demonstrating increased Tlr4 gene and protein expression levels in parallel with

increase in evoked pain sensitivity in several models, raise the possibility that was not

tested before, that overexpression of Tlr4 may also correlate with spontaneous

neuropathic pain levels in the Neuroma model. Mechanisms involving Tlr4 are shown in

the following schematic diagram (Scheme 2).

Scheme 2: TLR4 is a potential master switch in pain regulation after nerve injury (Bottom

rectangles are adapted from (Liu et al., 2012b) with permission # 3378941061859). TLR4 is

expressed in neuronal and non-neuronal cells, on primary afferents, macrophages and Schwann

cells in the periphery and post-synaptically on 2nd

-order neurons, excitatory and inhibitory

interneurons, pericytes (Peri), microglia and astrocytes in the spinal and trigeminal dorsal horns

(Liu et al., 2012b). As products of nerve injury, necrotic cells release HSP60, and extracellular

matrix component, fibronectin, which are the ligands of TLR4 receptors (‘DAMPs’) released

after tissue/nerve injury and stress. Neuronal TLR4 increases the excitability of nociceptors by

increasing TRPV1 activity and TRPV-1 dependent release of CGRP. TLR4 on macrophages in

the PNS and microglia in the spinal cord, clear up the myelin debris of necrotic primary afferents

and interneurons and promote nerve regeneration in the periphery. In the spinal cord, TLR4

induces the activation of microglia and astrocytes and the production of proinflammatory

cytokines (INFLAM), leading to the development and maintenance of neuropathic pain. TLR4

signals through the adaptor protein, myeloid differentiation primary response protein 88

(MyD88), which leads to the translocation of NFκB to the nucleus, its binding to the DNA,

ending in subsequent gene expression levels. Simultaneously, MAPK signalling pathways are

activated, such as ERK, p38, and c-Jun N-terminal kinase (JNK), leading to the phosphorylation

and activation of transcription factors (not shown) to bind DNA and initiate gene transcription

followed by protein synthesis of numerous genes. Activation of TLR signalling cascades

produces a wide array of inflammatory mediators, including cytokines and chemokines, such as

TNFα, IL-1β, IL-6, BDNF, PGE2, CCL2 (MCP-1), CXC-chemokine ligand 1 (CXCL-1) and

CXCL-10, as well as reactive oxygen/nitrogen intermediates such as NO. Microglial activation

via TLR4 signalling is dependent upon GM-CSF released from astrocytes, ex vivo. GM-CSF,

IL-1β and IL-6, released from endothelial cells induce neuronal excitability. TLR4 signalling in

neurons, macrophages, and glia leads to increase in excitatory (INFLAM) and decrease in

inhibitory (BDNF) synaptic transmission in PNS and spinal cord neurons, which ultimately lead

to central sensitization and ongoing pain. Endo = Endothelial cells

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macrophage

TLR4

Nerve injury

Debris clearance

Necrotizing afferentsNecrotizing interneurons

TLR4 ligands (Fibronectin; HSP-60)

Periphery Spinal dorsal horn

Autotomy PainInhibitory interneuron

Microglia

TLR4

TLR4

INFLAM

Astrocyte

Asc

end

ing

pai

n

pat

hw

ays

Neuronal hyperexcitability

INFLAM

GM-CSF

Microglia Astrocyte

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5.1.3 Tlr4 blockade reverses established neuropathic pain

behaviour

Studies on Tlr4 knockout and mutant mice, and other studies using Tlr4 blocking agents show

that absence, blocking or elimination of Tlr4 expression reverses established neuropathic pain

states (Hutchinson et al. 2008; Kuang et al. 2012; Lan et al. 2010; Lewis et al. 2012; Liu et al.

201; Wu et al. 2010). For example, following a CCI nerve injury in the rat, an established

neuropathic pain behaviour was reversed by intrathecal TLR4 receptor antagonists (Hutchinson

et al., 2008) and intrathecal Tlr4 small interfering RNA injection (siRNA) (Kuang et al., 2012;

Wu et al., 2010). Intrathecal Tlr4 injection of siRNA in the rat also prevented the initial

development of bone cancer pain on day 4 post-induction of the model, and when administered

on post-inoculation day 9 it attenuated (but not completely blocked) the well-established bone

cancer pain behaviour (Lan et al., 2010; Liu et al., 2010).

To summarize, TLR4 and its associated signalling components contribute to pain

hypersensitivity. Blocking TLR4 signalling pathway prevents the initial development of

neuropathic stimulus-induced pain such as allodynia and hyperalgesia in mice and rats, and

reverses these abnormal sensory hypersensitivities in these models (Bettoni et al., 2008; Cao et

al., 2009; Hutchinson et al., 2008; Lan et al., 2010; Liu et al., 2010; Saito et al., 2010; Tanga et

al., 2005; Wu et al., 2010). Tlr4, the gene encoding TLR4, and its expressed protein, have never

been associated with spontaneous neuropathic pain behaviour (autotomy) in rodent models of

neuropathy. Therefore, it was of interest for us to show whether this gene plays role in autotomy

behaviour in mice of certain genetic backgrounds. The following section introduces the C3H

mouse strain, which is widely used to study Tlr4 function in various phenotypes, including

neuropathic pain.

5.1.4 C3H/HeJ vs. C3H/HeN mice

Several inbred C3H mouse strains are commercially available, including C3H/HeJ and

C3H/HeN, which have been studied previously by others for autotomy behaviour. C3H/HeJ

mice carry a genetic mutation in a non-synonymous SNP located in the Trl4 gene that is absent

from C3H/HeN mice. It is possible that this mutation alters the way in which denervated mice of

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the two strains respond to neuropathic pain, displayed by differences in their autotomy levels.

Autotomy behaviour in these strains was not compared as of yet in the same lab at the same

time, although other mouse models for pain behaviour, i.e. L5 spinal nerve transection in the

SNL (‘Chung’) model, have shown that C3H/HeJ mutant mice have reduced pain sensitivity

levels compared to the wild-type C3H/HeN following nerve injury (Cao et al., 2009; Tanga et

al., 2005). In another model that induces allodynia via intrathecal LPS induction, investigators

showed that TLR4 dependence of allodynia exists in C3H/HeN-induced mice but is lacking in

C3H/HeJ-induced mice (Sorge et al., 2011). This difference in pain behaviour was strain

specific, as it related only to male mice and was absent in female C3H/HeN-induced and

C3H/HeJ-induced mice (Sorge et al., 2011). Table 13 lists the phenotypic differences between

C3H/HeJ (TLR4-deficient) and C3H/HeN (TLR4 wild-type) mice.

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Phenotypes References

TLR4-deficient TLR4 wild-type

AFS Median = 1, AUC Median = 24.5 (Blech-Hermoni, 2005)

Average AFS = 10,

90% of mice

autotomized

(Minert et al., 2007a;

Rubinstein et al., 2003)

Average AFS = 7.5 (Persson et al., 2009a)

Folloing total denervation: average AFS = 9,

Following partial nerve ligation

(SNL): 30-35% of B/C3H/HeJ mice developed

thermal and mechanical hypersensitivity

compared to A/J mice.

(Mogil et al., 1999a)

Attenuated hypersensitivity to mechanical allodynia

and thermal hyperalgesia, compared to wild-type,

after CCI or spinal nerve L5 transection (SNL)

(Bettoni et al., 2008;

Cao et al., 2009; Tanga

et al., 2005)

Decreased expression of spinal microglial

markers and proinflammatory cytokines,

compared to wild-type, following CCI or

spinal nerve L5 transection (SNL)

(Bettoni et al., 2008;

Cao et al., 2009; Tanga

et al., 2005)

Reversal of mechanical hypersensitivity and

diminished glial activation markers after

resolution of peripheral inflammation

(Christianson et al.,

2011)

Decreased allodynia induced by intrathecal

LPS in male C3H/HeJ vs. C3H/HeN mice

(Sorge et al., 2011)

Resistance to atherosclerosis (Wang et al., 2007)

Lower levels of pro-inflammatory cytokines

TNFα and IL-6 in lung tissue in vivo after

bacterial infection

(Hassan et al., 2012)

Increased risk of intestinal damage and

microbial infection during acute graft-versus-

host-disease (GVHD)

(Imado et al., 2010)

Weigh less, but

consume more food,

leaner body mass

and fat mass; more

severe cancer

cachexia, but

smaller tumors;

IL1β is elevated in

serum of tumor-

bearing mice

(Cannon et al., 2007)

Endotoxin hyporesponsive (tolerant) (Baker et al., 1991;

Berenson et al., 2001;

Chung et al., 1998)

Table 13: Documented phenotypic differences between TLR4-deficient and TLR4 wild-type mice

(http://jaxmice.jax.org/strain/000659.html).

136

5.1.5 Tlr4 and Csf2rb1 in the inflammatory process

It has been shown that GM-CSF, one of CSF2RB1’s ligands, is responsible for microglial

activation (Duport and Garthwaite, 2005; Giulian and Ingeman, 1988; Hutter et al., 2010; Lin

and Levison, 2009; Maresz et al., 2005; Mukaino et al., 2010; Nakajima et al., 2006; Parajuli et

al., 2012; Quan et al., 2009; Sheikh et al., 2009; Suzuki et al., 2008; Volmar et al., 2008). More

recently, GM-CSF-induced microglial activation was shown to be dependent on TLR4 signalling

(Parajuli et al., 2012). GM-CSF enhances both mRNA and surface expression of TLR4 in brain-

derived isolated microglia, peaking at 48 hours after stimulation and remaining stable for 72 hrs.

GM-CSF-induced expression of TLR4 increases the production of LPS-mediated IL-1β, IL-6,

TNFα, and NO, via NFκB (Parajuli et al., 2012), associated neuropathic pain (Kuang et al., 2012;

McMahon et al., 2005; Milligan and Watkins, 2009). We showed that the receptor for GM-CSF,

CSF2RB1, is also associated with neuropathic pain. Csf2rb1 mRNA levels were up regulated in

the spinal cord following hind paw denervation and correlated with autotomy behaviour

(Chapters 3 & 4). Therefore it is suggested that co-expression of Tlr4 and Csf2rb1 may be

necessary to promote signalling events that are mediated by NFκB, such as the production of

inflammatory mediators associated with neuropathic pain. We hypothesize that the up-regulation

of Csf2rb1 in the spinal cord, which is associated with autotomy following denervation, may be

correlated with Tlr4 expression in mice. To test this hypothesis we compared the gene

expression levels of Csf2rb1 in the spinal cord before and following hind paw denervation, in

TLR4 wild-type vs. TLR4-deficient mice.

5.1.6 pStat3

Stat3 is a signal transducer activated upon phosphorylation by receptor-associated kinases in

response to cytokines and growth factors (Yuan et al., 2004). Stat3 is a transcription factor in the

Jak/Stat3 intracellular signalling pathway that once activated by IL-6, gp130 and Jak2 it initiates

in various cell types, including cells in the peripheral nervous system such as DRG satellite cells,

and the CNS, such as microglia and astrocytes, the transcription of cytokines and inflammatory

mediators Il-6, Mmp9, Ccl2, Il-1β, Tnfα, and p38 (Yu et al., 2009). When this happens following

peripheral nerve injuries, the release of these mediators in the spinal and trigeminal dorsal horns

has been shown to lead to allodynia and hyperalgesia (Dominguez et al., 2008, 2010; Dubový et

137

al., 2010; Kohli et al., 2010; Maeda et al., 2009; Tang et al., 2012). The active, phosphorylated

form of Stat3 (i.e., pStat3) is present in microglia and astrocytes of the spinal cord after SNL

injury (Dominguez et al., 2008; Tang et al., 2012), and in microglia (Dominguez et al., 2010) and

satellite cells of the DRG (Dubový et al., 2010) after CCI injury. The Jak-Stat3 pathway also

initiates nerve injury-induced astrocyte proliferation in the dorsal horn after SNI injury (Tsuda et

al., 2011). Blocking signal transmission in the Jak-Stat3 pathway, with leptin suppressors or

with the suppressor of cytokine signalling 3 (SOCS3), can reverse mechanical allodynia and

thermal hyperalgesia following PNS and CNS damage (Dominguez et al., 2008, 2010; Lim et al.,

2009; Maeda et al., 2009; Tang et al., 2012; Tian et al., 2011; Tsuda et al., 2011). Transgenic

mice that express the gene for human sickle hemoglobin (HbS; a protein that causes in humans a

syndrome of painful sickle cell anemia) show musculoskeletal and cutaneous mechanical and

heat allodynia (Kohli et al., 2010). These behavioural abnormalities were associated with

increased spinal pStat3 (activated Stat3) signalling, and an accompanying increase in IL-6, TLR4

levels (Kohli et al., 2010).

Stat3 also plays a role in bone cancer pain rodent models. This effect is mediated by the release

of G-CSF or GM-CSF from tumor-affected tissues. Binding of G-CSF or GM-CSF each to its

own receptor on sensory nerve fibres and cell bodies in the PNS was associated with sensory

hypersensitivity manifesting in mechanical allodynia and thermal hyperalgesia (Schweizerhof et

al., 2009). In cultured sensory DRGs, activation of the Jak/Stat pathway by G-CSF or GM-CSF

lead to phosphorylation of Stat3 and its translocation to the cell nucleus, where it initiates the

transcription of nociceptive transducers such as Trpv1, Nav1.8 and potassium channels involved

in regulating excitability of sensory nerves, such as Kcnd2 (Kv4.2) (Schweizerhof et al., 2009).

Exposure to G-CSF or GM-CSF sensitizes sensory nerve fibres to nociceptive stimuli, which can

be observed by electrophysiological recordings from single afferents ex vivo as well as by

behavioural responses to noxious stimuli in vivo (Stösser et al., 2011). Blocking G-CSF or GM-

CSF signalling by receptor neutralizing antibodies or by signalling inhibitors leads to a relief in

bone cancer pain in rodent models (Stösser et al., 2011). It has also been shown that Stat3 (not

directly through G-CSF or GM-CSF, but through Jak2 and/or NFκB, which signal downstream

of CSF2RB1) induces in cancer cells the transcription of inflammatory mediators such as Mmp9,

Il-6 Il-1β and Ccl2 (Yu et al., 2009), known to play a role in pathological pain (Qian et al., 2011;

Soria-Castro et al., 2010). These evidence link indirectly the GM-CSF receptor beta

138

(CSF2RB1), the gene product focused in this dissertation, to Stat3 signalling. While this

evidence relate to the role of Stat3 signalling following its activation by ligands of CSF2RB1 in

peripheral nociceptors, there is no report documenting the role of Stat3 signalling in CNS

spontaneous neuropathic pain following peripheral nerve injury. On this basis, we raised the

hypothesis that GM-CSF released from the terminals of injured nerves in the CNS, astrocytes,

blood cells (to name a few; see Scheme 4, Chapter 6) into the spinal cord following hindpaw

denervation, binds to its common receptor recognition subunit alpha (CSF2RA), on the surface

of expressing cells and ’ignite’ an intracellular signalling mechanism via the active subunit of the

common receptor beta (CSF2RB1), and Stat3 within the same cells. The transcription and

release of inflammatory mediators from these cells into the extracellular space may interact with

more of the same cells in a positive feedback loop (Ogura et al., 2008), or may excite second

order neurons, and promote autotomy (Scheme 4, Chapter 6). We further hypothesize a similar

pathway occurring in the hippocampal dentate gyrus, since we detected CSF2RB1+ cells in that

region and associated it with high autotomy. These hypotheses were tested here in the following

sets of experiments. First, we induced autotomy and sham operations in A and B mice and

excised their lumbar spinal cords and brains for histology after perfusion fixation of the CNS.

Next, we immunolabelled the sections with pStat3 antibodies (selective for activated Stat3) and

with various markers of neurons and glial cells. Finally, we analyzed the pStat3 labelled sections

in the spinal cord and brains, and associated them statistically with CSF2RB1 data.

5.1.7 C3H/HeJ and C3H/HeN mice in the Neuroma model

C3H/HeJ and C3H/HeN mice have been used by other investigators interchangeably to study

autotomy behaviour following peripheral neurectomy, not noticing the genetic difference

between the strains and unaware of the Tlr4 mutation and its reported impact on stimulus-

induced neuropathic pain, including us (Blech-Hermoni, 2005; Minert et al., 2007a; Mogil et al.,

1999a; Persson et al., 2009a; Rubinstein et al., 2003). Thus, on this background it was of interest

to test whether this single point mutation in the Tlr4 gene causes mice to develop a different

course of autotomy than other known “high” autotomy wild-type mouse strains including

C3H/HeN. To test the hypothesis that absence of TLR4 modifies autotomy behaviour in mice,

we monitored and compared this behaviour in mutated C3H/HeJ mice vs. wild-type C3H/HeN

mice.

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5.2 Results

5.2.1 Autotomy behaviour in denervated C3H/HeN (Tlr4 wild-type)

and C3H/HeJ (Tlr4-deficient) mice

C3H/HeN and C3H/HeJ mice were denervated, sham-operated or left intact, and were followed

up for autotomy behaviour up to day 14 and 21 PO, respectively. As expected, none of the

sham-operated or intact mice expressed autotomy. As well, most wild-type mice (C3H/HeN)

have already expressed high autotomy before day 14, and had to be euthanized and perfused on

or before that day and their spinal cords were removed for gene expression analysis. Because

most denervated C3H/HeJ mice did not develop autotomy in the first 14 days PO, the follow up

was continued until day 21 PO, to test whether autotomy will develop albeit a delay. However,

mice perfused prior to day 21 PO retained their individual autotomy scores as part of their groups

scores until the end of the autotomy follow up period (i.e., PO day 21). Thus, on day 21 PO

autotomy scores in denervated mice of both strains varied from 0-11. Figure 30 shows the PO

course of autotomy behaviour in denervated wild-type mice compared to TLR4-deficient mice.

Average autotomy scores of the two strains diverged soon after the denervation procedure.

TLR4 wild-type mice showed a significant increase from day 4-14 PO (Figure 30A). Fifty

percent of wild-type mice (10 out of 20) expressed autotomy by day 14, their last observation

day, compared to 21% of TLR4-deficient mice (6 out of 28) at a significance level of P<0.003

(Figure 30B).

The average autotomy onset day for denervated wild-type and TLR4-deficient mice is shown in

Figure 31. The average onset day differed significantly in the C3H/HeN compared to that of

C3H/HeJ mice (7.1±1.3 vs. 9.8±0.91, P<0.05). The severity of autotomy at onset day,

characterized by the average score of autotomy at its onset, also differed significantly (Figure

32). Thus, mice carrying the Tlr4 mutation expressed a reduced average score at onset compared

to the wild type mice (1.3±0.35 vs. 12.6±0.72; P<0.04).

140

Figure 30: Course of autotomy levels in C3H/HeN mice carrying the wild-type Tlr4 gene

sequence and C3H/HeJ mice carrying the mutant gene followed up daily up to day 14. (A) Daily

average autotomy scores of C3H/HeJ mice (N=28) were significantly lower than those of

C3H/HeN (N=20). ANOVA for repeated measurements with Tukey’s post hoc test were

performed for all 14 days, yielding a value of P<0.005 for comparisons carried out for PO days

4, 12-14, and a value of P<0.0001 for PO days 5-11, (B) Percent mice with an autotomy score

above 6 on day 14 PO is shown for wild-type and TLR4-deficient mice. This difference was

significant at P<0.003.

Figure 31: Average autotomy onset day in denervated C3H/HeN and C3H/HeJ mice, shown in

grey and red, respectively as noted for TLR4 wild-type and TLR4-deficient mice. Average

autotomy onset day of the two strains differed significantly (2-tailed independent T-test, P<0.05).

-1

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141

Figure 32: The average score of autotomy at its onset in denervated C3H/HeN and C3H/HeJ

mice (N=20 and 28) following sciatic and saphenous neurectomy was significantly lower for

mice of the C3H/HeJ strain , and ranged from 1-7, compared to the score in C3H/HeN mice

which ranged from 1-8 (2-tailed independent T-test; P<0.003).

5.2.2 Csf2rb1 expression patterns in C3H/HeN and C3H/HeJ

mice

As shown in Figure 33, levels of Csf2rb1 expressed in the spinal cord of nerve injured mice of

the C3H/HeN (carriers of the Tlr4 wild-type sequence of the gene) were significantly higher than

those of naïve and sham controls of this strain (1.36±0.08 vs. 0.96±0.13, P<0.001; vs. 1.05±0.06,

P<0.002, respectively), and was even significantly elevated in mice expressing NLS compared to

naïve and sham controls (1.35±0.05 vs. 0.96±0.13, P<0.009; vs. 1.05±0.06, P<0.02). There was

no significant difference in Csf2rb1 mRNA levels in the spinal cord between denervated mice of

this strain that expressed NLS and those that expressed MHS. The expression of Csf2rb1 in the

two strains was not significantly different when comparing the two naïve groups, the two sham

groups, the two low-autotomy groups, or the two high autotomy groups of the two strains.

Within the C3H/HeJ strain (mice carrying the Tlr4 mutation) Csf2rb1 gene expression levels also

did not change significantly following denervation. The same outcome was evident when

P<0.003

0

0.5

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1.5

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TLR4 wild-type TLR4-deficient

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Autotomy onset score in C3H/HeN and C3H/HeJ mice

142

comparing MHS-C3H/HeJ, mice that developed high autotomy scores, and NLS-C3H/HeJ

groups with naïve and sham-operated mice of this strain.

Autotomy onset day and score at onset were not significantly correlated with gene expression

levels of Csf2rb1 in the two strains (Figure 34). Csf2rb1 gene expression levels were

comparable regardless of the time mice began to develop autotomy. Similarly, Csf2rb1 gene

expression levels were similar regardless of the degree of self-mutilation mice had at onset and

up to day 14 and 21 PO in C3H/HeN and C3H/HeJ mice, respectively.

Figure 33: Csf2rb1 gene expression levels in denervated C3H/HeN and C3H/HeJ mice.

Average Csf2rb1 mRNA levels relative to Hprt reference gene (see Appendix IV) of naïve (N=4

and N=6), sham-operated (N=8 and N=6), denervated with NLS (N=4 and N=5) and denervated

with MHS (N=12 and N=4) TLR4 wild-type and TLR4-deficient mice, respectively. Csf2rb1

expression did not change significantly among the TLR4-deficient mice by sham operation or

denervation, whether resulting in NLS or MHS. In the TLR4 wild-type mice, however, Csf2rb1

levels were significantly elevated after nerve injury (but not after sham operation) in mice that

expressed NLS as well as in those with MHS. Significantly elevated spinal levels of Csf2rb1

were also noted when comparing the two denervated groups against the sham operated group

(One-way ANOVA with post hoc LSD test).

P<0.01P<0.001

P<0.05

P<0.005

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TLR4-deficient

143

Figure 34: Correlation of Csf2rb1 gene expression levels (in denervated C3H/HeN and

C3H/HeJ mice) with the onset day of autotomy and autotomy onset scores. (A) Average Csf2rb1

mRNA levels (relative to Hprt reference gene) are represented for NLS/Late onset C3H/HeJ and

C3H/HeN mice, connoted by TLR4-deficient and TLR wild-type, respectively, which developed

an autotomy score of 0 on days 8-21 and 8-14, respectively, post-operation (N=5 and N=4),

MHS/Late onset C3H/HeJ and C3H/HeN mice, which developed an autotomy score of 5-7 on

day 11-15 post-operation (N=1 and N=2, respectively), and MHS/Early onset mice, which

developed autotomy scores of 1-8 on day 2-6 post-operation (N=3 and N=10, respectively).

Csf2rb1 expression was not significantly regulated between the groups of mice (one-way

ANOVA followed by LSD’s post hoc test). (B) Average Csf2rb1 mRNA levels (relative to Hprt

reference gene) are represented for C3H/HeJ and C3H/HeN denervated mice, which did not

develop autotomy and remained with an onset score of 0 up to day 21 and 14 post-operation,

respectively (N=5 and N=4), denervated mice, which developed MHS or NLS and their onset

score was 1 (N=2 and N=2), and MHS mice, which developed high autotomy and their onset

score was ≥2 (N=2 and N=10, respectively). Csf2rb1 expression was not significantly regulated

between the mice groups (one-way ANOVA followed by LSD’s post hoc test).

In conclusion, autotomy behaviour data show that denervated C3H/HeN mice that are known to

carry a normally functional Tlr4 gene responded to denervation with moderate to high scores of

autotomy with an early onset compared to denevated C3H/HeJ that are known to carry a non-

functional Tlr4 gene. Moreover, while denervation and autotomy in the wild-type mice was

associated with an increase in spinal Csf2rb1 gene expression levels, this increase was lacking in

denervated TLR4-deficient mice.

B

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144

5.2.3 CSF2RB1+ cells in the central canal of C3H/HeN mice

Next, we were interested to see whether or not CSF2RB1 protein is expressed in the spinal cords

of wild-type and mutant C3H mice, and to determine whether these cells change in number and

morphology following nerve injury in these mice. As in the A and B mice, CSF2RB1+ cells of

C3H/HeN and C3H/HeJ mice were located around the central canal. However, no significant

differences in the number of these cells in MHS- and sham-operated C3H/HeN mice were

detected, as shown in Figure 35.

Figure 35: CSF2RB1 immunostaining in the central canal of C3H/HeN mice. Average number

of cells per 50μm thick transverse slice having processes that extend laterally from the cell body

at the central canal for ≥20 μm into lamina X of the spinal cord are presented for sham-operated

and denervated MHS mice; ≥6 sections per mouse. Each column represents data for 1 mouse, 2

mice per treatment.

5.2.4 Tlr4 gene regulation in A vs. B mice

The results we got for the two C3H strains prompted us to ask whether Tlr4 also regulates

differentially the expression of Csf2rb1 in A and B mice following denervation. Tlr4 gene

expression levels were determined in the lumbar DRGs associated with the injured sciatic and

saphenous nerves, as well as in the spinal cords of these mice, and were compared to naïve and

0

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Sham 1 Sham 2 High Aut 1 High Aut 2

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CSF2RB1+ cells in C3H/ HeN mice

145

sham operated mice. The expectation was that mRNA levels of Tlr4 will be higher in A mice

compared to B mice, before and/or after peripheral nerve injury, and that this over expression

will lead to more receptor abundance and function, driving pain mechanisms in A but not B

mice. In the case of TLR4-mutant mice, mRNA levels of Tlr4 gene and its protein product are

diminished both in PNS and CNS tissue. However, in A and B mice we could not predict

whether the functional difference would occur in the periphery (DRG) or in the CNS (spinal

cord), or both, and due to the fact that we had previous data on the transcript abundance in both

tissues from our expression profiling arrays (Chapter 3), we analyzed these data from both DRGs

and spinal cords of A and B naïve, sham-operated and NLS and MHS mice.

In support of the hypothesis, we found that Tlr4 mRNA levels in the DRGs and spinal cords of A

mice were elevated 2-fold compared to the levels of B mice for the same groups (naïve, sham-

operated, and denervated whether with or without MHS) (Figure 36). Tlr4 mRNA levels in the

ipsilateral DRGs of naïve A and B mice were 0.53±0.60 and 0.28±0.02, respectively (P<0.001,

one-way ANOVA with LSD post hoc test). In sham-operated A and B mice these levels were

also significantly different 0.59±0.06 and 0.31±0.05, respectively (P<0.0001, one-way ANOVA

with LSD post hoc test). Tlr4 mRNA in denervated NLS-A mice was 0.62±0.05 compared to

0.28±0.04 in NLS-B mice (P<0.0001, one-way ANOVA with LSD post hoc test). Finally,

denervated MHS-A mice had 0.54±0.01 compared to denervated NLS-B mice (P<0.001, one-

way ANOVA with LSD post hoc test). In the DRG of A and B mice, denervation that led to

NLS or MHS was not associated with a change in mRNA levels of Tlr4 compared to sham-

operated same-strain groups or the same-strain naïve groups (Figure 36A).

Similar 2-fold differences in Tlr4 gene expression levels were observed in ipsilateral spinal cords

of A vs. B mice (Figure 36B). Sham-operated and denervated NLS-A mice had >2-fold

difference in Tlr4 expression levels compared to the B mice with the same phenotypes (sham

0.20±0.03 and 0.08±0.05, P<0.03; denervated NLS 0.21±0.045 and 0.09±0.04, P<0.03,

respectively). As well, Tlr4 gene expression levels were >2-fold in the denervated MHS- A mice

compared to the denervated NLS- B mice (0.23±0.04 vs. 0.09±0.04, P<0.01, respectively). Tlr4

mRNA levels were not significantly higher in naïve A vs. B mice. As in the DRG, ipsilateral

spinal Tlr4 expression levels did not correlate the type of treatment mice received, since

expression levels in sham-operated mice, and nerve injured mice with NLS or with MHS, did not

146

differ from those of the naive group. Likewise, spinal Tlr4 expression levels of nerve-injured

mice with NLS or with MHS did not differ from those of the sham operated mice. The same lack

of change was seen in B mice groups. The only differences were across the strains.

Figure 36: Tlr4 gene expression levels in DRG and spinal cord of A vs. B mice. Average Tlr4

mRNA levels (relative to those of a mouse reference gene pool) are shown in (A) for DRGs of

the naïve (N=5 and N=5), sham-operated (N=5 and N=5), denervated with NLS (N=3 and N=5)

A and B mice, respectively, and denervated A mice with MHS (N=5), and in (B) for the

ipsilateral spinal cord of naïve (N=5 and N=5), sham-operated (N=5 and N=5), denervated with

NLS (N=5 and N=5) A and B mice, respectively, and denervated MHS-A mice (N=5). One-way

ANOVA with LSD post hoc test was performed.

5.2.5 Tlr4 gene polymorphisms in MHS vs. NLS mice

Next, we were interested to determine whether differences in pain behaviour between autotomy

susceptible and autotomy resistant strains may be explained by polymorphic contrasts within the

Tlr4 locus. Using the MGI website

(http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=snpQF) we found, 35 SNPs in

Tlr4 (29% out of all 122 reported SNPS within Tlr4 from the MGI website) between A and B

mice that contrast in autotomy levels, located in coding, untranslated (UTR) and intronic regions

of the gene (Table 14), including 2 non-synonymous, 2 synonymous, 3 UTR and 28 intronic

mutations. When using the same SNP browser to compare SNPs in other strains having known

levels of autotomy (Blech-Hermoni, 2005; Devor et al., 2007; Mogil et al., 1999a), contrasting

those that typically express MHS with those expressing NLS autotomy scores, such as BALB/cJ

0

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A

B

A

0

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HighAutotomy

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A

B

BP<0.05 P<0.05P<0.001

P<0.0001 P<0.0001 P<0.001P<0.01

147

(MHS) vs. AKR/J and DBA/2J (NLS), we found 20 SNPs out of all 122 reported SNPs within

Tlr4 (16%). These were located in exons (i.e., 4 non-synonymous and 2 synonymous), in UTR

(n=3) and in intronic regions of the gene (n=11 SNPs). Twenty-one SNPs were identified

between high (BALB/cJ) and moderate autotomy strains (129S1/SvImJ and 129X1/SvJ; 17%).

These were 5 non-synonymous SNPs, 2 synonymous SNPs, 3 UTR SNPs and 11 SNPs in

intronic regions of the gene. Thus, there are a large number of SNP variations in the sequence

code of Tlr4 that might correlate the contrast in autotomy behaviour between A and B mice, and

possibly between other MHS vs. MLS strains. These polymorphic regions may alter Tlr4 mRNA

stability or TLR4 protein structure that would have functional relevance for autotomy behaviour

in the A strain compared to the B strain (as reported above the Tlr4 gene expression levels

difference across the two strains). Some of these SNPs may adversely affect the function of the

TLR4 receptor making the gene in C3H/HeJ mice dysfunctional.

Table 14: Tlr4 SNPs that contrast between A and B mice and are located in coding regions

(synonymous and non-synonymous SNPs), intronic and un-translated regions. Some of these are

also polymorphic between other strains known to express NLS vs. MHS of autotomy, such as

BALB/cJ, AKR/J and DBA/2J vs. 129X1/SvJ and/or 129S1/SvImJ, respectively. Some SNPs

regulated between all of the above MHS vs. NLS strains. Other SNPs regulated only between

some of the above strains.

5.2.6 pStat3 in dorsal horn of A vs. B mice

Unlike CSF2RB1 expressed in the central canal of the spinal cord, pStat3 was not expressed

there, only to a minimal extent. pStat3 was expressed in unidentified cells within the whole

spinal cord, including the dorsal horn. As can be seen in Figure 37, GFAP labelling did not

coincide with pStat3, therefore all we can state at this time is that pStat3 is not expressed in

astrocytes. The expression levels of pStat3 in the ipsi- and contralateral dorsal horns of

denervated mice were not significantly different (Student’s t-test). However, significant

Gene

mRNA UTR Coding Synonymous Coding Nonsynonymous Intronic

Total SNPs

A vs. BHigh vs.

LowHigh vs.

IntTotal SNPs

A vs. BHigh vs.

LowHigh vs.

IntTotal SNPs

A vs. BHigh vs.

LowHigh vs.

IntTotal SNPs

A vs. BHigh vs.

LowHigh vs.

Int

Tlr4 12 3 3 3 13 2 2 2 11 2 4 5 86 28 11 11

148

differences were observed between sham operated and denervated mice of the same strain (AS

8.3±1.8 vs. NLS-A 22.2±3.8, P<0.001; AS 8.3±1.8 vs. MHS-A 19.1±3.1, P<0.004, respectively),

and between A and B denervated mice (NLS-B 11.3±1.3 vs. NLS-A 22.2±3.8, P<0.007; NLS-B

11.3±1.3 vs. MHS-A 19.1±3.1, P<0.03, respectively). Figure 37 shows photomicrographs of

transverse sections of the L4-5 segments of the spinal cord with a focus on the dorsal horn

region, immunolabelled with pStat3 and GFAP antibodies. The Figure clearly shows an elevated

expression in pStat3 in the dorsal horn of denervated A mice, significantly more so than the

sham-operated A mice, and the denervated B mice. There was no significant difference in pStat3

expression between denervated A mice with (MHS) and without (NLS) autotomy.

Figure 37: Photomicrographs of the spinal cord of denervated and sham operated A mice

labelled immunohistologically for pStat3. pStat3 (red) was highly expressed in the L4-5 spinal

dorsal horn of MHS-A mice (A), compared to sham-operated A mice (B-C). pStat3 is evident

throughout the grey matter, but note that it was absent around the central canal (arrow) in A

sham mouse (D). pStat3 did no co-localize with astrocytes (GFAP; green) and also not with

most neuronal nuclei (NeuN; green), but only co-localized with a small subpopulation of neurons

(B & E). (F) Cell nuclei in the spinal dorsal horn, immunolabelled with pStat3 were counted in

an area of 100μm x 100μm (0.01mm2) on gray scale images to accentuate pStat3 labelled in

white over a dark gray/black background, as shown in (B). Data is shown for 6-8 sections, 1

A

FED

CB

0

5

10

15

20

25

30

pST

AT3

+ ce

lls /

10

mm

²

pSTAT3+ cells in ipsilateral dorsal horn

P<0.001

P<0.004

P<0.007

P<0.03

A

pSTAT3GFAP

ED

CB

pSTAT3NeuN

pSTAT3NeuN

pSTAT3NeuN

pSTAT3

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mouse per group; One-way ANOVA with LSD post hoc test; Magnification, 20X; Scale bar 80

μm.

5.2.7 pStat3 in the dentate gyrus of A vs. B mice

In order to detect whether the Stat3 pathway in the dentate gyrus plays role in autotomy

behaviour, as we found for CSF2RB1, we looked at its active form, pStat3, using

immunostaining analysis. Strikingly, there was a significant difference in the expression of

pStat3 between the right and left dentate gyrus of denervated A mice expressing high autotomy

scores compared to denervated low autotomy B mice. There was a 2-fold difference in pStat3

expression levels in these MHS-A mice between the left and right sides, which was not seen in

any studied low autotomy groups, in which the fold change remained around 1 between the

dentate gyri on the two sides (Figure 38). pStat3 did not co-localize with the astroglial marker

GFAP (Figure 38B-C). We could not perform a double labelling of pStat3 with CSF2RB1, since

the two antibodies came from the same species. But labelled cells in the dentate gyrus

polymorph layer with either pStat3 or CSF2RB1 looked identical in morphology.

5.2.8 pStat3 in the peri-ventricular nucleus of A mice

A significant labelling of pStat3 was also observed around the brain ventricles of denervated and

sham operated A mice, a region that comes in contact with cerebrospinal fluid (Figure 39). A

small population of these cells also reacted with GFAP, indicating that some astrocytes in the

peri-ventricular region express pStat3. pStat3 did not co-localize with neuronal nuclei (as

marked by NeuN; Figure 39B). Immunoreactivity with CSF2RB1 was also seen in this region in

both A and B mice (Chapter 4), suggesting that the two proteins may be co-expressed withtin the

same cells.

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Figure 38: Photomicrographs of the dentate gyrus of denervated and sham operated A mice

labelled immunohistologically for pStat3. pStat3 (red) is expressed by cells in polymorph and

granule layers of the dentate gyrus. It did not co-localize with GFAP (astrocytes, green; A & B),

or with NeuN (neuronal nuclei, green; D). pStat3 expression in the left hemisphere of A high-

autotomy mouse is shown in (A) compared to the right hemisphere of the same mouse where

lower pStat3 expression was detected (B) and compared to a hemisphere of an A sham mouse

where lower pStat3 expression was also valid (D). (C) Fold change data is shown for 1-2 mice

per treatment type. Number of cells in both hemispheres, per section, was counted in an area of

50μm x 50μm (2,500μm2), 4-6 sections per mouse. Ratios between right and left hemispheres

are presented as a fold change. One-way ANOVA with Tukey’s post hoc test. Magnification -

20X; Scale bar - 80 μm.

C

BA

pSTAT3GFAP

pSTAT3GFAP

D

pSTAT3NeuN

0

0.5

1

1.5

2

2.5

3

A sham A low-autotomy

A high-autotomy

B low-autotomy

Fold

ch

ange

of

pST

AT3

+ ce

lls in

left

/rig

ht

he

mis

ph

ere

s

Increase in dentate gyrus pSTAT3+ cells correlates with autotomy

P<0.0001P<0.0001 P<0.0001

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Figure 39: Photomicrographs of the brain ventricles of denervated and sham operated A mice

labelled immunohistologically for pStat3. pStat3 immunopositive cells are seen around the brain

ventricles and throughout the peri-ventricular region. pStat3 co-localized with a small

population of astrocytes in a MHS-A mouse (Shown in yellow; arrows in A, C) but not with

neurons shown for an A sham mouse in (B). Magnification – 20X; Scale bar – 80 μm.

5.3 Discussion

5.3.1 Tlr4 contributes to chronic neuropathic pain

In the present study we showed for the first time that Tlr4 is a gene whose product contributes to

the maintenance of spontaneous chronic neuropathic pain in the Neuroma model of autotomy.

This is based on the observations that C3H/HeJ (TLR4-mutant) mice were less susceptible to

develop autotomy compared to C3H/HeN (wild-type) animals, manifested by the lower number

of mice that autotomized on day 21 post-denervation, longer onset times and lower autotomy

scores at onset compared to wild-type mice. These results suggest that Tlr4 is a gene that

correlates with the maintenance of spontaneous neuropathic pain in the mouse. In addition to

Tlr4 it is possible that there might be other genes having SNP variations between C3H/HeJ and

C3H/HeN, and these genetic differences may play a role in spontaneous neuropathic pain. More

research is needed to answer this question.

These results are compatible with previous studies that compared Tlr4 mutant and wild-type

mice in models of stimulus-evoked chronic neuropathic pain. Tanga et al. (2005) compared Tlr4

knockout and point-mutant mice that underwent a L5 nerve transection in the SNL model and

A B

pSTAT3NeuN

pSTAT3GFAP

C

pSTAT3GFAP

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reported observing a significantly attenuated behavioural hypersensitivity and a decreased

expression of spinal microglial markers, reduced glial activation and reduced expression of pain-

related cytokines. In another study using the CCI model, the authors have shown reduced

thermal hyperalgesia and mechanical allodynia on days 4 and 8 post-injury in Tlr4 knockout

compared to wild type mice (Bettoni et al., 2008). Thus, we can conclude that Tlr4 gene may be

important in maintaining both stimulus-evoked and spontaneous pain-related behaviours.

5.3.2 Tlr4 gene is differentially expressed in A vs. B mice

The autotomy-contrasting mouse strains A and B, which we used in studies 1 and 2 of this thesis

to seek candidate genes for autotomy and study the role of CSF2RB1, showed a significant

contrast in Tlr4 gene expression levels, as follows. Denervated B mice, which did not develop

autotomy after total hindpaw denervation, had significantly lower Tlr4 mRNA levels in DRG

neurons and in the lumbar spinal cord, as compared with denervated A mice, which expressed

moderate-high autotomy scores following the same denervation procedure. This contrast in Tlr4

gene expression levels between A and B strains was also demonstrated when comparing their

two sham operated groups (i.e., AS vs. BS) as well as the denervated groups of these strains

(MHS-A and NLS-A vs. NLS-B mice). These results suggest that levels of expressed Tlr4 may

be important in regulating pain after tissue and nerve injury in A mice, and not in B mice,

peripherally (in DRG neurons) and centrally (in the spinal cord), or that lower Tlr4 gene

expressed in B mice relative to A mice in these tissues may have protected them from autotomy

behaviour.

The current results also demonstrate that constitutive Tlr4 mRNA levels in the spinal cord of

intact A and B mice are not significantly different; suggesting that in these mice Tlr4 in the

spinal cord is not involved in the triggering mechanisms of chronicity at the time of nerve injury,

when the animal is intact. This conclusion is supported by findings of other groups that showed

that loss of Tlr4 gene did not change acute pain behaviour, and did not alter acute pain behaviour

of the intact contralateral paw, after an ipsilateral nerve injury (Bettoni et al., 2008; Cao et al.,

2009). However, our results also showed that Tlr4 expression levels of intact A and B mice in

the DRG is significantly different, and this may propose a peripheral mechanism involving Tlr4

at the time of nerve injury. Further studies are needed to assess in which cell types within the

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DRG Tlr4 gene is expressed, to better understand whether or not an ‘injury discharge’

mechanism is involved. Within DRG tissue are DRG neurons, satellite cells, and also

macrophages (Hu and McLachlan, 2003), which are the potential cells to express Tlr4. Since

TLR4 is expressed on macrophages and is also found on the cell membrane of trigeminal

nociceptive neurons (Wadachi and Hargreaves, 2006), the gene may be transcribed within the

DRGs, and then transported to the cell membrane, or it may be transcribed in the macrophages or

both. Thus both or one of these cell types may be potential targets for producing and relaying

‘injury discharge’ to the CNS, associated with TLR4. Additional studies are needed to answer

these questions.

Experiments carried out in silico, confirmed that the Tlr4 gene is polymorphic in contrasting

autotomy strains, including in A and B mice. Therefore, the alterations in Tlr4 gene expression

levels between A and B mice (or between other autotomy contrasting strains) can be explained,

in part, by differences in DNA sequence between MHS and NLS strains. Currently, there is no

functional information available for these SNPs specifically, but other SNPs in Tlr4 have been

associated with disease. A study by Yamakawa et al.(2013) shows that a human polymorphism

in the Tlr4 gene causes LPS hyporesponsiveness, and is associated with a variety of infectious

and non-infectious diseases (Yamakawa et al., 2013). Numerous studies in humans and fewer

studies in mice demonstrated that polymorphisms in the Tlr4 locus between individuals and

strains, respectively, exist, and these modify the responses to inflammatory (Liu et al., 2011),

autoimmune (Leichtle et al., 2011; Sivula et al., 2012) and neurodegenerative disease (Yu et al.,

2012).

The data presented here suggest a correlation between Tlr4 expression and autotomy behaviour

in wild-type vs. TLR4-mutant mice, as well as in A vs. B mice, demonstrated by the differences

in autotomy levels, day of onset and onset scores following hind paw denervation, and by

differences in Tlr4 mRNA levels in autotomizing A vs. non-autotomizing B mice. Further

studies are needed to show whether blocking the Tlr4 gene expression or the function of the

TLR4 protein in A mice could potentially reduce autotomy following hindpaw denervation in the

Neuroma Model.

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5.3.3 Proposed mechanisms for Tlr4 in neuropathic pain

In the present study we showed that Tlr4 gene expression levels in the spinal cord and DRGs

correlate with autotomy behaviour in denervated A mice and with the absence of this behaviour

in denervated B mice. We also showed that the presence of functional TLR4 receptors correlates

with autotomy in Tlr4 wild-type mice and that absence of TLR4 modulates autotomy behaviour

in Tlr4 mutant mice. We suggest that in the PNS and spinal cord of A and C3H/HeN mice,

TLR4 mediates macrophage/glial activation, which is significantly higher in these mice

compared with B and C3H/HeJ mice. The proposed enhanced macrophage/glial activation in the

PNS and spinal cords of A and C3H/HeN mice may initiate the transcription and production of

cytokines and inflammatory mediators and other factors which sensitize pain pathways. These

substances may facilitate excitation of nociceptors in the PNS and second order neurons in the

spinal dorsal horn, thereby contributing to peripheral and central sensitizations. In addition,

some anti-inflammatory mediators and neurotrophins (such as BDNF) may facilitate blocking of

inhibitory interneurons in the spinal dorsal horn, a mechanism which is also known to contribute

to central sensitization.

There is a large body of evidence linking microglial activation to TLR4 activation, and vice

versa (reviewed in (Guo and Schluesener, 2007; Nicotra et al., 2012). In essence, Tlr4 is a

documented key regulator for initiating and/or maintaining pain, by enhancing the production of

inflammatory mediators and factors that promote pain. Simultaneously, TLR4 may be involved

in other mechanisms including phagocytosis in debris clearance of necrotic cells in the periphery

and spinal cord, which are remnants of dying nociceptors, by-products of the denervation and

second order spinal neurons. Although not shown before, such dying neurons may occur higher

up supraspinally along pain pathways, stirring up the phagocytic action of glia and inflammatory

cells, which we saw in those supraspinal structures as manifested in CSF2RB1 expressing cells.

This mechanism may, in addition to producing sensitization of pain pathways directly, also

enhance processes of regeneration and axonal sprouting in the neuroma and from collaterals of

intact afferents in the periphery as well as within the spinal cord, including collateral sprouting of

central terminals of intact neighbouring cells into spinal termination sites that do not normally

process these inputs. In the periphery, axonal sprouting within the neuroma and assembly of

sodium channels in the sprouts’ membranes leads to ectopic activity of action potentials that

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relay nociceptive messages through the spinal cord and to the brain. In the spinal cord, axonal

sprouting of nociceptors into laminae that do not normally process pain inputs, send nociceptive

messages to the brain.

Following nerve injury TLR4 signalling can be activated by various ligands (i.e., necrotic cells,

HSP60, fibronectin) in numerous cell types in peripheral and central nervous systems (Schwann

cells, macrophages, neurons, pericytes and glia) to produce and maintain pain (See section 5.1.1

Tlr4). Therefore, mice that lack the functional receptor (i.e., C3H/HeJ, the Tlr4 mutant mice), or

have fewer copies of the receptor (e.g., B mice) compared with mice having the functional

receptor (i.e., C3H/HeN; Tlr4 wild type) and in greater numbers (i.e., A mice) are more

susceptible to autotomy behaviour.

5.3.4 Csf2rb1 gene expression levels in wild-type and TLR4-

deficient mice

The Csf2rb1 gene expression data in denervated C3H/HeN and C3H/HeJ mice shows that this

gene is up regulated after denervation in the wild-type mice, but not in TLR4-mutant mice,

suggesting that a functional Tlr4 is necessary for the induction of Csf2rb1 expression levels

following denervation, and that the two genes may be involved in the same pathway.

In Chapter 4 the data showed that the cytokine receptor CSF2RB1, encoded by its gene on

Pain1, correlates with MHS of autotomy in denervated A mice, and that its protein co-localizes

in specialized ependymal cells/radial glia/tanycytes around the central canal of the spinal cord

and in the brain in the dentate gyrus and peri-ventricular and arcuate nuclei of the hypothalamus.

We also showed that compared to denervated mice of the B strain, the number of such

specialized cells around the spinal central canal of autotomizing A mice increases, and their

morphology changes by hypertrophy that manifests in elongation of their processes.

Additionally we showed that following denervation in mice of the A and B strains, the number of

CSF2RB1+ ependymal cells/radial glia/tanycytes in the central canal region is significantly

increased compared to sham-operated and naïve mice of both strains. The present study

demonstrated co-localization of CSF2RB1 in ependymal cells/radial glia/tanycytes in wild-type

mice of the C3H/HeN strain, which extended into the grey matter of the spinal cord and reached

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the pial surface. However, our data failed to show an increase in the number of these cells

(immunolabelled with CSF2RB1) in denervated MHS mice compared to sham-operated wild-

type mice. These data suggest that, compared to autotomy-resistant B mice, both A and

C3H/HeN autotomy-susceptible strains have longer processes of CSF2RB1-expressing

ependymal cells/radial glia/tanycytes which may play role in the modulation of pain in these

strains.

5.3.5 pStat3 expression in the spinal cord of A vs. B mice

In the present study we determined the expression of phosphorylated Stat3 (pStat3) in the spinal

dorsal horn by immunohistochemical analysis and correlated it with the levels of neuropathic

pain behaviour. The activated form of the protein, pStat3, was expressed in the spinal dorsal

horn following hindpaw denervation demonstrated by a higher number of labelled cells in

denervated A mice, which are genetically susceptible to express autotomy, compared to B mice

that are genetically resistant to express this behaviour following the same denervation procedure.

Similar low numbers of pStat3-labelled cells were evident after sham-operation in the A strain.

These results suggest that Stat3 signalling in the spinal cord is correlated with autotomy

behaviour.

To understand better which cell types are responsible for the change in expression of pStat3 in

the spinal dorsal horn after denervation and during autotomy we labelled the cells with the

astrocyte marker GFAP or with a neuronal nucleus marker NeuN, testing whether they co-

localize with pStat3 in the same cells, based on previous knowledge that astrocytes and neurons

originally differentiated from ependymal cells/radial glia/tanycytes (Barnabé-Heider et al.,

2010). Our hypothesis was that CSF2RB1+ spinal central canal ependymal cells/radial

glia/tanycytes that have a long process into the spinal dorsal horn, become activated after

peripheral injury by a signal that is still unknown. When activated they sensitize pain pathways

in the dorsal horn that in turn, produce a spontaneous nociceptive message to the brain where

spontaneous pain is perceived, motivating the animal to express autotomy in A and C3H/HeN

mice. Also possible is that ependymal cells/radial glia/tanycytes in their known capacity as stem

cells would multiple and differentiate into astrocytes and/or neurons, which would in an

unknown way contribute to pain behaviour. Either way, be it via neoneurogenesis and/or

neoastrogliogenesis, or hypertrophy of processes in existing ependymal cells/radial

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glia/tanycytes, our data are compatible with CSF2RB1 in these cells as a necessary component

that activates in these cells perhaps via Jak/Stat3 the signalling that triggers transcription of pain

genes to maintain the state of chronic pain. However, in contrast to the ‘stem cell hypothesis’,

pStat3 did not co-localize with astrocytes, and only co-expressed in a few neurons. It is possible

that these signalling events may occur in microglia, which expresses pStat3 in mice strains

studied in our experiments. These events may be mediated by increased CSF2RB1 levels in

these glia activating Jak and triggering transcription and synthesis of inflammatory mediators of

pain maintenance. However, we did not co-localize CSF2RB1 with the microglial marker OX-

42 in the spinal cord. Interestingly, other investigators were able to show co-localization of the 2

proteins in cell cultures and in brain tissue (Parajuli et al., 2012).

Further experiments are needed to validate and extend our findings in the spinal dorsal horn with

additional markers for microglia, such as Iba-1, Integrin alpha M (ITGAM), and F4/80, which

are used interchangeably in similar studies. These antibodies may or may not show different

results in co-expression with pStat3 presented here.

5.3.6 Proposed mechanism for pStat3 and CSF2RB1 in the spinal

cord

Jak/Stat3 signalling may be triggered through binding of any of the 3 ligands (IL-3, IL-5 and

GM-CSF) of CSF2RB1 in the spinal dorsal horn. In addition to the Jak/Stat3 pathway that is

likely initiated by CSF2RB1 in DRG neurons (as introduced above in Section 5.1.7 ‘pStat3’),

other Stat pathways are known to be initiated by CSF2RB1, including Stat1, Stat3, Stat5 and

Stat6, which depend on Jaks or Src kinases and the cell type in which signalling is initiated

(Reddy et al., 2000). Both Jak and Src kinase have a role in phosphorylating, and thereby,

activating Stats as well as other proteins intracellulary, such as the intracellular domain of

CSF2RB1, which is phosphorylated by Jak2. Once activated, pStats enter the nucleus and

initiate transcription of several genes. In BaF3 cells, CSF2RB1 activates the transcription of c-

Fos, c-Jun and c-Myc (Itoh et al., 1996; Watanabe et al., 1995) (Scheme 6), which are

immediate-early genes that have a known role in initiating pain mechanisms (Munglani and

Hunt, 1995). Other genes include Trpv1, Scn10a, Kcnd2 and Kcnk2, which are transcribed via

CSF2RB1/Jak/Stat3 signalling in nociceptive DRG neurons associated with bone cancer pain

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and most likely in other chronic pain syndromes as well (Stösser et al., 2011). We can speculate

from these previous reports that CSF2RB1/Jak/Stat3 in the dorsal horn may be activated in (i)

post-synaptic cells that express c-Fos and c-Jun 14 days after SNL (Munglani and Hunt, 1995),

similar to our Neuroma model, in which mice were evaluated for protein expression on day 14

post- hindpaw denervation, or (ii) microglia which express pStat3 correlated with neuropathic

pain following peripheral nerve insults. These 2 scenarios are discussed in greater detail in

Chapter 6.

5.3.7 Hippocampal pStat3 expression is associated with autotomy

In Chapter 4 of this thesis report, we showed that the expression of CSF2RB1 in the

hippocampal dentate gyrus is associated with autotomy behaviour. The pStat3 labelled round

cells did not react with NeuN (a label of neurons) nor with astroglial marker GFAP. Additional

experiments are needed to confirm if pStat3+ cells are microglial or ependymal. Here we show

that pStat3+ cells, which numbers correlate autotomy behaviour, appear in similar morphologies

and localization in the dentate gyrus as are the CSF2RB1+ cells in that region. It is possible that

the same cells are labelled for both CSF2RB1 and pStat3 in the hippocampal dentate gyrus, and

that these two molecules operate in the same cellular pathway, but we did not carry out this

experiment as of yet.

In contrast to the findings with CSF2RB1, where we showed a bilateral equal increased

expression in the dentate gyrus, hypothalamic peri-ventricular and arcuate nuclei of MHS mice,

pStat3 expression was more dominant in one hemisphere compared to the other and correlated

with autotomy. This side difference did not correlate with sham- or hind paw denervation

operation. Whether this unilateral increased signal of pStat3 in the dentate gyri is correlated with

chronic neuropathic pain needs more research. There are many connections between the 2 brain

hemispheres by way of the corpus callosum, commissures, and decussations at every level below

the brain down to the spinal cord or brainstem nuclei, which makes isolation between the 2 sides

of the CNS almost impossible. Therefore, pain pathways may be interconnected between the

two sides of the CNS and expression levels of pain genes and proteins should be analyzed

carefully.

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Studies have shown a hemispheric dominance in levels of gene and protein expression after

ipsilateral injuries to the nerve or CNS that are compatible with known anatomical decussations.

For example, when compared to sham-operated animals, the CCI or SNI nerve injuries in the rat

were accompanied by an over-expression of cytokines that have a role in pain behaviour, such as

IL-1β and IL-6, in the dentate gyri bilaterally, more prominently on the contralateral side (del

Rey et al., 2011). Another group of investigators showed in the rat that transient forebrain

ischemia (which sometimes leads to brain injury) caused an increased expression of Stat3 and

pStat3 in nuclei of the dentate hilar region starting from day 1 to day 14 post-treatment (Choi et

al., 2003). Shortly after hypoxic/ischemic brain injuries (24–48 h), pStat3 is only observed in the

ipsilateral side (mainly in the hippocampal fissure and corpus callosum) of mice, while

inflammatory mediators such as IL-6, IL-1β and TNFα were highly expressed in the ipsilateral

hemisphere up to 7 days post-injury (Shrivastava et al., 2013). No expression in these cytokines

was detected at 14 days post-injury in these mice. Shrivastava et al. (2013) co-localized pStat3+

nuclei in a small subpopulation of reactive astrocytes and microglia in the ipsilateral hemisphere

up to 7 days post-injury, specifically in the Cornu Ammonis 1 (CA1) and hippocampal fissure

regions. This study suggests that pStat3 may be an important facilitator of pro-inflammatory

production soon after injury and may mediate the proliferation and activation of glial cells in the

hippocampus. Once glial cells are activated, they may carry on different cellular signalling

events independent of pStat3. However, since we detected our pStat3 in cells 14 days post-

injury, we suggest that pStat3 in the hippocampus has a significant role in maintaining pain at 14

days following hindpaw denervation.

pStat3 in the above studies was localized in astrocytes (Choi et al., 2003; Shrivastava et al.,

2013), unlike our results, which did not co-localize pStat3 with GFAP (a marker of astrocytes).

The discrepancy may be due to the different type of nerve injury or strain of mice. Because we

did not carry out co-localization experiments of pStat3 with OX-42 or Vimentin, which label

microglia or ependymal cells, respectively, we suggest that pStat3 molecule could be localized to

one of these cell types within the dentate gyrus in mice of the Neuroma model.

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5.4 Conclusions

In the present study we showed that the gene Tlr4 and the transcription factor, pStat3, are

associated with spontaneous neuropathic pain in the Neuroma mouse Model. Autotomy

behaviour was more profound in the TLR4 wild-type mice, showing increased levels of spinal

Csf2rb1 mRNA following hindpaw denervation, compared to the TLR4-mutant mice which had

delayed autotomy and an unchanged spinal Csf2rb1 gene expression level following the same

denervation. pStat3 immunoreactivity was detected in the spinal dorsal horn of mice and

correlated with autotomy behaviour in the ipsilateral side, and in the hippocampal dentate gyrus,

correlating with autotomy. pStat3 was also evident in the brain ventricles, but was lacking

around the spinal central canal. More studies are needed to determine the exact mechanisms of

TLR4 and pStat3 in the spinal cord and brain to bring about spontaneous neuropathic pain.

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Chapter 6

General Discussion and Conclusions

The main results presented in this dissertation identified candidate genes for spontaneous

neuropathic pain in the mouse following peripheral nerve injury using expression profiling of the

whole genome as well as in the region of Pain1, a quantitative trait locus on mouse chromosome

15 known to harbour such gene(s). We used genome-wide expression arrays to identify genes in

DRGs and spinal cord, the expression of which is changed by hindpaw denervation and the new

levels of the protein regulate autotomy behaviour. Whole genome expression studies have been

used extensively in many rodent models of neuropathic pain, in order to identify such genes and

the molecular and physio-pathological mechanisms that they encode, which drive the animal to

express genetically associated neuropathic pain levels.

Most of these rodent models used stimulus-evoked pain behaviour, testing the partially

denervated limbs mainly for allodynia and hyperalgesia in the still partially innervated receptive

fields of the injured nerves, but none has done it as of yet for spontaneous neuropathic pain

behaviour which is the symptom most bothering pain patients and for which identified genes

would be treatment targets (Coyle, 2007; Kim et al., 2009b; Ko et al., 2002; Kõks et al., 2008;

Lacroix-Fralish et al., 2006; Nesic et al., 2005; Rodriguez Parkitna et al., 2006; Sun et al., 2002;

Valder et al., 2003; Wang et al., 2002a). This dissertation reports on experiments that have used

the Neuroma model in the mouse to focus on the identification of candidate genes controlling

spontaneous pain levels, because this model mimics well the genetic and neuropathological

events that are believed to occur in humans who suffered limb amputation and develop

spontaneous post-amputation chronic pain, or in patients who had post-surgical pain (anesthesia

dolorosa).

6.1 Mechanisms involving the Colony-Stimulating Factor 2

Receptor Beta 1, CSF2RB1

Using the non-biased approach of whole-genome expression profiling in inbred lines of

mice with known contrasting spontaneous neuropathic pain behaviour following an

identical nerve injury, we identified in the mouse spinal cord Csf2rb1 as a candidate gene

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associated with this behaviour and having a major correlation with this trait’s variance.

In the following sections we discuss the literature about Csf2rb1 and its ligands in the

intact nervous system and in response to neural insults, as well as the mechanisms

involved.

6.1.1 The GM-CSF cytokine and its receptors in the nervous

system

GM-CSF has been linked in the literature to many pain mechanisms following nerve injury, by

way of their effect as glial activators (Duport and Garthwaite, 2005; Giulian and Ingeman, 1988;

Hutter et al., 2010; Lin and Levison, 2009; Maresz et al., 2005; Mukaino et al., 2010; Nakajima

et al., 2006; Quan et al., 2009; Sheikh et al., 2009; Suzuki et al., 2008; Volmar et al., 2008) and

causing neuronal excitation (Schweizerhof et al., 2009). GM-CSF has also been implicated in

several human neurological disorders and animal models of disease, such as Alzheimer disease

(Danielyan et al., 2010; Tarkowski et al., 2001; Volmar et al., 2008), bone cancer pain

(Schweizerhof et al., 2009), in the EAE model (an inflammatory demyelinating disease similar to

multiple sclerosis) (Kroenke et al., 2010), Hypoxia-ischemia (Clarkson et al. 2007), malignant

gliomas (Rafat et al., 2010), and x-adreno-leuko-dystrophy, a neurodegenerative disease (Ferrer

et al., 2010). Receptors for GM-CSF (i.e., Csf2ra and Csf2rb1, and -2, where -1 is also known as

Il3rβ and Il5rβ) have also been implicated in many neurological and pain syndromes, such as

bone cancer pain (Csf2ra; Schweizerhof et al. 2009), bipolar disorder (Csf2rb1; Moskvina et al.

2009), depression (Csf2rb1; Chen et al. 2011), diabetes mellitus (Il3rβ; Loesch et al. 2010),

malaria (Csf2rb1; Medana et al. 2009), and Schizophrenia (Csf2rb1; Carter 2009; Moskvina et

al. 2009; Chen et al. 2011). Thus, not surprisingly, GM-CSF and its receptors may be involved

in cellular mechanisms in the nervous system related to impaired cell production, activation,

function and eventually apoptosis and cell death.

Under normal conditions, GM-CSF is expressed in numerous cell types, including monocytes,

macrophages, fibroblasts and endothelial cells, in addition to T-cells (Fleetwood et al., 2005;

Franzen et al., 2004; Hamilton, 2002, 2008; Whetton and Dexter, 1989). In the nervous system,

GM-CSF is mostly produced and released from astrocytes, in addition to other cells, namely

endothelial cells (reviewed by Franzen et al. 2004), and pericytes (co-localized with IL-1β and

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IL-6; Fabry et al. 1993), whereby it crosses the blood-brain barrier as well as the blood-spinal

cord barrier (McLay et al., 1997). Scheme 3 illustrates a model of the cell types in body tissues

in the periphery expressing GM-CSF and its receptors in the intact peripheral and central nervous

system. CSF2RB1 is involved in peripheral mechanisms of pain (Schweizerhof et al., 2009;

Stösser et al., 2011). However, the focus of this dissertation is on CSF2RB1 in the CNS,

correlating with our findings that CSF2RB1 in the spinal cord and brain are associated with

autotomy behaviour. Therefore, detail is given foremost to mechanisms in the spinal cord.

Scheme 4 summarizes major events that occur following peripheral nerve injury, which involve

GM-CSF and CSF2RB1. Following peripheral nerve injury (PNI in the Scheme), GM-CSF is

secreted from injured nerves (Mirski et al., 2003), terminating in the spinal or trigeminal dorsal

horn, and is responsible for the excessive multiplication, hypertrophy and activation of microglia

and astroglia in the CNS (Fleetwood et al., 2005; Franzen et al., 2004; Mirski et al., 2003). GM-

CSF, IL-3 and IL-5 get secreted from hematopoietic cells that pass through the blood-brain

barrier (Schäbitz et al., 2008), and these participate in activating CSF2RB1-expressing cells in

the spinal cord, microglia, astrocytes, nociceptive terminals at their central synapses and second

and third order neurons in the dorsal horn, as well as on inhibitory interneurons. Multiple

intracellular signalling pathways in these cell types are triggered upon CSF2RB1 ligand binding.

These include: (i) the Jak/Stat pathway, (ii) the MAP kinase pathway, and (iii) the PI3 kinase

pathway (Hamilton, 2008; Kaushansky, 2006). These pathways lead to the transcription of

messengers of certain genes and protein synthesis including mediators of inflammation,

neurotrophins, ion channels, etc. In glia, these pathways release cytokines and chemokines that

contribute to membrane excitability of neurons nearby. GM-CSF is considered a critical

mediator in the development of chronic inflammation (Reddy et al., 2009), and also regulates the

composition of the glial scar after injury to the CNS (Reddy et al., 2009). Up until here we

mentioned mainly GM-CSF as the ligand of CSF2RB1, however, as mentioned above, IL-3 and -

5 also utilize this receptor. This raises the question which cell type expresses these interleukins

and under which conditions are they released. To date, neither IL-3 nor IL-5 cytokines were

shown to be released by damaged nerve cells, but they do get released from immune cells in

response to infection or injury. IL-3 stimulates the production and activation of mast cells and

basophils, which are important cell regulators of the TH2 inflammatory responses (Galli et al.,

2005). The Th2-type cytokines include interleukins 4, 5, 10 and 13 (Berger, 2000). IL-5 is

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mainly eosinophil-specific (Lopez et al., 1988) and thus linked to diseases where eosinophils are

the main cells elevated participating in pathological processes, such as some forms of asthma

(Humbert et al., 1997).

A study on cultured mouse embryonic cells and fibroblasts identified a novel TNF receptor-

associated factor 6 (TRAF6) binding domain in mediating NFκB signalling through GM-CSF

receptor (Meads et al., 2010). NFκB is activated in the mouse spinal cord following sciatic nerve

transection and is expressed in neurons on the ipsilateral side of the spinal cord 3-5 days post-

injury (Pollock et al., 2005), and in microglia and astrocytes following PNI. NFκB triggers gene

expression, including of the inflammatory mediators Il-1, Il-6, Tnfα, and monocyte chemo-

attractant protein-1 (Mcp-1), which are all implicated in pain (Qian et al., 2011; Soria-Castro et

al., 2010).

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Scheme 3: Normal expression of GM-CSF/ IL-3/IL-5 (Rohde et al., 1994), CSF2RA and

CSF2RB1 receptor complex in the intact peripheral nervous system and spinal dorsal horn. GM-

CSF and IL-3 are normally produced by T-cells, monocytes, endothelial cells, pericytes and

mostly by astrocytes in the intact CNS (Fabry et al., 1993). IL-5 is produced by eosinophils.

Under normal conditions, when the tissues are healthy, there are only very low plasma

concentrations of GM-CSF, IL-3 and IL-5. These cytokines can cross the BSCB (Schäbitz et al.,

2008) and contribute to the activation of CSF2RB1-expressing cells in the spinal cord. Main

mechanisms initiated by the receptor complex are hematopoietic cell survival (via IP-3 kinase

activation) and apoptosis suppression (Williams et al., 1990). Increased cytokine concentrations,

such as during an inflammatory response, promote both cell survival and proliferation

(Guthridge et al., 2006).

Periphery Spinal dorsal horn

GM-CSF

R

Inhibitory interneuron

R

R

T

T

T

GM-CSFATPGlut

Microglia

RT

T

T

R

Pre-synaptic nociceptive neuron

Post-synaptic cell

Schwann cell

GM-CSF/IL-3/IL-5 receptor

Receptor ligands (GM-CSF, IL-3, IL-5)

T-cell

Monocyte

R

T

IL-3IL-5

IL-3IL-5

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Scheme 4: A suggested model for CSF2RB1, CSF2RA and their ligands following peripheral

nerve injury (PNI) in the Neuroma Model. PNI (shown in red) induces production and release of

GM-CSF from injured sciatic nerve (necrotizing afferents) (Mirski et al., 2003) and fibroblasts in

the periphery (Be’eri et al. 1998; Ebadi et al. 1997; Franzen et al. 2004; Saadea et al. 1996), and

from astrocytes and endothelial cells (Endo) in the spinal dorsal horn (Franzen et al., 2004), to

promote a local inflammatory response. GM-CSF promotes myelin, axonal and neuronal debris

clearance by macrophages in the periphery and microglia in the spinal cord. GM-CSF in the

spinal cord increases microglial activation, thereby releasing neuroinflammatory mediators that

increase and membrane excitability of primary afferent terminals, projected post-synaptic

neurons in the spinal dorsal horn and local interneurons. IL-1β released from microglia/pericytes

and IL-6 released from pericytes contributes to neuronal hyperexcitablity of post-synaptic cells.

Upon the binding of GM-CSF to its receptor on nociceptive afferents, the Jak/Stat pathway is

activated and gene expression of ion channels, such as Trpv1, Scn10a, Kcnd2 and Kcnk2 (not

shown) (Stösser et al., 2011) is promoted which facilitates excitability of the post-synaptic cell.

The increase in neuronal hyperexcitability facilitates by ascending pain pathways to the

perception of pain (autotomy) in the brain. Invading monocytes (Inv Mono) also respond to

GM-CSF by binding to its receptor, which leads to autoimmune inflammation.

macrophage

Nerve injury

Debris clearance

Periphery Spinal dorsal horn

Autotomy PainInhibitory interneuron

IL-1β

GM-CSFAstrocyte

Asc

en

din

g p

ain

p

ath

way

s

Neuronal hyperexcitability

GM-CSF/IL-3/IL-5 receptor Receptor ligands (GM-CSF, IL-3, IL-5)T-cellMonocyte

R

T

GM-CSF

R

T

T

T

R

Microglia

R

R

Microglia

R

GM-CSF

Jak-Stat3

Microglial activation

Nectotizing afferentsNecrotizing interneuronsFibroblasts

T

T

T

InvMono

R

Autotimmuneinflammation

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6.2 CSF2RB1 is expressed in Ependymal cells/radial

glia/tanycytes in the CNS

The results of experiments carried out in Chapter 4 have identified the cells expressing

CSF2RB1 in the spinal cord central canal region as a type of glia cell known as ependymal cells,

or radial glia (Doetsch, 2003; Jacquet et al., 2009; Kirsch et al., 2008; Knabe et al., 2005). On

this basis, we propose that these cells are one of the cellular players in CNS pain mechanisms

that correlate spontaneous pain manifested by autotomy behaviour in some denervated A strain

mice. Our results also showed that glia cells in select brain regions, including the hippocampal

dentate gyrus, the peri-ventricular nucleus and arcuate nucleus of the hypothalamus, and some

glia cells located in the pial surface of the spinal cord and brain, and dorsal columns, also express

CSF2RB1. Many of the latter cells share the same cellular morphological appearance as those of

the spinal central canal radial glia cells, i.e., having a bipolar thin spindle shaped elongated soma,

with one short process extending to spaces containing cerebro-spinal fluid (CSF) and another

very elongated process extending to the opposite direction, ranging over long distances into grey

matter nearby in regions known to process pain input such as the spinal dorsal horn,

hippocampus and hypothalamus.

Tanycytes are characterized by the presence of elongated processes that extend deeply into the

subjacent neuropil. These processes might entrap a capillary or terminate on a neuron or an

astrocyte (Tang and Sim, 1997). Both tanycytes and ependymal cells were found to have typical

physiological characteristics of glial cells: high resting membrane potential, responsiveness to

extracellular K+, and extensive dye coupling indicative of a cellular network and gap junctions.

Tanycytes and ependymal cells function as mechanical and chemical barriers in the CNS, and

protect it from potentially hazardous materials that might be in the CSF. Ependymal cells also

contain special enzymes, one of which is monoamine oxidase, which inactivates biogenic amines

including serotonin, epinephrine, norepinephrine, and dopamine. This may be important in the

context of pain mechanisms, since these amines normally function in the CNS via inhibitory and

facilitatory descending pain pathways. Thus, an increased population of ependymal cells in the

CNS, as we detected in our A mice positive for CSF2RB1, may encourage biogenic amine

inactivation, and diminish inhibitory descending action, leading to disinhibition and more pain,

and an increased drive for autotomy. This hypothesis may apply to ependymal cells located in

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both the spinal cord and brain regions engaged in descending pain modulation, such as PAG

nucleus. Due to the morphological characteristics found in our CSF2RB1+ cells around the

central canal, we propose that these cells are ependymal cells/radial glia/tanycytes. Some

investigators suggest that these types should be universally named ependymal cells (Meletis et

al., 2008).

Results of the present study show that, while the short process of the ependymal cells line is in

the central canal lining, their very elongated process projects dorsally, or ventrally, or laterally.

The latter terminates in the neuropil around the central canal or extends into the dorsal and

ventral horns. Some of these processes make end feet on capillaries in the neuropil. Electron

microscopic analysis of the ultra-structure of the dorsal horn labelled with antibodies against

CSF2RB1 could elaborate more on the targets of these processes, because at the fluorescent light

microscopic level that we used for the present study it was impossible to follow these fine

processes further to their termination sites. Nevertheless, fine reaction product was seen

throughout the neuropil attesting to the abundance of the termination sites, literally, throughout

the grey matter, and indirectly pointing at the importance of their possible function(s) in health

and post-injury, and diseases. Although speculative, their position with “one foot in the cerebro-

spinal fluid” and one in the neuropil suggests that their function is related to passing CSF to glia

and/or neurons.

6.2.1 CSF2RB1+ ependymal cells/radial glia/tanycytes

surrounding the spinal central canal and in spinal lamina X

Previous authors have already classified the cell types located around the central canal of the

spinal cord. The technique used by investigators to catalogue types of cells in the nervous

system includes silver impregnation methods such as the Golgi stain that selectively labels only

some of the cells in neural tissues under study (but those that it does stain get labelled in their

entirety). One type of cell around the central canal, the ependymal cell, looks identical to those

we labelled in this study as CSF2RB1+ central canal cells (Scheme 5, taken from Gray 1918).

The original description associated with this cell type describes the central canal region as: “It is

filled with cerebrospinal fluid, and lined by ciliated, columnar epithelium, outside of which is an

encircling band of gelatinous substance, the substantia genlatinosa centralis. This gelatinous

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substance consists mainly of neuroglia, but contains a few nerve cells and fibres; it is traversed

by processes from the deep ends of the columnar ciliated ependymal cells which line the central

canal (Fig. 667)” (Gray, 1918).

Scheme 5: Ependymal and neuroglial cells surrounding the central canal of the spinal cord

(Gray, 1918)

The gene expression validation experiments of Csf2rb1, described in Chapter 4, further

associated this gene with autotomy levels in some denervated A mice. Spinal cord Csf2rb1

mRNA levels linearly correlated with high autotomy scores, and follow up CSF2RB1

immunolabelling experiments that we performed, further suggested that Csf2rb1 is indeed

expressed in ependymal cells of the central canal. The central canal region of the spinal cord is

localized within lamina X, a region that used to be named substantia genlatinosa centralis,

where glia and neurons are located.

Primary afferents terminating in lamina X originate from numerous peripheral sources including

among others, unmyelinated axons of the sciatic nerve (Wang et al., 1994a). Interestingly,

within lamina X there is a bundle of primary afferent axons running rostrocaudally ventral to the

central canal, known as the ventromedial afferent bundle (VMAB) (Nadelhaft and Booth, 1984).

This is a unique bundle of afferents that express neurochemicals such as substance P and CGRP,

the nerve growth factor receptors p75 and trkA, the vanilloid receptor VR1, and the GM1

ganglioside, and binding the plant lectin IB4 (Gibson et al., 1981; Michael et al., 1997; Ramer et

al., 2001; Wang et al., 1994a). The expression of these molecules within the DRG is frequently

used to divide sensory neurons into subpopulations that minimally overlap (Snider and

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McMahon, 1998). For example, in rodents small and thinly myelinated or unmyelinated lumbar

DRG neurons are subdivided into 40% expressing substance P and CGRP, and 30% expressing

IB4. The remaining 30% of DRG neurons are large myelinated axons, and can be labelled with

the B fragment of cholera toxin (CTB), which binds to the GM1 ganglioside. Thus the VMAB

contains all primary sensory afferent subtypes. Wall et al. identified a dense bundle of CGRP and

IB4 positive cells in VMAB at all spinal segments, but this bundle was the largest at L3-L5

(Wall et al., 2002), indicating that these cells may be primary afferents of the sciatic nerve. The

fact that some of these afferents express IB4 and others express CGRP characterizes them as the

two subpopulations of nociceptive neuron (Nagy and Hunt, 1982; Silverman and Kruger, 1990),

and their termination in the VMAB proposed this area as another area in the spinal cord that

receives and processes pain information from the peripheral target, such as the hindpaw. This is

important because the ependymal cells that we found to associate with autotomy in this region

may crosstalk with these nociceptors as glia do in the dorsal horn, thereby provoking the

ascending neurons in this region to transmit pain information into the brain.

Although traditionally lamina X has not been defined as a primary pain input processing area

per se, numerous reports have suggested that second and third order lamina X neurons may be

engaged in processing nociceptive inputs, harbouring molecules, including peptides such as

substance P, cholecystokinin, metenkephalin, neurophysin, oxytocin, adrenocorticotrophic

hormone, thyroid-releasing hormone, and vasoactive intestinal peptide, which were also found in

lamina X with similar concentrations to those found in laminae I and II (Honda and Lee, 1985;

Matsushita, 1998; Nicholas et al., 1999; Phelan and Newton, 2000), receptors such as for NK1,

NK2, opioids, substance P, and galanin and neurotransmitters including GABA and glycine

(Matsushita, 1998), glutamate (Phelan and Newton, 2000), acetyl choline (Phelan and Newton,

2000), and 5HT (Honda and Lee, 1985) all known to be related directly or indirectly with pain.

Axons originating in lamina X neurons ascend to higher brain centres that process pain-related

inputs (Menétrey et al., 1982; Nahin et al., 1983; Wall et al., 2002). Some neurons in this region

have a small receptive field whereas that of others is very large encompassing a whole

dermatome and a few having an even much larger field, suggesting that neurons in Rexed’s

Lamina X may have a generalized integrative function, which is still poorly understood (Honda

and Lee, 1985; Honda and Perl, 1985; Nahin et al., 1983; Ness and Gebhart, 1987; Wall et al.,

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2002). Since many of the afferents terminating in this region harbour properties that are

compatible with them being nociceptive afferents, and axons originating from this area ascend to

higher pain-related brain centres, it is possible that pre- and post-synaptic cells in lamina X are

critical for pain perception. CSF2RB1+ ependymal cells/radial glia/tanycytes may associate with

the excitability of second order neurons in Lamina X. Since ependymal cells/radial

glia/tanycytes are known to be in contact with the CSF in the central canal (LaMotte, 1987), we

speculate that their possible role in the autotomy contrast between some denervated A and all

denervated B mice may be related to this as of yet unknown function. More work is needed to

elucidate this hypothesis.

Evidence for GM-CSF and IL-3 originating from fibroblasts, endothelial cells, T-cells,

pericytes, microglia and astrocytes following peripheral nerve injury have been discussed above

in section 6.1.1. It may be that some or all of these cells release inflammatory cytokines

responsible to activate CSF2RB1 in ependymal cells/radial glia/tanycytes. Regardless of their

source, however, since our findings indicate that denervated A mice have significantly more

ependymal cells/radial glia/tanycytes extending laterally into the grey matter than denervated B

mice, but not more cells that extend dorsally or ventrally into the white matter, they may carry

different functions depending on the direction to which they extend. Several studies have

described intense proliferation of ependymal precursors during embryonic development or

induced by an external stimulus, such as spinal cord compression or physical activity (Cizkova et

al., 2009; Sevc et al., 2009, 2011), which occurs not in the dorso-ventral poles but rather in the

dorso-lateral walls (Sevc et al., 2009). This may be important in that the processes that extend

dorso-laterally, toward laminae I and II of the spinal cord may serve as ‘ladders’ for CSF2RB1+

ependymal cells/radial glia/tanycytes that differentiate following hind paw denervation into

astrocytes, neurons and oligodendrocytes, where they can contribute in the mechanisms of pain.

This model suggests the same role that radial glia have in the developing brain and spinal cord,

where they serve as “ladders” to enable other primordial cells to crawl on them on their way to

their targets in the cortex.

It is possible that during the post-operative period, binding of GM-CSF, IL-3 and IL-5 to

CSF2RB1 receptors on ependymal cells/radial glia/tanycytes that extend laterally in A mice is

part of a mechanism associated with maintaining the hypersensitive state of CNS pain pathways,

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resulting in some denervated A mice expressing considerably more neuropathic pain-like

behaviour than denervated B mice. It is suggested that mediators released from the injured

nerve, which terminates in the spinal cord, would activate more cells in the ipsi-lateral side,

compared to the contra-lateral side. We did not see a visible difference in the number of these

cells that extend laterally between the ipsi- and contralateral spinal cord. Further analyses are

needed to determine whether or not a significant difference is detected between the two sides,

which may assist in explaining the mechanism of these cells in pain processing.

6.2.2 Metabotropic glutamate receptor 1 alpha (mGluR1α) is

expressed in ependymal cells/radial glia/tanycytes of the spinal

central canal

Ependymal cells/radial glia/tanycytes of the central canal express metabotropic glutamate

receptor 1 alpha (mGluR1α), one of the 8 metabotropic glutamate receptors that play a role in

excitatory neurotransmission, neuronal plasticity and neurotoxicity in the CNS (Nakanishi et al.,

1994). If these cells were only playing a role in production and transport of CSF to and from the

central canal, why should they also express these receptors, unless they also play a role in

excitatory neurotransmission, and/or neuronal plasticity, and/or neurotoxicity? Thus, mGlur1α

may contribute to the excitatory responses evoked by stimulation of synaptic afferent inputs,

leading to long-term increases in synaptic efficacy (Baude et al., 1993). This may be relevant to

injury discharge which comprises a high frequency input (Cohn and Seltzer, 1991; Seltzer et al.,

1991b, 1991c). mGluR1α immunoreactivity was found in ependymal cells/radial glia/tanycytes

of the medulla oblongata and the spinal cord (Tang and Sim, 1997).

6.2.3 Endothelin B receptor is expressed in ependymal cells/radial

glia/tanycytes of the spinal central canal

The Endothelin B receptor (ETBR) immunostaining in the rat is very similar to the CSF2RB1

labelling that we observed throughout the spinal cord of mice, especially those in the central

canal region and in the white matter (Peters et al., 2003). This receptor, which localized in

ependymal cells of naïve rats (Peters et al., 2003), is involved in mediating reactive gliosis of

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ependymal cells 1-2 weeks after brain injury in the rat (Koyama et al., 1999). This study

suggests that CSF2RB1 in this region of the spinal cord may be involved in the activation of

ependymal cells/radial glia/tanycytes after peripheral nerve injury. More importantly, ETBR has

been implicated in neuropathic pain models. ETBR is elevated in rat spinal nerves and

contributes to cold allodynia after SNL (Werner et al., 2010). Additionally, ETBRs contribute to

orofacial mechanical allodynia induced by unilateral constriction of the infraorbital nerve in rats

(Chichorro et al., 2006). Although ETBRs are thought to play a role in elevating pain levels by

operating on spinal neurons, as suggested from the above studies, it may also have a role in

increasing neuropathic pain indirectly via increased expression in central canal ependymal

cells/radial glia/tanycytes that also express CSF2RB1.

6.3 Co-localization of CSF2RB1 and the neural stem cell marker

Vimentin in the spinal cord

We proposed that CSF2RB1 is expressed by ependymal cells/radial glia/tanycytes in the

central canal region of the spinal cord based on their unique morphology as well as co-

localization of the protein marker Vimentin, which is known to be expressed by these

cells (Bodega et al., 1994; Prieto et al., 2000). However, Vimentin is also expressed by

immature astrocytes and invading fibroblastoid cells, a component of the developing scar

tissue (Conrad et al., 2005; Schwab et al., 2005), macrophages (Kim et al., 2003; Shin et

al., 2003), invading meningeal cells (Wang et al., 1997) and cells surrounding blood

vessels (Farooque et al., 1995). Indeed we co-localized CSF2RB1 and Vimentin in cells

lining blood vessel walls in the spinal cord. But the only cell type in the central canal

that looks like those expressing CSF2RB1 are ependymal cells/radial glia/tanycytes.

These cells were present in intact mice, hence they cannot be considered

“reactive/responsive” or “invading” at the constitutive stage. Thus, CSF2RB1 expressing

cells are stem cells reservoir of the spinal cord, aside from astrocytes and

oligodendrocytes (Barnabé-Heider et al., 2010). In response to spinal cord injury,

ependymal cells are known to proliferate dramatically, producing oligodendrocytes, and

more abundantly astrocytes, that migrate to the site of injury and make a substantial part

of the glial scar (Barnabé-Heider et al., 2010; Meletis et al., 2008). It has been

documented long ago and is now well established that peripheral nerve injury causes

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degeneration of central terminals of primary afferents in the dorsal horn of the spinal cord

(Arvidsson et al., 1986). This debris may release chemicals that trigger ependymal cells

to multiply and become astrocytes. Two weeks after spinal cord injury astrocytes

constitute most of the newly formed cells followed by ependymal cells and

oligodendrocyte progenitors (Barnabé-Heider et al., 2010). By 4 months after the injury,

there is a reduction in the number of progeny of all three cell types, and at this stage the

largest number of new glial cells derived from ependymal cells followed by astrocytes

and oligodendrocytes (Barnabé-Heider et al., 2010). Of special note, ependymal cells

may also differentiate into neurons. In the EAE model of multiple sclerosis, ependymal

cells of the spinal cord seem to be restricted to their own lineages and do not proliferate

into reactive astrocytes that express GFAP (Guo et al., 2011). So it seems that the signal

for proliferation of these progenitor ependymal cells depends on the type of pathology.

We are the first to suggest the hypothesis that peripheral nerve injury is yet another such

signal that triggers the proliferation of these cells. If established by future experiments,

ependymal cells may be seen as having a role of providing the spinal cord with glia cells,

that in addition to clearing up the debris of degenerating afferents and second order

neurons in the spinal cord following peripheral nerve injury, may be associated with

sensitization of ascending pain pathways, thereby facilitating the process of development

of neuropathic pain. Additionally, by way of neo-neurogenesis, ependymal cells may

replenish the dorsal horn with the partially depleted pain suppressing interneurons that

die out by excitotoxicity and apoptosis in response to the nerve injury (see Introduction in

Chapter 1) (Dubner and Ruda, 1992; Suzuki and Dickenson, 2000; Woolf and Salter,

2000; Zimmermann, 2001).

Interestingly, Vimentin induces microglial activation in murine primary cultures and

enhances the expression of LPS-induced inflammatory genes such as Tnfα and iNos in

these cells (Jiang et al., 2012). These studies suggest that our Vimentin+/CSF2RB1+

cells in the central canal region may be associated with microglial activation and

subsequent TLR4 signalling, particularly following hind paw denervation. We showed

that these ependymal cells/radial glia/tanycytes multiplied significantly following the

denervation in both A and B mice. Indeed, CSF2RB1 (via GM-CSF) has been shown to

increase TLR4 and microglial activation ex vivo (Parajuli et al., 2012). Additionally, the

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impairment of microglial activation in Vim-/-

mice was associated with reduced neuronal

toxicity in ischemic brains (Jiang et al., 2012). These data may suggest that our

Vimentin+/CSF2RB1+ cells detected in the central canal may also be involved with

neuronal excitotoxicity, a trigger for central sensitization and maintained pain. As there

are no indications from the literature that ependymal cells/radial glia/tanycytes play a

direct role in neuroplasticity, another interesting finding is that TLRs 2 and 7, rather than

TLR4, were found to be over expressed in ependymal cells of murine cerebellar white

matter after parasite infection (Mishra et al., 2006). This indicates a possible role of

ependymal cells/radial glia/tanycytes in inflammatory processes mediated by TLR

signalling.

6.4 The hippocampus in processing nociceptive input

A substantial body of evidence indicates that (i) the hippocampus processes pain-related inputs,

(ii) some hippocampal neurons respond exclusively to noxious stimuli, and (iii) long-term

anatomical changes occur in neurons of the hippocampal dentate gyrus following noxious

stimulation (reviewed in Liu & Chen 2009). NMDA receptor antagonist drugs administered to

the hippocampus interfered with long-term potentiation, learning, and memory in hippocampus

neurons. When applied to the spinal cord these drugs prevented similar long-term

neurophysiological changes elicited by noxious stimulation. McKenna and Melzack showed that

blocking NMDA receptors in the dentate gyrus reduced nociceptive behaviour in an animal

model of sub acute inflammatory pain, the Formalin Pain Model (McKenna and Melzack, 2001).

The experience of pain is currently perceived as the result of complex processing of nociceptive

inputs by the nervous system using three complementary approaches, including sensory-

discriminative, affective-motivational and cognitive-evaluative aspects (Melzack and Casey,

1968; Price, 1999). Thus, the experience of pain results from processing inputs in a neuro-matrix

of widely distributed and hierarchically interconnected neural networks in the brain of which the

hippocampus plays an important role (Bushnell, 2006; Melzack, 2005, 2008). The hippocampus

has been traditionally known for its role in learning and memory, affect and motivation. Thus,

involvement of the hippocampal formation in pain is connected to aspects of learning avoidance

behaviour, anchoring past painful experiences in memory, and labelling a painful event as having

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a negative valence. Like in other senses, the experience of pain is compared against stored past

pain experiences in an attempt to seek the most adaptive response and the best behavioural

approach to cope with the stimulus. But in recent years cumulative efforts from many

laboratories have allowed a clearer dissection of the roles of the hippocampal formation in pain

processing, integrating behavioural, electrophysiological, molecular/biochemical, structural and

functional brain imaging evidence to support the role played by the hippocampal formation in the

affective/motivational aspects of pain perception (Al Amin et al., 2004; Echeverry et al., 2004;

Favaroni Mendes and Menescal-de-Oliveira, 2008; Khanna et al., 2004; Lathe, 2001; McKenna

and Melzack, 1992, 2001; Soleimannejad et al., 2006, 2007; Yamamotová et al., 2007; Zhao et

al., 2009). Melzack and Casey proposed that the limbic forebrain structures, including the

hippocampal formation, play important roles in the ‘aversive drive and affect that comprise the

motivational dimension of pain’ (Melzack and Casey, 1968). Based on our findings, we

speculate that compared to denervated mice that do not express autotomy, the increased

expression of CSF2RB1+ in the dentate gyrus of denervated A mice that express high-autotomy

suggests that these mice may experience an increased negative valence to the nociceptive inputs

arriving to the brain from neuromas, DRGs and sensitized pain pathways in the CNS, which

increases their pain experience possibly by also increasing the fear of pain that is produced by

their inability to stop and/or avoid the pain. Compared to NLS mice, the enhanced pain

experience in denervated MHS-A mice may additionally be associated with a higher volume and

frequency of ectopic activity in injured primary afferents from neuromas, DRGs and due to

increased input volume arriving to the brain via sensitized pain pathways in the CNS.

In addition, it has been shown previously in intact mice that decreased levels of hippocampal

CSF2RA and GM-CSF is associated with a reduce capacity for memory formation (Krieger et

al., 2012). Thus, high autotomy in some denervated A mice may be related to their enhanced

capacity to memorize the negative valence associated with the pain input. It has also been shown

that the number of GM-CSF immunoreactive neurons expressed in the dentate gyrus are reduced

in patients with Alzheimer’s disease who suffer from increasing memory deficits (Ridwan et al.,

2012), suggesting a role of GM-CSF signalling in memory. Thus, it may also be that those A

mice who prior to the injury had more CSF2RB1+ cells compared to other A mice, lead them to

develop a stronger impact of the pain enormity in their memory and that this is part of the

emotive drive that causes them to label the pain as more hurtful. Further research is needed to

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explain the differences in A mice that express autotomy vs. A mice that do not express autotomy

following hindpaw denervation, trying to tag them ahead of the denervation in some way that

would predict who is at risk for expressing high levels of autotomy after nerve injury. For

example, testing levels of pain avoidance in these mice when they are still intact, before their

denervation, may indicate whether some of these mice have a negative valence to the pain

experience. Such studies could then be followed by trials of pre-emptive analgesic approaches in

mice found at high risk, also in an attempt to suppress the negative valence in these mice,

followed by hindpaw denervation.

6.4.1 Processing chronic pain inputs in the hippocampus

We have shown that CSF2RB1+ cells in the granule layer of the hippocampal dentate gyrus are

correlated with levels of autotomy behaviour in some denervated A mice. Previous studies also

suggested that the hippocampus is associated with neuropathic pain and that abnormal

hippocampal function may contribute to the sensation of chronic pain or even be causative in

producing such a sensation (for a review see Liu & Chen, 2009). Rats that had a nerve injury

and expressed neuropathic pain-like behaviour showed a bilateral hippocampal increase in the

transcription factor NFκB and this effect was mediated by activation of NMDA receptors (Chou

et al., 2011). NFκB normally facilitates the transcription of numerous inflammatory cytokines

that play a role in neuropathic pain, including Il-1β, Il-6, Tnfα and Mcp-1 (Qian et al., 2011;

Soria-Castro et al., 2010). Rats that prolonged nerve injury in the CCI and SNI neuropathic pain

models had increased levels of hippocampal cytokines and neurotrophic factors such as IL-1β,

IL-6, NGF and GDNF (Al-Amin et al., 2011), enhanced regulation of hippocampal gene

expression of cytokines and the neurotrophin Bdnf (Duric and McCarson, 2005; Hu et al., 2010;

Uçeyler et al., 2008), and impaired neoneurogenesis in the hippocampus (Duric and McCarson,

2006; Terada et al., 2008). Del Rey et al. showed that sustaining a nerve injury in the SNI and

CCI models caused an up-regulation of hippocampal IL-1β, which was closely correlated with

the level of mechanical allodynia in both Sprague Dawley and Wistar Kyoto rat strains (del Rey

et al., 2011). In vivo and in vitro LTP induction in the hippocampus resulted in a long lasting

increase of IL-1β and IL-6 expression (Balschun et al., 2004; Schneider et al., 1998).

Furthermore, blockage of IL-1 signalling impaired the maintenance of LTP (Schneider et al.,

1998), while blockage of endogenous IL-6 prolonged it (Balschun et al., 2004). It is noteworthy

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that decreased levels of hippocampal GM-CSF did not alter stimulus-evoked pain behaviour in

control mice, as observed by paw withdrawal latencies in the Hargreaves test (Krieger et al.,

2012), indicating that signalling via GM-CSF and its receptors may be modified following nerve

injury, thereby taking part in neuropathic pain behaviour. Since CSF2RB1 promotes NFκB

activation (Meads et al., 2010), we propose the hypothesis that neuropathic pain as induced in the

Neuroma Model may be related to the activation of the CSF2RB1/NFκB signalling pathway in

the hippocampal dentate gyrus. NFκB activation in CSF2RB1+ dentate cells may lead to the

transcription and release of inflammatory mediators, such as Il-1β, and Tnfα, associated with

pain in the dentate gyrus of A mice. Future functional experiments with CSF2RB1are needed to

attest these hypotheses.

6.5 Which cell types express CSF2RB1 in the hippocampus?

Unlike the spinal cord, where CSF2RB1 was co-expressed with Vimentin in cells of the central

canal, we were unable to co-localize this receptor with Vimentin+ glia cells in the dentate gyrus,

nor with neurons or other glia. Only a few Vimentin+ cells at the boundaries of polymorph and

granule dentate layers were co-localized with CSF2RB1. Since the hippocampus is one of the

brain sites for neoneurogenesis, yet as Vimentin did not labels most of the CSF2RB1 cells, it is

possible that in the hippocampus CSF2RB1 cells express Nestin, another neural stem/progenitor-

associated marker, rather than Vimentin (Busch et al., 2010; Mothe and Tator, 2005; White et al.,

2010) (see below). As we reported in Chapter 4, CSF2RB1 cells in denervated MHS-A mice

multiplied in numbers, attesting to their capacity for glioneogenesis. While not studied in detail,

we did not detect a strong increase in the numbers of NeuN, GFAP and OX42 labelled cells, this

may point at the fact that the increase in CSF2RB1 cells was not accompanied by a similar

increase in the number of neurons, astrocytes or microglia in the dentate gyrus. Maybe lacking

Vimentin signifies that their capacity to multiply is only limited to becoming their kind (i.e.,

CSF2RB1 cells). Thus, maybe brain CSF2RB1 cells do not function as a reservoir for the de

novo production of neurons and other types of glia but only have a selective glioneogenetic

capacity to multiply specifically CSF2RB1 cells. This should be tested in future research.

In vivo intra-peritoneal administration of 200 ng GM-CSF to BALB/c, C57BL, and C3H/HeJ

promotes increased production of granulocytes and macrophages (Metcalf et al., 1987), and

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CSF2RB1 is involved in proliferation and differentiation of granulocyte-macrophage progenitors

(Brown et al., 2012). Similar mechanisms may be involved in the CNS with the function of

CSF2RB1 in differentiation of neural stem cells into other cell types. In embryonic and adult

brain, radial glial and some astroglia are neural stem cell-like and generate neurons and glial

cells (Doetsch, 2003). Once they differentiate into a new cell type, such as astrocytes, CSF2RB1

expression may activate the new cells and initiate the process of inflammation. We suggest that

neurons, astrocytes and ependymal cells in the dentate gyrus of autotomizing A mice may

differentiate from CSF2RB1+ neural stem cells, and once differentiated may contribute to the

role of pain in these mice.

CSF2RB1 operates in tandem with another subunit of the ‘common receptor’ – CSF2RA.

Indeed, other investigators showed that CSF2RA1 is expressed in the sub granular zone of the

dentate gyrus and in cells, which send elongated processes into the granular cell layer that

resemble migrating neural stem cells (Krüger et al., 2007). Moreover, hippocampal adult neural

stem cells in culture that are CSF2RA+ co-localized with Nestin; the Csf2ra gene was expressed

in neural stem cells from the hippocampus and the sub ventricular zone (Krüger et al., 2007). In

another study, the dendritic layer in the hippocampus showed weak to moderate CSF2RA

staining in neurons (Ridwan et al., 2012). Unlike healthy humans, Alzheimer patients’ granule

cells of the dentate gyrus were only slightly labelled with CSF2RA and GM-CSF (Ridwan et al.,

2012). In the mouse, in situ hybridization to Csf2rb2 showed a robust labelling in many neurons

in the granule layer. We did not localize CSF2RB1 in neurons, since none of the neurons

labelled by NeuN showed co-localization of CSF2RB1 (Dahlberg et al., 2003). This would

suggest that the GM-CSF receptors do not co-localize to the same cells, each relaying a specific

effect to its own cell type, glial (in the case of CSF2RB1) and neuronal (in the case of CSF2RA).

Interestingly GM-CSF and CSF2RA were also very faintly detectable in astrocytes, ependymal

cells and cells of the choroid plexus of all brain regions of Alzheimer patients (Ridwan et al.,

2012).

Although we have not localized CSF2RB1 to microglia in areas of the CNS, namely spinal cord

and brain, previous reports have demonstrated the expression of CSF2RA in brain-derived

microglia (Parajuli et al., 2012). According to Parajuli et al. (2012), both Csf2ra and Csf2rb1

gene transcripts are strongly expressed in brain-derived microglia, however their expression is

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much weaker in astrocytes and neurons isolated from brain. Parajuli et al. located CSF2RA, but

not CSF2RB1, to microglia by immunohistochemical staining, but not to isolated astrocytes or

cortical neurons. The fact that Parajuli et al. could label CSF2RA in microglia, but neither they

nor we could label CSF2RB1 in these cells, may suggest that the receptor complex CSF2RA/B1

is expressed on microglia, but suggests that the binding properties of immunolabelled antibodies

to the receptor β subunit may be disrupted when it is bound to the cell surface in microglia.

The granulocyte colony-stimulating factor (G-CSF), another hematopoietic growth factor with a

high analogy to GM-CSF, promotes the differentiation of neural precursors in adult brain and is

a part of the regeneration of the brain after insults (Kirsch et al., 2008). In adult Wistar rats, the

receptor for G-CSF is expressed in many brain areas, as well as in the walls of blood vessels,

muscles and their respective precursors, and neurons (Kirsch et al., 2008). The expression of the

G-CSF receptor in the developing CNS was most prominent in ependymal cells/radial

glia/tanycytes, co-expressed with the stem cell marker Nestin (Kirsch et al., 2008). The results

of Kirsh et al. may be relevant to us if CSF2RB1+ cells in the brain (that failed to co-localize

with Vimentin) would in fact be found to co-localize with Nestin.

Taken together, these results show that CSF2RB1 is present in the dentate gyrus, and is

correlated with denervation, as seen in the increased number of cells in the polymorph layer, and

with autotomy behaviour, as seen in the increased number of processes in the granule layer.

6.6 The involvement of CSF2RB1 in neuroprotection

6.6.1 Cytoprotection involves CSF2RB1 operating in a complex

with the erythropoietin receptor (EpoR)

While our findings associate autotomy levels with Csf2rb1 gene expression levels in the spinal

cord and the presence of CSF2RB1 protein in ependymal cells/radial glia/tanycytes of the central

canal, other glia in the dorsal columns and white matter of the spinal cord, glia in the

hippocampal dentate gyrus and hypothalamic peri-ventricular and arcuate nuclei, the mechanism

by which these cells produce neuropathic pain remains unknown.

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It is possible that CSF2RB1 may have pleiotropic effects in various parts of the nervous system,

not all of which have a role in chronic pain or if having roles in chronic pain – not necessarily

having the same role or even the same direction. It is not impossible to have a gene product

playing an antinociceptive role in one location of the pain pathway and the opposite direction in

another location. For example, it has been reported that CSF2RB1 is involved in a cytoprotective

mechanism by interacting with erythropoietin (Epo). Epo binds to a heterodimeric receptor

consisting of the Epo receptor (EpoR) and CSF2RB1 (i.e., IL-3Rβ) (Blake et al., 2002; Jubinsky

et al., 1997; Leist et al., 2004).

EpoR and IL-3Rβ also co-localize in neurons of the spinal cord where they play a

neuroprotective role in the mouse (Brines et al., 2004). Numerous other studies have shown the

localization of EpoR (i.e., without IL-3Rβ) in spinal cord neurons (Yoo et al., 2009), in

specifically GABAergic neurons (Won et al., 2007), and in neural progenitor cells ( Wang et al.

2010), as well as oligodendrocyte progenitor cells (Cho et al., 2012), and radial glia cells that

transform into astrocytes (Knabe et al., 2005). In these studies EpoR has an activating effect on

cell differentiation and growth both in neonates and in adults recovering from nerve injury. In a

rat model of spinal cord injury IL-3Rβ expression (regardless of the presence of EpoR) is

increased in neurons near the injury site compared with uninjured animals (King et al., 2007).

While an injured spinal cord is not like a spinal cord responding to peripheral nerve injury, there

are some similarities, because after peripheral nerve injury primary afferents and second order

neurons degenerate in the spinal cord, which triggers gliosis to clear the debris. While in our

experiments CSF2RB1 was not expressed in neurons, it is possible that ependymal glia and the

other glia that expressed CSF2RB1 also expressed EpoR. In this case, such glia could function

in cytoprotection. However, having more CSF2RB1 was associated in out experiments with

increased autotomy, a behaviour unrelated to cytoprotection but to the opposite effect - of

excitotoxicity and apoptosis in the spinal cord after nerve injury that results in disinhibition of

pain pathways. Thus, we do not believe that cytoprotection is a mechanism related to the effect

of CSF2RB1 in our phenotype. The spinal cord central canal CSF2RB1+ ependymal cells we

co-labelled with Vimentin increased in number after the nerve injury. They may also express

EpoR, and these two receptors may act in these cells together to promote cell proliferation and

differentiation.

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6.6.2 The neuroprotective cytokine erythropoietin is a ligand for

CSF2RB1

The cytokine Epo exerts generalized protective and trophic properties that have been

demonstrated in various tissues, including neural tissue (Bernaudin et al., 1999; Brines et al.,

2000; Grasso et al., 2009; King et al., 2007; Liao et al., 2008). When peripherally administered,

Epo can cross the blood-brain barrier, and stimulates neoneurogenesis, neuronal differentiation,

and activates brain neurotrophic, anti-apoptotic, anti-oxidant and anti-inflammatory signalling.

These mechanisms trigger tissue protective effects in disorders of the nervous system. Epo is

neuroprotective in several disorders, including stroke (for review see Hasselblatt et al. 2006),

peripheral nerve injury (for review see Höke & Keswani 2005), contusion or compression of the

spinal cord (Cetin et al., 2006; Gorio et al., 2002; Grasso et al., 2006; Leist et al., 2004). In some

occasions, Epo binds to the EpoR- CSF2RB1 receptor complex (EpoR- CSF2RB1) and activates

it. This complex receptor is up-regulated after tissue injury (Brines and Cerami, 2008; Brines et

al., 2004). Swartjes et al. showed that an Epo catabolite produces long-term relief of allodynia in

rats and mice following a SNI injury by activation of CSF2RB1 (Swartjes et al., 2011).

Knockout mice that lacked a functional CSF2RB1 did not recover from allodynia (Swartjes et

al., 2011). These results are not compatible with our findings in that CSF2RB1 plays in these

cited papers a protective role against neuropathic pain. We demonstrated that expression levels

of the Csf2rb1 gene and its expressed protein are correlated positively with neuropathic pain

levels, since high autotomy mice showed over expression of this gene compared to groups of

mice that did not express the behaviour. As argued in section 6.6.1, CSF2RB1 expression may

be tissue- and cell-specific, and depending on its site of expression it may undertake different

signalling pathways, leading to different cellular functions, such as neuroprotection or

neurodegeneration and pain.

6.7 CSF2RB1 operating with Receptor d’origine nantais (RON)

CSF2RB1 in the CNS can sometimes signal via the receptor d’origine nantais (RON) and its

signalling components including its ligand macrophage stimulating protein 1 (MSP). The

MSP/RON pathway is involved in several important biological processes, including

macrophage/microglial activity and morphological changes, neurite extension, cellular migration

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and wound healing (Funakoshi and Nakamura, 2001; Suzuki et al., 2008; Tsutsui et al., 2005;

Wang et al., 1996, 2002b). RON (in human) and its murine homologue Stem-cell derived

Tyrosine Kinase (STK) is a receptor with an extracellular α-chain, that binds MSP, and a

transmembrane β-chain with intrinsic tyrosine kinase activity (Wang et al., 1994b, 1995) that

initiates intracellular signalling via other proteins and receptors, such as CSF2RB1. When RON

signals via CSF2RB1, it promotes cellular ‘morphological changes’ (Mera et al., 1999). When

CSF2RB1 signals independently (through its own ligands), it promotes pathways of

’morphological changes’ and ‘cellular growth’(Mera et al., 1999). Since CSF2RB1 is involved

in numerous biological processes, such as autoimmunity and, inflammation normal and

malignant hematopoiesis, and autoimmunity (reviewed by Hercus et al. 2013), it is possible that

some of the pathways that lead to these processes depend on RON. RON, like CSF2RB1, is

expressed in various tissue and cell types, including peripheral macrophages, DRG neurons and

glial cells (Suzuki et al., 2008). There is currently no information about whether RON is

expressed on ependymal cells. It is possible that cross-talk between RON and CSF2RB1

mediates processes of inflammation in these cells, which could ultimately lead to pain.

A few studies dedicated to MSP and RON in the CNS are available, and demonstrate the

significance of their signalling in tissues such as the spinal cord and brain. Studies showed that

both in the presence and in the absence of RON, microglial activation is enhanced, suggesting

that CSF2RB1 may be involved via cross talk with RON and independent of RON in promoting

microglial activation . In the presence of RON, microglial activation and migration are enhanced

(Suzuki et al., 2008). MSP acting on brained-derived murine microglia, induces inflammatory

cytokine production of IL-6, GM-CSF, iNOS, and to a much lesser extent IL-1β and TNFα

(Suzuki et al., 2008). In the absence of RON, microglial activation in the spinal cord is

enhanced, and the pro-inflammatory cytokines IL-1β and TNFα are up-regulated (Tsutsui et al.,

2005). Since CSF2RB1 (via GM-CSF) increases TLR4 and microglial activation ex vivo

(Parajuli et al., 2012), these studies suggest that microglial activation may be mediated by

CSF2RB1 in the spinal cord in the Neuroma model used in the present study.

In addition to the mechanisms described above which involve CSF2RB1 and TLR4 microglial

activation is that of proliferation induced by GM-CSF in microglia (Suzumura et al., 1996) . In

RON deficient KO mice, after EAE induction, there is deterioration in demyelination, axonal

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injury and neuroinflammation, suggesting that RON signalling via CSF2RB1 is important in

these processes.

6.8 pStat3 in the spinal cord

PStat3 has been correlated with neuropathic pain, demonstrated by its expression in spinal cord

glia and certain cell types in the PNS of neuropathic pain animal models (Dominguez et al.,

2008, 2010; Dubový et al., 2010; Kohli et al., 2010; Maeda et al., 2009; Stösser et al., 2011;

Tang et al., 2012). PStat3 in astrocytes and/or microglia in the spinal dorsal horn of rodents in

neuropathic pain models enhanced the expression of Il-6, Il-1β, Tnfα, Nmdar, Ccl2 and Atf3.

Our results show that pStat3 did not co-localize with GFAP, the marker of astrocytes, even

though previous studies have shown that SNL and SNI in rats, and SCI in mice induces pStat3 in

astrocytes of the spinal cord (Herrmann et al., 2008; Tang et al., 2012; Tsuda et al., 2011). This

discrepancy with our results could be with the nature of the species and mouse model used.

Whereas SNL in the rat induces pStat3 in astrocytes and microglia (Dominguez et al., 2008;

Tang et al., 2012), the study by Dominguez et al. (2008) failed to show the expression of pStat3

in astrocytes, indicating discrepancies within the same species and model exist. In another study

by Dominguez et al. (2010), CCI in the rat induced pStat3 in microglia, but once again, failed to

show the expression of this molecule in astrocytes.

It is noteworthy that intrathecal LPS produces mechanical allodynia in the rat by activating Stat3

in spinal astrocytes, and blockade of spinal pStat3 can attenuate mechanical allodynia,

correlating with decreased astrocyte activation in the dorsal horn (Liu et al., 2013). LPS elevated

the chemokines Cxcl3cl1, Cxcl10, Cxcl5 and Ccl20 in the spinal dorsal horn, and pStat3

inhibited the levels of these chemokines, with decreased pain behaviour (Liu et al., 2013). Since

LPS is induced by TLR4, we propose that this mechanism is regulated also via TLR4,

presumable in astrocytes.

Based on previous reports we illustrate a model of CSF2RB1/pStat3 signalling in cells in the

PNS and CNS, where these proteins play role in neuropathic pain states (Scheme 6). PNI

induces the production and binding of cytokines to CSF2RB1 and initiate intracellular signalling

via Jak2, Src and PI3K, the kinases that phosphorylate and activate CSF2RB1. Jak2

phosphorylates Stat3 and pStat3 is translocated to the nucleus and initiates gene transcription. At

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the same time, Src phosphorylates and activates MAPK and ERK1/2. Downstream target

kinases of MAPK, JNK and p38 get activated upon phosphorylation by MAPK. All these

kinases contribute to the phosphorylation and activation of pStat3 in the nucleus. Once activated

and bound to the DNA, pStat3 induces the transcription of pain-associated genes. These genes

are cell-specific and include: Il-6, Ccl2, Il-1β, Tnfα, Nmdar, Atf3 (expressed in glia and

macrophages), Mmp9, Cox-2, iNos (expressed in glia, macrophages and dorsal horn neurons) and

Trpv1 Scn10a Kcnd2 Kcnk2 (expressed in DRG neurons). Independently of pStat3, pERK1/2

promotes the gene transcription of c-Fos and c-Jun. An alternative pathway to pStat3 on

CSF2RB1 expressing cells is initiated by PI3K and promotes gene transcription of pro-

inflammatory genes via NFκB.

PNI

CSF2R

Astrocyte & Microglial ProliferationG

liaM

acro

ph

ages

Glia

Mac

rop

hag

esD

ors

al h

orn

neu

ron

sD

RG

neu

ron

s

Cell GrowthSurvival

Differentiation

MAPK

Il-6Ccl2Il-1βTnf-αNmdarAtf3

Mmp9Cox-2iNos

Trpv1 Scn10a Kcnd2 Kcnk2

Neuropathic pain

Gene Expression

α α

ββ

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Scheme 6: Adapted from (Qiagen): CSF2RB1 intracellular signalling through Jak/Stat3, PI3K

and Src. As shown, upon binding of cytokines (GM-CSF, IL-3 or IL-5) to the 2α receptor

subunits on the extracellular cell surface, there are multiple pathways that lead from the cell

membrane to the nucleus via the activation of the receptor β1 subunit by Jak2, PI3K, or Src/Ras,

that continue through the protoplasm to affect cell survival (and astro- microglial proliferation)

by pStat3, or continue to the nucleus where transcription factors such as Stat3, ERK1/2 and

NFκB activate changes in gene expression. Pain-associated genes include pro-inflammatory

mediators, receptors and transcription factors (expressed in glia, macrophages and dorsal horn

neurons) and ion channels (expressed in DRG neurons), and bring about neuropathic pain by

increasing the membrane hyper excitability of neurons in the PNS and spinal cord.

6.10 pStat3 in the peripheral nervous system (PNS) contributing

to neuronal hyperexcitablility may be associated with CSF2RB1 in

A mice

Previous studies have shown the involvement of Stat3 signalling with or without CSF2RB1

activation in the PNS, and associated it with chronic neuropathic pain. Stat3 is over-expressed in

ganglionic satellite glial cells in the rat following sciatic CCI injury, and is activated by IL-6

(over-expressed in both L4-5 DRGs and satellite glial cells) binding to its receptor in these cells.

pStat activation in satellite glial cells was accompanying the ensuing allodynia and hyperalgesia

(Dubový et al., 2010). In another model, the partial sciatic nerve ligation (PSL), pStat3 is

maximally expressed in macrophages of the injured nerve at 3 hours PO, and continues to be

expressed in these cells up to day 7 PO, but is absent in sham-operated mice (Maeda et al.,

2009). Since injury in the PSL model produces neuropathic pain for 7 weeks or longer (Seltzer

et al., 1990), it is suggestive that pStat3 in the PNS is involved in the initiation of pain, rather

than its maintenance. pStat3 activity in peripheral macrophages releases inflammatory mediators

(Maeda et al., 2009) that contribute to membrane hyper-excitability of nociceptive neurons.

Additionally, pStat3 activity in nociceptive DRG neurons, mediated by CSF2RB1, promotes

membrane hyperexcitability by initiating gene transcription of ion channels and trans locating

them to the cell membrane (Stösser et al., 2011). These reports hint at the possibility of

CSF2RB1/pStat3 playing roles in initiating autotomy in A mice, and not in B mice. This is

consistent with our findings in DRG neurons where we showed a difference in constitutive levels

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of Csf2rb1 gene in intact A mice vs. B mice, and proposed then that these transcript differences

at the time of nerve injury may be associated with injury discharge propagation into the spinal

dorsal horn in the first 24 hours or so after the denervation. Thus, depending on the cell type and

the location in the nervous system (i.e., in the periphery or the spinal cord), mechanism involving

pStat3 may carry different roles initiating or maintaining chronic neuropathic pain.

6.11 The pain-associated genes induced by pStat3 in the PNS

and CNS

In some cases, the same pain genes elicited by Jak/Stat3 following PNI, are expressed in both

peripheral and central cell types. For instance, PSL injury induces the expression of genes

promoting pain such as Mmp-9, Cox-2 and iNos, via pStat3 in macrophages of the injured sciatic

nerve (Maeda et al., 2009). However, these genes are also regulated in the spinal cord after

peripheral nerve insults. MMP-9 is constitutively expressed in spinal neurons and microglial

cells, and is immediately up-regulated after PSL injury, returning to baseline levels at day 3

(Liou et al., 2013). COX-2, while not present in dorsal root ganglion cells, exists in spinal cord

neurons, including those in the superficial dorsal horn (Willingale et al., 1997).

Immunoreactivity and mRNA levels of iNOS in dorsal horn neurons of the spinal segments L4–

L5 were up regulated in CCI animals 14 days after the surgery (Martucci et al., 2008). The

above indications from the literature suggest that pStat3 in our MHS-A mice may induce the

expression of Mmp-9, Cox-2 and iNos genes in spinal post-synaptic neurons through the

activation of immediate early genes c-Fos and c-Jun. Alternatively, pStat3 in autotomizing mice

may activate the transcription of Mmp-9, Cox-2 and iNos genes in microglial cells, already

discussed above.

6.12 pStat3 is expressed in the sub ventricular zone of the brain

but not around the spinal central canal

It is noteworthy that CSF2RB1+ radial glia surrounding the central canal did not express pStat3

constitutively and did not start expressing this protein post-operatively in denervated mice,

indicating that these cells carry mechanisms independent of Jak/Stat3. It is more likely that in

these radial glia cells, CSF2RB1 activates the Jak/Stat5 pathway initiating cell proliferation.

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Studies on hematopoietic cells show that a mechanism involving IL3 and its receptor, IL3R (also

known as CSF2RB1) initiates proliferation and promotes cell survival through the Jak/Stat5

pathways (reviewed in Reddy et al. 2000). Alternatively, it may also be that CSF2RB1 in these

glia initiate another pathway, such as PI3K/NFκB or the Ras/Raf ERK pathway (Hamilton, 2008;

Kaushansky, 2006; Schweizerhof et al., 2009; Zhuang et al., 2005), which result in activation of

transcription factor(s) that increase gene expression of multiple pain genes, like Il-1β, Il-6 and

Tnfα (via the former pathway), or c-Fos and c-Jun (in the latter pathway), to name a few

(Scheme 6). C-Fos expression is increased 4-fold in the central canal of mice in response to

formalin-induced acute pain (Palkovits et al., 2007). This finding supports the idea that post-

synaptic cells in lamina X have similar characteristics as the post-synaptic cells in the dorsal

horn. Further studies necessitate determining whether c-Fos and c-Jun play role in neuropathic

pain conditions after peripheral nerve injury in lamina X neurons, and whether CSF2RB1 plays

role in this mechanism.

pStat3 and CSF2RB1 both showed intense immunolabelling in the arcuate nucleus of the

hypothalamus, the peri-ventricular nucleus and the third ventricle. The areas of the ventricles

also include the choroid plexus which is made up of ependymal and epithelial cells and functions

as a blood-cerebrospinal fluid barrier. If pStat3 in also expressed in ependymal cells, then

CSF2RB1 may function with this transcription factor in ependymal cells surrounding the

ventricles. We have not yet carried out experiments of co-localization with pStat and Vimentin.

As in the central canal region, c-Fos is up regulated in murine brain ventricles in a model of

acute pain. A single formalin injection into the hind paw elicits strong c-Fos mRNA and Fos

protein expression in pain-related brain areas, in the circumventricular organs, in the choroid

plexus and in tanycytes and ependymal cells of the third ventricle (Palkovits et al., 2007). C-Fos

mRNA was also seen throughout the damaged neocortex in neurons and in non-neural brain cells

(e.g., glia, pia, ependymal) within a short time after induction of brain ischemia or trauma in the

human, rat and mouse (Wessel et al., 1991; Dragunow et al., 1990). The over expression of c-

Fos was also observed in the hippocampal dentate gyrus granule layer and thalamic nuclei after

brain injury in mice (Wessel et al., 1991; Dragunow et al., 1990). The induction of c-Fos in

these cells may be related to their proliferation or to growth factors production as previously

suggested (Dragunow and Robertson, 1988; Mocchetti et al., 1989), and may be mediated in part

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by pStat3 and CSF2RB1 signalling. NMDA-receptor-mediated events lead to c-Fos induction in

neocortical neurons after injury (Dragunow et al., 1990). Thus, c-Fos has significant roles in the

spinal cord and brain, especially in areas where we detected CSF2RB1 and pStat3 expression. It

is possible that CSF2RB1- and pStat3-expressing cells in these areas have a role in pain

modulation, which may involve the activation of c-Fos. Interestingly, c-Fos and c-Jun genes are

activated by CSF2RB1 in hematopoietic BaF3 cells, and induce their proliferation (Watanabe et

al., 1993). It is likely that CSF2RB1 ependymal cells in the brain areas detected has roles in

proliferation, particularly through c-Fos activation.

As in the central canal region, where CSF2RB1+ ependymal cells extended processes deep into

the grey matter of the spinal cord, CSF2RB1+ ependymal cells send long processes deep into

brain regions of the hypothalamus and the PAG, 2 brain areas that processes pain information by

ascending and descending modulation. In these regions we propose that CSF2RB1 activates

ependymal cells and initiates intracellular signalling events that lead to the release of cytokines,

chemokines and other pain related molecules. These factors in the extracellular space can

sensitize neighbouring neurons of the ascending modulation in the hypothalamus and the

descending inhibitory modulation in the PAG. Whether excitatory transmission or inhibitory

modulation is associated with CSF2RB1 in these areas is a new area of research to be explored.

6.13 Study design considerations in gene expression studies

In a non-biased approach that used whole genome expression profiling, we shortlisted candidate

genes for autotomy behaviour in the mouse using a unique study design that included a few

criteria discussed below.

6.13.1 Pooled vs. individualized arrays

Murine studies mostly focused on pooled brain tissue after nerve injury (Kõks et al., 2008), drug

treatments (Chetcuti et al., 2008; Korostynski et al., 2007), or on naïve animals (not associated

with nerve injury) (Ghate et al., 2007). One example of a non-pooling study sought oxidative

stress genes in the hippocampus and cortex following traumatic brain injury in Apolipoprotein E

(APOE) transgenic mice (Ferguson et al., 2010). In this particular study, non-pooling was

crucial because stress-associated genes were primarily considered. As we reasoned previously in

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the Methods Section 2.4.1, genes encoding stress proteins can change their expression levels not

only due to a particular treatment used but can also be affected by environmental factors that are

not associated with the treatment. Similarly, when seeking for pain genes in the Neuroma

Model, stress-related genes associated with the nerve injury that may be important in pain

mechanisms, should be distinguished from those that alter expression levels during tissue

harvesting. These genes can only be detected as outliers of a group using the non-pooling

approach.

Other studies that used the pooled approach and may therefore misinterpret their results in

certain genes are listed. In one study on glutamate transgenic mice vs. wild-type mice, three

mice per group were used to assess hippocampal gene expression levels (Wang et al., 2010a).

Other murine studies were performed on DRGs after nerve injury (Méchaly et al., 2006), or on

DRGs isolated from naïve animals (not associated with nerve injury) (LeDoux et al., 2006). A

more recent study compared the gene expression levels of DRGs via an unknown approach,

pooling/non-pooling, following axotomy (Persson et al., 2009b). Another study on Mmp9

knockout vs. wild-type mice did not indicate whether they used pooling or not for their genome

wide expression profiling in normal vs. sciatic nerve and DRG (Kim et al., 2012). Murine

studies in spinal cord were performed in herpes zoster-associated neuropathic pain (Takasaki et

al., 2012). Up to date, no comparative gene expression studies were carried out on spinal cord

tissue in mice following peripheral nerve injury.

6.13.2 The number of arrays per group

The higher the number of arrayed individuals the greater the statistical power there is to

find genes that significantly change their expression levels after nerve injury at a

−1.5≥fold change ≥1.5 and at a significance level of p<0.05, after correction of the p

values by FDR set at ≤0.05. However, the higher the number of arrays used in a study,

the costlier the project. The convention for neural tissue is 3 arrays per group (Géranton

et al., 2007; Di Giovanni et al., 2005; Griffin et al., 2007; Kim et al., 2009b; Korostynski

et al., 2007; LeDoux et al., 2006; Nesic et al., 2005; Sun et al., 2002; Valder et al., 2003;

Vega-Avelaira et al., 2009; Wang et al., 2010a; Zhang et al., 2004), however, other

studies used fewer arrays per group (Costigan et al., 2002; Coyle, 2007; Ghate et al.,

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2007; Lacroix-Fralish et al., 2006; Rodriguez Parkitna et al., 2006; Takasaki et al., 2012)

and even fewer studies used more than 3 arrays per group (Chetcuti et al., 2008; Di

Giovanni et al., 2003; Kõks et al., 2008). To improve the chances of finding genes that

are significantly regulated by the studied trait we studied 5 animals per group.

6.13.3 Type of group comparisons and choice of the

reference (control) group

Several studies compared nerve-injured animals to naïve animals (Costigan et al., 2002;

Griffin et al., 2007; Rodriguez Parkitna et al., 2006). In this inferior type of comparison,

it is impossible to separate gene expression levels related to injury response and the

recovery thereafter (i.e., production of injury discharge and/or the impact that this

message of occurred injury has on its targets, degeneration of nerve fibres and removal of

debris, inflammation at the cut end of the nerves, regeneration and sprout formation, and

tissue healing) from genes directly associated with neuropathic pain behaviour, having

had a nerve injury. It is, therefore, undesirable to compare naïve vs. nerve-injured groups

when studying neuropathic pain-associated genes. Rather, to identify genes whose

products drive animals to expressing high levels of neuropathic pain-related behaviour -

expression levels of DRGs (Persson et al., 2009b; Valder et al., 2003; Vega-Avelaira et

al., 2009; Wang et al., 2002a), spinal cords (Di Giovanni et al., 2003; Lacroix-Fralish et

al., 2006; Liu et al., 2012a; Wang et al., 2002a; Zhang et al., 2004), and brain tissue

(Kõks et al., 2008), the correct comparisons should be between nerve-injured animals

who express high levels of the pain behaviour and: (i) sham-operated animals of the same

genetic background, (ii) nerve-injured animals of the same genetic background who

express low/no levels of the pain behaviour, (iii) nerve-injured animals from another

genetic background who are known to express no/low levels of the pain behaviour, (iv)

sham-operated animals of that second strain, and (v) naive animals of both strains.

The study of Kim et al. (2009b) compared gene expression levels in the DRGs on the

injured ipsilateral side with the DRGs on the uninjured contralateral side of the same

animal, based on previous findings indicating that gene expression profiles were similar

between tissues contralateral to nerve ligation and those from sham animals of the same

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genetic background (Wang et al., 2002a). There are no known direct interactions between

injured peripheral neurons on the affected side and those contralaterally in the same

spinal segments or the trigeminal brainstem nuclei through their central terminals.

However, indirect bilateral interactions may be possible via CNS interneurons that cross

contra laterally or systemically through the circulation. Indeed, chronic pain may

manifest not only ipsilaterally, referred to the side of nerve injury, but also

contralaterally, expressed as ‘mirror-image’ pain. It is likely that such pain results from

genes abnormally regulated in the DRGs on the uninjured side. However, it is not correct

to regard the contralateral side as a control even if the animal does not show behavioural

changes contralaterally, because genes contralaterally may be abnormally regulated even

if this does not manifest in neuropathic sensory abnormalities.

Likewise, it is not optimal to compare gene expression levels in the two sides of the

spinal cord or trigeminal brain stem nuclei in the same animal, in light of the known side-

to-side neural connections that exist between neurons of the same segments. To detect

genes whose expression is associated with processing sensory inputs, including

nociceptive domains, Sun et al. (2002) compared gene expression levels in the dorsal

horn with that of the ventral horn, which is associated with integrating sensory inputs and

motor outputs. The results of such a comparison are even more puzzling.

These discrepancies in some of the published reports that studied the regulation of gene

expression levels in DRGs and the spinal cord point out the need for an optimal study

design and the significance of selecting the correct groups to be compared in such a

study. In our study we compared the ipsilateral DRGs and spinal cord segments to those

of the sham-operated mice of the same genetic background.

Two studies done in rats following spinal cord injury and partial hindpaw denervation in

the SNL model, respectively (Coyle, 2007; Nesic et al., 2005), compared spinal cord gene

expression levels between nerve-injured animals expressing neuropathic pain versus

nerve-injured animals that did not express the pain phenotype. The pain phenotype in

both studies was mechanical allodynia. This group comparison most closely resembled

our study design in that it compared animals within the same genetic background and

eliminated the influence of most non-genetic environmental factors by comparing

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animals having the same inciting event. In our experiment, gene expression levels in A

mice that expressed high autotomy levels, were compared to those of mice of the same A

strain who did not express autotomy or expressed very low autotomy levels following the

same nerve injury, and to sham-operated A mice. Moreover, Nesic et al. (2005) were one

of the few who grouped animals according to their pain levels, retaining the levels of

individual animals, as was done in the present study rather than pooling their mRNA as

was done in most other studies.

6.13.4 Arrayed neural tissues

Our study carried out expression profiling analyses on two types of neural tissues (i.e.,

DRGs and spinal cords) from the same animals. To date, previous reports that studied

more than neural tissue type (LeDoux et al., 2006; Rodriguez Parkitna et al., 2006; Wang

et al., 2002a) have used the pooled approach. In one proteomic profiling study that

compared both sciatic nerve and dorsal horn tissues, the authors neglected to mention

whether pooling or individual arraying was used in their expression profiles (Liu et al.,

2012a). Another research group performed DRG (Xiao et al., 2002) and spinal cord

expression profiling (Zhang et al., 2004) in two separate studies, also by pooling the

samples. Studies using individual tissue arrays of only 3 animals per group were carried

out on mouse brain (Kõks et al., 2008), rat spinal cord (Nesic et al., 2005), and rat DRG

(Valder et al., 2003) tissues.

To summarize, we believe that our study improved on study design over most published

previous studies, in: (i) using individual specimens to compare gene expression levels in

individuals rather than pooling the mRNA, (ii) comparing two neural tissues of the same

animals, enabling correlation of the expression across animals of the same groups and

across groups, (iii) selecting better specific group comparisons that included naive, sham-

operated and hindpaw-denervated animals in the two strains of contrasting pain

behaviours, and (iv) comparing mice of the same genetic background having had the

same nerve injury bur responding with contrasting pain behaviour. In this design within-

strain comparisons enabled us to identify genes that interact with the environment (gene-

by-environment interactions, ‘GXE’) associated with the studied pain phenotype,

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whereas across-strain comparisons permitted the findings of genetic factors related to

chronic pain behaviour.

6.13.5 Statistical considerations

We conducted a number of statistical analyses of gene expression profiles, looking for

genes across the whole genome that correlate the contrast in pain behaviour, with a

special focus on genes in the Pain1 region. The more comparisons we made, the higher

the likelihood that some of the genes identified as significantly regulated by pain were

false positive identifications (‘type 1 error’), and that those identified as not significant –

might have been wrongly labelled as such and were actually false negative identifications

(‘type 2 error’). In an attempt to limit this likelihood one needs to correct for inflation of

the alpha level due to multiple comparisons by using the Bonferroni adjustment factor, or

other, less conservative methods, such as the False Discovery Rate (Benjamini and

Hochberg, 1995). The considerably more conservative of the two approaches was the

Bonferroni method because when dividing the level of 0.05 by the number of probes in

the array we used in our study (N=44,000), the outcome yields an α=E-06 as the minimal

p value needed to reject the null hypothesis that the expression of a gene under study was

invariable across the compared groups. Any p value above this threshold would have

been considered insignificant in a genome-wide analysis. However, as our hypothesis

was that the gene under study should be located within Pain1, dividing 0.05 by the

number of probes in Pain1 genes (N=238) should have yielded p≤0.0002 as the criterion

of significance. This p-value was used as the criterion of significance to seek candidate

pain genes in Pain1. We were unable, using a cut off of FDR<0.05 to locate any gene in

the spinal cord that is significantly associated with the trait, nevertheless, decided to

proceed with further analyses without considering the FDR correction of the alpha level.

6.13.6 Group comparisons

Another factor of importance when seeking candidate genes using a whole genome

expression array was to decide on the cut-off level of fold changes considered

meaningful. Although a p<0.05 alone would result in many false-positives, the

combination of fold-change and p-value thresholds could eliminate most false positives

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that are obtained only with p<0.05 (Di Giovanni et al., 2005). Since we analyzed our

data in 3 group comparisons, all genes found to be significant at p<0.05 and −1.5≥fold

change were considered candidate genes. This set of criteria has also been used in other

studies (Costigan et al., 2002; Coyle, 2007; Di Giovanni et al., 2005; Szpara et al., 2007).

In one of these studies (Szpara et al., 2007) an FDR test was run as well, but results of the

FDR were not used in filtering data for clustering or further analysis, as was done in the

present study.

6.14 Limitations of the studies

In this study we used 2 strains that are susceptible to develop autotomy (A and C3H) and 2

strains that are resistant to this behaviour (B and AKR), and looked at the Csf2rb gene

expression levels in these strains. Tlr4 gene was compared in the two C3H strains, C3H/HeN

and C3H/HeJ. PStat3 was studied in the A vs. B strains. In order to get a more general

perspective of these genes on autotomy behaviour, this necessitates using more contrasting

strains to study the following genes, especially in immunohistochemical techniques in which

mostly A and B mice were used.

For immunohistochemical labelling of pStat3, there is a need for a greater N to signify the

findings in the spinal dorsal horn and brain regions, to be able to assess if and what is the

difference between high autotomy and low autotomy mouse groups. Another limitation in the

immunohistochemical studies was the inability to co-label pStat3 cells in the spinal dorsal horn

and in the brain areas, as well as to co-label CSF2RB1 in the dentate gyrus, and to assess in

which cell types these gene products are located in these areas. More antibodies other than the

ones used in the present study to label microglia (i.e., Iba-1, Itgam) and neural stem cells such as

Nestin are needed to complete the study. The fact that we could not co-localize CSF2RB1 in

some of the dentate gyrus cells, via GFAP and Vimentin markers, could be due to very intensely

labelled CSF2RB1 masking a weaker label of the other markers. It is also possible that neural

stem cells positive for CSF2RB1 were beginning to differentiate into astrocytes, ependymal cells

and neurons at the time the animals were perfused for the study, which is why we could not

detect the receptor in these latter cells.

196

One of the future avenues of research could be focusing on regions we found harbouring cells

expressing CS2RB1 and using double or triple labelling to map the same tissue sections for GM-

CSF, IL-3, IL-5, and their receptors (CSF2RB1, CSF2RB2 and CSF2RA). We did not co-

localize CSF2RB1 to neuronal nuclei, marked by NeuN only to a very small number of neurons

(which may be an artefact due to z-stack imaging), and it is possible that membrane-bound, as

opposed to nuclear CSF2RB1 is found on neurons of our mice. Another possibility is to label the

CSF2RB1 cells found in white matter of the dorsal horn with a double label for axons, to exclude

the possibility that they localize in neurons. These glial cells did not co-localize with Vimentin,

indicating that they are not the same ependymal cells/tanycytes/radial glia found in the central

canal.

Imaging of the fluorescence was observed using a conventional epifluorescence microscope.

However, the use of confocal microscopy could have accentuated the origins and end points of

CSF2RB1-labelled processes, especially in the spinal central canal region, dorsal horn, and

dorsal columns as well as in the brain.

Immunolabelling of TLR4 is necessary in A vs. B mice, to detect where this receptor is

expressed in the spinal cord and brain, and to assess whether or not differences exist between the

strains/autotomy levels. Using a better gear (fluorescent microscope with laser light) could have

helped detect where the CSF2RB1+ processes around the central canal terminate, whether in the

neuropil or elsewhere.

As mentioned in the Methods, the experimenter was blinded while autotomy phenotyping to the

identity of the group under observation, i.e., not knowing if the operated animal was denervated

or sham-operated. However, differences do exist between the two operations, in that the

denervated paw is also paralyzed, hence dragged by the animal. This can easily be observed by

the experimenter, thereby readily distinguishing between a sham and a denervated mouse.

However, autotomy behaviour is very clear when seen in an animal, and its presence or absence

is easy to quantify. Nevertheless, on some rare occasions it may be difficult to decide whether

an injury is caused by an intended self-mutilation or is the result of mechanical wounding due to

dragging of the paw on the floor. In summary, scoring of autotomy was performed using an

acceptable method that when using blinding techniques can minimize rater bias.

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6.15 Future direction of research

This dissertation reports on 3 main genes that correlated spontaneous pain behaviour in the

mouse, Csf2rb2, Tlr4 and Stat3. The main focus was in the Pain1 gene Csf2rb1 which we

correlated with autotomy in both the spinal cord and brain. It is not yet known in which

mechanism Csf2rb1 is involved and where is it primarily affecting the pain phenotype in the

mouse. Is Csf2rb1’s function dominating in the spinal cord or in the brain to maintain pain

behaviour? More experiments are needed to attest these possibilities.

The present study concentrated mostly on gene expression and immunohistochemical techniques

to seek for correlations between a gene of interest and autotomy behaviour. Note however, that

all of the observations reported herein, were of correlations and not mechanistic. Additional

functional studies are needed to assess whether the studied genes contribute to the development

and/or maintenance of chronic neuropathic pain. As such, there is a need to block a gene of

interest (i.e., Csf2rb1, Tlr4, Stat3) in specific areas such as the spinal cord, hippocampus, brain

ventricles, by way of injecting siRNAs, specific antibodies, or peptides that block the function of

the protein, and after complete hind paw denervation detect the differences in autotomy

behaviour between animals that received the block and those that did not. These studies should

be performed in strains that are susceptible to develop autotomy in a penetrance of 50% or more,

such as the A or C3H/HeN strain.

In situ blockade assays for Csf2rb1 are more preferable than using an available Csf2rb1

knockout mouse to study autotomy behaviour for several reasons. First, the commercially

available Cre-Lox conditional knockout mice from the Jackson Laboratories were produced in

C57BL6/J mice, which are known to be resistant to autotomy behaviour following hindpaw

denervation (Devor 2005). Knocking out this gene is expected to make these animals resistant

to autotomy following hindpaw denervation. Therefore, a conditional knocked out Csf2rb1 gene

in the C57BL6/J background would not be informative because these mice would not autotomize

regardless of the mutation (given their strain resistance to autotomy behaviour). To be

informative after hindpaw denervation, this knockout should have been introduced to A/J mice,

so that a wild-type mouse that usually autotomizes following hindpaw denervation would

expresses no autotomy, or lower autotomy levels, when this gene is knocked out. Finally,

198

developmental knockouts (i.e., mice whose gene is already knocked out at an embryonic stage)

are problematic because the animal may develop compensatory mechanisms, which would

enable it to function normally despite lacking functional copies of that gene, and therefore, an

adult knocked out mouse might not show the desired phenotype.

Alternative complementary studies of inducing receptor activity in autotomy resistant strains,

such as B and AKR, are beneficial. The ligand for CSF2RB1, GM-CSF can be injected into

these mice before and following hind paw denervation to assess whether or not autotomy

behaviour is induced in these strains, by activating the receptor. Alternatively, LPS can be

injected in these mice to assess whether TLR activation produces autotomy.

Results of CSF2RB1 in the dentate gyrus showing increased expression with autotomy behaviour

in A mice suggested a negative valence in these mice. We hypothesized that A mice probably

cannot avoid their pain in the way that B mice can. Therefore, in order to test this hypothesis, A

and B mice can be placed in chambers for pain avoidance testing to see whether there is a

difference in their avoidance behaviour. These tests can be performed after CSF2RB1 block in

A mice, for instance, or alternatively after GM-CSF induction in B mice, to see whether Csf2rb1

plays role in the avoidance.

Another step to determine whether Csf2rb1 is indeed an important neuropathic pain gene is to

study its sequence in human neuropathic pain patients vs. that of patients who suffered the same

injury yet did not develop neuropathic pain. Indeed, preliminary genotyping of a few Csf2rb1

SNPs in humans, have been carried out in our lab and I took part in this study. The pilot results

identified a candidate haplotype in 350 women with post-mastectomy neuropathic pain

syndrome that was different from the haplotype of pain-free women following the same

operation. However, we were unable to replicate the same haplotype in a smaller cohort of leg

amputees with and without phantom limb pain. These preliminary studies were lacking because

we genotyped only a few SNPs along the gene to adequately map it for polymorphisms.

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6.16 Conclusions

The present study aimed to identify novel genes associated with chronic neuropathic pain and the

mechanisms they encode in a mouse model of spontaneous pain, imitating phantom limb pain in

human amputees, and patients who sustained a complete cut nerve but the limb itself remained

part of the body (‘anesthesia dolorosa’). Using a non-biased approach, we identified a novel

gene named Csf2rb1 in a region on chromosome 15 of the mouse that was mapped in previous

studies Pain1. This gene encodes a cytokine receptor of 3 ligands IL-3, IL-5 and GM-CSF. We

identified two additional genes, named Tlr4 and Stat3, encoding the Toll-like receptor 4, and the

Signal transducer and activator of transcription 3, respectively. These genes propose new pain

mechanisms in the CNS, involving specialized glial cells in the spinal cord and brain that were

never implicated in neuropathic pain before. Our major findings regarding these 3 genes are

listed below.

1. We were able to substantiate in part the candidacy of Csf2rb1 as a gene for autotomy in

Pain1 and identified cells that express it in the spinal cord as ependymal cells/radial

glia/tanycytes. We propose that these cells in the central canal region may contribute to

the hyperexcitability of ascending nociceptive neurons (including those originating in

Layer X - the VMAB), thereby delivering pain inputs to higher brain centres.

2. We found that these cells proliferate post-injury and propose (although without

supporting evidence as of yet) that they may function as a reservoir of spinal stem cells

that respond to injury in the production of neurons and/or glia cells in the spinal cord and

white matter, in the form of ependymal cells/radial glia/tancyte gliosis. Such a

proliferation and differentiation in the dorsal horn may contribute to the maintenance of

neuropathic pain.

3. We were able to show a marked immunolabelling of both CSF2RB1 and pStat3 in the

peri-ventricular zone, the arcuate nucleus and the hippocampal dentate gyrus, where

expression of both proteins correlated with autotomy behaviour in some denervated A

mice. We suggest that these areas, shown previously to be significant in neuropathic pain

behaviour, may be important in maintaining spontaneous neuropathic pain. Additionally

200

we showed a moderate expression of CSF2RB1 in the dorsal horn, and of pStat3 that

increased in mice with high-autotomy levels. Whether the main mechanism for Csf2rb1

is spinal or at higher brain centres, such as the hippocampus and hypothalamus, is yet to

be discovered. However, our findings show that over-expression of Csf2rb1 and/or its

product is correlated with high autotomy levels in the spinal cord, and brain of some

denervated A mice, not in B mice.

4. We show for the first time that TLR4 regulation is associated with spontaneous

neuropathic pain. TLR4-deficient mice, carrying a dysfunctional TLR4 receptor, had a

delayed autotomy behaviour compared to wild type mice having the normal receptor.

Consistent with this finding, Tl4 gene expression levels were differentially regulated in

DRGs and spinal cords of autotomy susceptible A vs. autotomy resistant B mice. Spinal

Csf2rb1 gene in TLR4-deficient mice failed to up-regulate following hindpaw

denervation, whereas in TLR4 wild-type mice Csf2rb1 mRNA levels up-regulated after

the same denervation procedure.

5. We propose cellular mechanisms in pain pathways, which involve Csf2rb1, Tlr4, and

pStat3 that ultimately lead to the sensitization of neurons in the spinal cord and brain via

cytokines, chemokines, and other pain-related molecules.

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238

Appendix I

Genome wide candidate genes associated with autotomy in the DRGs. The expression of these

genes differentially contrasted between A high vs. A sham, A low, and B low autotomy mice at a

p-value<0.05 and a -1.5>fold change>1.5

239

p-value FC p-value FC p-value FC

--- 2610200G18RikMus musculus adult male tongue cDNA, RIKEN full-length

enriched library, clone:22.00E-07 2.1 0.00074 1.6 0.00884 -1.8 ↕

Chr 1chr1:108538339-

108538280Bcl2

Mus musculus 13 days embryo male testis cDNA, RIKEN

full-length enriched library0.01048 -1.5 0.00938 -2.6 0.03584 -1.9 ↓

Chr 1chr1:109352466-

109352525Serpinb2

Mus musculus serine (or cysteine) peptidase inhibitor,

clade B, member 2 (Serpin9.67E-05 3.3 0.00606 2.4 0.00418 2.9 ↑

Chr 1chr1:121245445-

121245386Inhbb Mus musculus inhibin beta-B (Inhbb), mRNA [NM_008381] 7.35E-08 2.8 2.86E-06 2.5 0.02656 1.5 ↑

Chr 1chr1:123265403-

123269353Ccdc93

Mus musculus coiled-coil domain containing 93 (Ccdc93),

transcript variant 3, mR0.01137 1.6 0.02907 1.6 0.01186 1.7 ↑

Chr 1chr1:133837210-

133837269Elk4

Mus musculus 16 days neonate thymus cDNA, RIKEN full-

length enriched library, cl0.03116 2.1 0.05113 -2.4 0.02691 2.4 ↕

Chr 1chr1:161916409-

1619164684930523C07Rik

Mus musculus 2 days pregnant adult female oviduct cDNA,

RIKEN full-length enrich0.01527 -1.9 0.01775 -2.0 0.01512 -1.8 ↓

Chr 1chr1:165138892-

165138833AK081699

Mus musculus 16 days embryo head cDNA, RIKEN full-

length enriched library, clone0.03718 -1.6 0.00241 -2.2 0.00325 -2.7 ↓

Chr 1chr1:168151835-

168151776D1Ertd471e

Mus musculus DNA segment, Chr 1, ERATO Doi 471,

expressed, mRNA (cDNA clone IMAG0.01154 -2.5 0.00531 1.6 0.00022 -4.0 ↕

Chr 1chr1:169684432-

169684491Lmx1a

Mus musculus LIM homeobox transcription factor 1 alpha

(Lmx1a), mRNA [NM_033652]0.00306 2.2 0.00645 2.2 0.00095 2.0 ↑

Chr 1chr1:172963507-

1729635661700009P17Rik

Mus musculus RIKEN cDNA 1700009P17 gene, mRNA (cDNA

clone MGC:74123 IMAGE:6774920.00205 -1.7 0.0029 -1.8 0.03063 -1.6 ↓

Chr 1chr1:173837985-

173837926Vangl2

Mus musculus loop tail associated protein, mRNA (cDNA

clone IMAGE:30545600), par0.006 -1.7 0.00463 -2.1 0.00095 -1.8 ↓

Chr 1chr1:177520104-

177520045Chml

Mus musculus 9 days embryo whole body cDNA, RIKEN full-

length enriched library, 0.04746 1.8 0.02216 1.5 0.00189 1.6 ↑

Chr 1chr1:183013430-

183013371Wdr26

PREDICTED: Mus musculus WD repeat domain 26,

transcript variant 1 (Wdr26), mRNA 0.00012 1.7 0.00023 1.7 0.00033 1.6 ↑

Chr 1chr1:183652496-

183652437Lbr Mus musculus lamin B receptor (Lbr), mRNA [NM_133815] 0.04287 1.5 0.03616 1.7 0.03223 1.6 ↑

Chr 1chr1:196514819-

196514878Plxna2 Mus musculus plexin A2 (Plxna2), mRNA [NM_008882] 0.00355 -1.9 0.00109 -2.2 0.03009 -1.5 ↓

Chr 1chr1:37206871-

37206930Cnga3

Mus musculus cyclic nucleotide gated channel alpha 3

(Cnga3), mRNA [NM_009918]0.03656 -1.8 0.00735 -2.3 0.03595 -1.7 ↓

Chr 1chr1:44064804-

44070854Bivm

Mus musculus basic, immunoglobulin-like variable motif

containing (Bivm), mRNA [0.03583 2.1 0.04015 2.3 0.00078 3.2 ↑

Chr 1chr1:46690040-

46690099BG082943

H3082F09-5 NIA Mouse 15K cDNA Clone Set Mus musculus

cDNA clone H3082F09 5', mRN0.00301 -2.2 0.04592 -1.8 0.00398 -2.4 ↓

Chr 1chr1:52066524-

52066583Stat1

Mus musculus 3 days neonate thymus cDNA, RIKEN full-

length enriched library, clo0.04731 -2.1 0.05204 -2.2 0.03696 -2.1 ↓

Chr 1chr1:93204297-

93204356BC056923

Mus musculus cDNA sequence BC056923 (BC056923),

mRNA [NM_173395]0.00019 3.5 0.01629 2.3 0.03172 1.8 ↑

Chr 2chr2:113808012-

113806082Aqr Mus musculus aquarius (Aqr), mRNA [NM_009702] 0.02304 1.7 0.02912 1.8 0.01246 1.8 ↑

Chr 2chr2:119045695-

119045754Chac1

Mus musculus ChaC, cation transport regulator-like 1 (E.

coli) (Chac1), mRNA [NM6.52E-05 1.9 0.01673 1.5 0.02038 -1.5 ↕

Chr 2chr2:121995549-

121995490Duoxa1

Mus musculus dual oxidase maturation factor 1 (Duoxa1),

mRNA [NM_145395]0.02468 -1.5 8.88E-13 -11.7 0.00091 -1.8 ↓

Chr 2chr2:12290412-

122903532310047O13Rik

Mus musculus 12 days embryo spinal cord cDNA, RIKEN

full-length enriched library0.00932 -1.6 0.04184 -2.7 1.85E-05 -2.7 ↓

Chr 2chr2:12930017-

12930076C1ql3

Mus musculus 0 day neonate eyeball cDNA, RIKEN full-

length enriched library, clo0.04169 1.8 0.04456 1.9 0.00687 2.0 ↑

Chr 2chr2:135760884-

1357609436330527O06Rik

Mus musculus RIKEN cDNA 6330527O06 gene

(6330527O06Rik), mRNA [NM_029530]0.01027 4.0 0.02848 3.7 0.01589 3.0 ↑

Chr 2chr2:143518220-

143518161Bfsp1

Mus musculus beaded fi lament structural protein in lens-

CP94 (Bfsp1), mRNA [NM_00.01048 -2.1 0.05226 -1.8 0.02418 -1.7 ↓

Chr 2chr2:146769105-

146769164Xrn2

Mus musculus 5'-3' exoribonuclease 2 (Xrn2), mRNA

[NM_011917]3.92E-05 1.5 0.01294 1.7 1.82E-05 1.5 ↑

Chr 2chr2:151800890-

151800949Srxn1

Mus musculus sulfiredoxin 1 homolog (S. cerevisiae)

(Srxn1), mRNA [NM_029688]7.06E-08 2.4 0.00012 1.8 7.02E-05 1.8 ↑

Chr 2chr2:165842112-

165842053AK038166

Mus musculus 16 days neonate thymus cDNA, RIKEN full-

length enriched library, cl0.02272 -2.3 0.00502 -3.2 0.007 -2.5 ↓

Chr 2chr2:172046374-

1720464332010011I20Rik

Mus musculus RIKEN cDNA 2010011I20 gene

(2010011I20Rik), mRNA [NM_025912]1.64E-09 2.3 0.00023 1.5 0.00031 1.9 ↑

Chr 2chr2:172165210-

1721652691700029J11Rik

Mus musculus adult male testis cDNA, RIKEN full-length

enriched library, clone:10.00885 1.5 0.00081 1.9 0.01194 1.5 ↑

Chr 2chr2:181318940-

181318881Btbd4

Mus musculus 0 day neonate kidney cDNA, RIKEN full-

length enriched library, clon0.02321 2.2 0.00935 2.8 0.01989 2.0 ↑

Chr 2chr2:33387100-

33386753Lmx1b

Mus musculus LIM homeobox transcription factor 1 beta

(Lmx1b), mRNA [NM_010725]0.01168 -1.5 0.01051 -1.6 6.94E-05 -2.1 ↓

Chr 2chr2:34765430-

34765371Traf1

Mus musculus Tnf receptor-associated factor 1 (Traf1),

mRNA [NM_009421]0.01873 -1.8 0.04355 -1.6 0.00618 -1.7 ↓

Chr 2chr2:3695767-

36958263110001A13Rik

Mus musculus RIKEN cDNA 3110001A13 gene

(3110001A13Rik), mRNA [NM_025626]2.77E-05 1.5 9.78E-05 2.0 0.00156 1.6 ↑

Chr 2chr2:63869523-

63869464AK035003

Mus musculus 12 days embryo embryonic body between

diaphragm region and neck cDN0.005 -3.1 0.04297 -2.4 0.00091 -4.6 ↓

Gene RegulationA high vs. A lowA high vs. A sham A high vs. B low

ChromGenomic

CoordinatesGene Name Description

240

Chr 2chr2:68911727-

68911786Lass6

Mus musculus longevity assurance homolog 6 (S.

cerevisiae) (Lass6), mRNA [NM_1722.52E-05 2.1 0.00121 1.8 2.63E-06 -2.7 ↕

Chr 2chr2:76855437-

768505602610301F02Rik

Mus musculus 0 day neonate lung cDNA, RIKEN full-length

enriched library, clone:0.03079 -1.9 0.01947 -2.1 0.05115 -1.7 ↓

Chr 3chr3:101589718-

101589777Igsf3

Mus musculus immunoglobulin superfamily, member 3

(Igsf3), mRNA [NM_207205]0.02011 -1.6 0.03171 -1.6 0.01351 -1.6 ↓

Chr 3chr3:101942973-

101942914BC037703

Mus musculus cDNA sequence BC037703 (BC037703),

mRNA [NM_172295]0.00332 -1.7 0.02241 -1.6 0.0226 -1.5 ↓

Chr 3chr3:104782221-

104782280Slc16a1

Mus musculus solute carrier family 16 (monocarboxylic

acid transporters), member0.00565 2.0 0.04573 1.7 3.61E-05 2.7 ↑

Chr 3chr3:137563799-

137563858Ddit4l

Mus musculus DNA-damage-inducible transcript 4-like

(Ddit4l), mRNA [NM_030143]0.04862 -1.8 0.00884 -1.6 0.00084 -2.9 ↓

Chr 3chr3:146078707-

146078766Mcoln3

Mus musculus 12 days embryo male wolffian duct

includes surrounding region cDNA,0.00019 4.8 0.01824 2.8 0.00018 4.5 ↑

Chr 3chr3:27321129-

27318861Ect2 Mus musculus ect2 oncogene (Ect2), mRNA [NM_007900] 0.04356 1.5 0.01931 1.7 0.03212 -2.0 ↕

Chr 3chr3:51492886-

51492827Ndufc1

Mus musculus NADH dehydrogenase (ubiquinone) 1,

subcomplex unknown, 1 (Ndufc1), 0.04556 -1.7 0.00764 -2.8 0.00869 -1.9 ↓

Chr 3chr3:88722014-

88724240Arhgef2

Mus musculus rho/rac guanine nucleotide exchange

factor (GEF) 2 (Arhgef2), mRNA 0.03031 1.8 0.0148 2.1 0.0154 1.8 ↑

Chr 3chr3:88797018-

887970772810403A07Rik

Mus musculus 12 days embryo spinal ganglion cDNA,

RIKEN full-length enriched lib0.04796 1.5 0.01641 -2.2 0.02114 2.3 ↕

Chr 3chr3:90129695-

90129636Ubap2l

Mus musculus ubiquitin associated protein 2-like

(Ubap2l), transcript variant 2,0.01495 1.5 0.01892 -4.2 0.03315 -3.1 ↕

Chr 4chr4:116473895-

116477403Zswim5

Mus musculus zinc finger, SWIM domain containing 5

(Zswim5), mRNA [NM_001029912]0.00108 -2.4 0.0223 -1.9 0.00126 2.5 ↕

Chr 4chr4:122794043-

122794102Pabpc4

Mus musculus poly A binding protein, cytoplasmic 4

(Pabpc4), transcript variant 0.0199 1.7 0.00303 2.0 0.01381 1.7 ↑

Chr 4chr4:126018254-

126018195Eif2c4

Mus musculus eukaryotic translation initiation factor 2C,

4 (Eif2c4), mRNA [NM_10.04612 -1.5 0.00799 -2.1 7.92E-05 -2.0 ↓

Chr 4chr4:131196947-

131196883Epb4.1

Mus musculus 10 days neonate cerebellum cDNA, RIKEN

full-length enriched library0.01879 2.3 0.02854 2.3 0.00164 2.8 ↑

Chr 4chr4:131572636-

1315725779530096D07Rik

RIKEN cDNA 9530096D07 gene

[Source:MarkerSymbol;Acc:MGI:2441753]

[ENSMUST0000004

0.00472 -1.4 0.0015 -1.8 0.00084 -1.5 ↓

Chr 4chr4:150960446-

150960505Tnfrsf25

Mus musculus tumor necrosis factor receptor

superfamily, member 25 (Tnfrsf25), m0.00215 -1.5 0.00024 -1.7 2.94E-05 -1.8 ↓

Chr 4chr4:154627261-

154627202A230069A22Rik

Mus musculus RIKEN cDNA A230069A22 gene

(A230069A22Rik), mRNA [NM_001033394]0.00325 2.6 0.04483 2.0 0.02445 2.3 ↑

Chr 4chr4:40459443-

404595022010003O02Rik

Mus musculus adult male small intestine cDNA, RIKEN full-

length enriched library0.01743 -1.5 0.00413 -2.9 0.00012 -2.3 ↓

Chr 4chr4:42776430-

42776371Ccl19

Mus musculus chemokine (C-C motif) l igand 19 (Ccl19),

mRNA [NM_011888]0.01625 -2.3 0.05161 -2.1 0.00704 -2.5 ↓

Chr 4chr4:43055216-

43055060B230312A22Rik

Mus musculus RIKEN cDNA B230312A22 gene

(B230312A22Rik), mRNA [NM_172691]0.00833 2.1 0.03375 1.9 0.04377 1.7 ↑

Chr 4chr4:49671132-

49671191Rnf20

Mus musculus ring finger protein 20 (Rnf20), mRNA

[NM_182999]0.02757 -1.5 0.02746 -1.5 0.00582 -1.7 ↓

Chr 4chr4:52499529-

52499588Smc2

Mus musculus structural maintenance of chromosomes 2

(Smc2), mRNA [NM_008017]0.00818 1.7 0.00403 1.9 0.00453 2.8 ↑

Chr 4chr4:64842834-

64842893Pappa

Mus musculus pregnancy-associated plasma protein A

(Pappa), mRNA [NM_021362]0.02753 1.8 0.01502 2.1 0.04897 1.7 ↑

Chr 4chr4:88338603-

883385444930553M12Rik

Mus musculus adult male testis cDNA, RIKEN full-length

enriched library, clone:40.00198 1.9 0.00021 2.5 0.00018 2.5 ↑

Chr 5chr5:116738100-

1167362301500001A10Rik Mus musculus mRNA for mKIAA1853 protein [AK129457] 0.02568 2.1 8.51E-05 1.9 0.00868 -2.2 ↕

Chr 5chr5:15037678-

15037619Speer4c

PREDICTED: Mus musculus spermatogenesis associated

glutamate (E)-rich protein 4c0.00084 2.9 0.03809 2.0 0.00194 -2.9 ↕

Chr 5chr5:26594067-

26594126LOC664837

PREDICTED: Mus musculus similar to RIKEN cDNA

5031410I06, transcript variant 2 (1.77E-08 3.8 0.00712 1.7 0.0473 -1.5 ↕

Chr 5chr5:27827153-

27827094Speer4b

Mus musculus spermatogenesis associated glutamate (E)-

rich protein 4b (Speer4b),0.00596 3.2 0.02735 2.8 0.04506 -2.3 ↕

Chr 5chr5:4250526-

4250585NAP067089-1 Unknown 0.04893 -1.7 0.01351 -2.1 0.05219 -1.6 ↓

Chr 5chr5:65910887-

659091719030416H16Rik

Mus musculus RIKEN cDNA 9030416H16 gene, mRNA

(cDNA clone MGC:29439 IMAGE:3964500.04501 1.6 0.05013 1.7 0.04813 1.5 ↑

Chr 5chr5:74909055-

74905333Lnx1

Mus musculus l igand of numb-protein X 1 (Lnx1), mRNA

[NM_010727]0.00529 -1.5 0.00312 -1.6 0.00132 3.7 ↕

Chr 5chr5:87638404-

87638345Tmprss11f

Mus musculus transmembrane protease, serine 11f

(Tmprss11f), mRNA [NM_178730]3.38E-05 2.7 1.17E-05 3.4 0.00039 -2.6 ↕

Chr 5chr5:92433181-

92433122Btc

Mus musculus betacellulin, epidermal growth factor

family member (Btc), mRNA [NM0.0301 -1.8 7.93E-11 -18.9 0.00331 -2.7 ↓

Chr 5chr5:93422014-

93421955Cxcl10

Mus musculus chemokine (C-X-C motif) l igand 10 (Cxcl10),

mRNA [NM_021274]0.05301 -2.4 0.00492 -4.5 0.00739 -3.3 ↓

Chr 6chr6:108516481-

108516540Itpr1

Mus musculus inositol 1,4,5-triphosphate receptor 1

(Itpr1), mRNA [NM_010585]5.80E-07 2.7 0.00112 1.5 0.00163 1.5 ↑

Chr 6chr6:117889534-

117889593Hnrpf

Mus musculus heterogeneous nuclear ribonucleoprotein F

(Hnrpf), mRNA [NM_133834]0.04362 1.6 0.01804 1.9 0.02914 1.6 ↑

Chr 6chr6:120759377-

120759436Slc25a18

PREDICTED: Mus musculus solute carrier family 25

(mitochondrial carrier), member0.02916 -1.9 0.03866 -1.9 0.01632 -1.9 ↓

Chr 6chr6:121854555-

121854614Mug1

Mus musculus murinoglobulin 1 (Mug1), mRNA

[NM_008645]0.0003 2.7 0.00227 2.5 0.00075 -2.3 ↕

241

Chr 6chr6:108516481-

108516540Itpr1

Mus musculus inositol 1,4,5-triphosphate receptor 1

(Itpr1), mRNA [NM_010585]5.80E-07 2.7 0.00112 1.5 0.00163 1.5 ↑

Chr 6chr6:117889534-

117889593Hnrpf

Mus musculus heterogeneous nuclear ribonucleoprotein F

(Hnrpf), mRNA [NM_133834]0.04362 1.6 0.01804 1.9 0.02914 1.6 ↑

Chr 6chr6:120759377-

120759436Slc25a18

PREDICTED: Mus musculus solute carrier family 25

(mitochondrial carrier), member0.02916 -1.9 0.03866 -1.9 0.01632 -1.9 ↓

Chr 6chr6:121854555-

121854614Mug1

Mus musculus murinoglobulin 1 (Mug1), mRNA

[NM_008645]0.0003 2.7 0.00227 2.5 0.00075 -2.3 ↕

Chr 6chr6:145821384-

145821325Bhlhb3

Mus musculus basic helix-loop-helix domain containing,

class B3 (Bhlhb3), mRNA [0.03399 2.1 0.01697 2.5 0.02001 -2.0 ↕

Chr 6chr6:29403235-

29403294Flnc

Mus musculus fi lamin C, gamma (actin binding protein

280), mRNA (cDNA clone IMAG0.0048 1.8 0.00546 2.0 0.00476 1.8 ↑

Chr 6chr6:29538054-

29536167Tnpo3 Mus musculus transportin 3 (Tnpo3), mRNA [NM_177296] 0.01213 1.9 0.03065 1.6 0.00141 2.3 ↑

Chr 6chr6:31450627-

31450568Podxl

Mus musculus podocalyxin-like (Podxl), mRNA

[NM_013723]0.00371 1.9 0.04623 1.5 0.02339 1.6 ↑

Chr 6chr6:31484657-

31484598AK081840

Mus musculus 16 days embryo head cDNA, RIKEN full-

length enriched library, clone0.04185 -1.5 0.04348 -1.6 0.00217 -2.1 ↓

Chr 6chr6:39532627-

39532686Ndufb2

Mus musculus NADH dehydrogenase (ubiquinone) 1 beta

subcomplex, 2 (Ndufb2), mRNA0.00308 -1.8 0.02497 -1.5 8.91E-05 -2.0 ↓

Chr 6chr6:50503201-

505031424921507P07Rik

Mus musculus RIKEN cDNA 4921507P07 gene

(4921507P07Rik), mRNA [NM_027564]0.00681 -2.2 0.02629 -2.0 0.00911 -2.0 ↓

Chr 6chr6:54199945-

541998869130019P16Rik

Mus musculus RIKEN cDNA 9130019P16 gene

(9130019P16Rik), mRNA [NM_198118]0.05313 1.7 0.01105 2.1 0.00087 2.4 ↑

Chr 6chr6:58607928-

58612858Abcg2

Mus musculus ATP-binding cassette, sub-family G

(WHITE), member 2 (Abcg2), mRNA 0.00776 1.5 0.01047 1.5 1.23E-05 2.3 ↑

Chr 6chr6:6459871-

6459930NAP051844-1 Unknown 0.01205 -1.7 0.01647 -1.7 0.00064 -1.9 ↓

Chr 6chr6:65386839-

65386898C130060K24Rik

Mus musculus 2 days pregnant adult female oviduct cDNA,

RIKEN full-length enrich0.00449 2.7 0.00119 -2.2 0.00873 -2.9 ↕

Chr 6chr6:73398382-

733983234931417E11Rik

Mus musculus RIKEN cDNA 4931417E11 gene

(4931417E11Rik), mRNA [NM_025737]0.05204 -1.6 0.01308 -2.1 0.01039 -1.9 ↓

Chr 6chr6:78358042-

78358101Reg1

Mus musculus regenerating islet-derived 1 (Reg1), mRNA

[NM_009042]0.0003 4.0 0.00228 3.5 0.00412 -3.5

Chr 6chr6:90386540-

90386481Ccdc37

Mus musculus adult male corpus striatum cDNA, RIKEN

full-length enriched library0.00921 -1.8 0.05402 -1.6 0.01066 -1.7 ↓

Chr 7chr7:102960190-

102960249Olfr584

Mus musculus olfactory receptor 584 (Olfr584), mRNA

[NM_147054]0.00031 -3.2 0.02751 -2.1 2.42E-05 -4.5 ↓

Chr 7chr7:113176968-

113176909Pth

Mus musculus parathyroid hormone (Pth), mRNA

[NM_020623]0.01344 1.8 0.04977 1.7 0.00212 -2.0 ↕

Chr 7chr7:131823690-

131823749Gpr26

Mus musculus G protein-coupled receptor 26 (Gpr26),

mRNA [NM_173410]0.00983 -2.2 0.04532 -2.0 0.01134 -2.2 ↓

Chr 7chr7:19062889-

19062830Pvr

Mus musculus 18 days pregnant adult female placenta

and extra embryonic tissue c0.00032 1.6 4.62E-05 1.9 0.00561 2.8 ↑

Chr 7chr7:27587534-

275875934732475C15Rik

Mus musculus RIKEN cDNA 4732475C15 gene

(4732475C15Rik), mRNA [NM_001024726]0.04421 2.1 0.00781 -1.7 0.00158 3.5 ↕

Chr 7chr7:30486569-

30487019Dmkn

Mus musculus dermokine (Dmkn), transcript variant 1,

mRNA [NM_028618]0.00138 2.1 0.002 2.2 0.02199 1.6 ↑

Chr 7chr7:35425399-

35425340Dpy19l3

Mus musculus 13 days embryo male testis cDNA, RIKEN

full-length enriched library0.01848 -1.7 0.01086 -1.9 0.03525 2.0 ↕

Chr 7chr7:43516868-

43516927Ceacam18

Mus musculus CEA-related cell adhesion molecule 1

(Ceacam18), mRNA [NM_028236]4.32E-09 3.3 7.35E-06 2.4 0.0002 1.5 ↑

Chr 7chr7:43562415-

43562474Klk14

Mus musculus kall ikrein related-peptidase 14 (Klk14),

mRNA [NM_174866]0.02523 -1.7 0.00418 -2.3 0.0002 -2.5 ↓

Chr 7chr7:48315423-

48315364Mrgprb1

Mus musculus MAS-related GPR, member B1 (Mrgprb1),

mRNA [NM_205810]0.03331 1.7 0.03246 1.8 0.00043 2.3 ↑

Chr 7chr7:62260223-

62260282Magel2

Mus musculus 12 days embryo spinal cord cDNA, RIKEN

full-length enriched library0.03782 -1.6 0.03046 -1.7 0.04074 -1.5 ↓

Chr 7chr7:68105625-

68105684Igf1r

Mus musculus insulin-like growth factor I receptor (Igf1r),

mRNA [NM_010513]0.02123 -1.6 0.00323 -2.0 0.02358 -1.7 ↓

Chr 7chr7:70227061-

70227002Nr2f2

Mus musculus 11 days embryo whole body cDNA, RIKEN

full-length enriched library,0.00173 -1.8 0.01045 -1.7 0.0018 -2.0 ↓

Chr 7chr7:79610909-

79610850Pex11a

Mus musculus peroxisomal biogenesis factor 11a

(Pex11a), mRNA [NM_011068]0.01909 -1.7 0.00052 -2.6 0.02393 -1.8 ↓

Chr 7chr7:97425627-

974255681810020D17Rik

Mus musculus RIKEN cDNA 1810020D17 gene

(1810020D17Rik), mRNA [NM_183251]0.00159 -1.6 0.02922 -1.5 0.05219 -2.4 ↓

Chr 8chr8:123052462-

1230524031700120B06Rik

Mus musculus RIKEN cDNA 1700120B06 gene

(1700120B06Rik), mRNA [NM_001033980]0.00032 -2.1 0.01797 -1.7 0.01131 -1.5 ↓

Chr 8chr8:27217710-

27217651Ddhd2

PREDICTED: Mus musculus DDHD domain containing 2

(Ddhd2), mRNA [XM_356065]0.02397 -1.5 0.00091 -2.0 0.02039 -1.4 ↓

Chr 8chr8:41102680-

41102621Msr1

Mus musculus macrophage scavenger receptor 1 (Msr1),

mRNA [NM_031195]0.00128 2.9 0.01343 2.4 0.02073 2.0 ↑

Chr 8chr8:45069326-

45069267Adam26a

Mus musculus a disintegrin and metallopeptidase domain

26A (testase 3) (Adam26a)0.02514 -1.5 0.03237 -1.7 0.00427 -1.8 ↓

Chr 8chr8:48345949-

48346008Irf2

Mus musculus interferon regulatory factor 2 (Irf2), mRNA

[NM_008391]0.03653 -1.5 0.00421 -2.0 0.03536 -1.5 ↓

242

Chr 8chr8:48345949-

48346008Irf2

Mus musculus interferon regulatory factor 2 (Irf2), mRNA

[NM_008391]0.03653 -1.5 0.00421 -2.0 0.03536 -1.5 ↓

Chr 8chr8:50368233-

50368292AK032387

Mus musculus adult male olfactory brain cDNA, RIKEN full-

length enriched library0.02994 -2.0 0.00325 -3.0 0.00776 1.6 ↕

Chr 8chr8:88323251-

88323310ENSMUST00000047705

RIKEN cDNA 4921524J17 gene

[Source:MarkerSymbol;Acc:MGI:1913964]

[ENSMUST0000004

0.02605 1.6 0.0483 1.6 0.03786 1.5 ↑

Chr 8chr8:97700999-

97701058Ccl17

Mus musculus chemokine (C-C motif) l igand 17 (Ccl17),

mRNA [NM_011332]0.05016 -1.5 0.00069 -2.4 0.00359 -1.9 ↓

Chr 8chr8:98128622-

98128563Cngb1b

Mus musculus cyclic nucleotide gated channel beta 1b,

mRNA (cDNA clone IMAGE:4507.36E-05 3.3 0.00075 3.0 0.00294 2.1 ↑

Chr 9chr9:107206448-

1072065076430571L13Rik

Mus musculus RIKEN cDNA 6430571L13 gene

(6430571L13Rik), mRNA [NM_175486]0.00015 -2.1 0.00952 -1.7 0.02058 -1.8 ↓

Chr 9chr9:108841747-

108841806Col7a1

Mus musculus procollagen, type VII, alpha 1 (Col7a1),

mRNA [NM_007738]8.32E-06 2.6 0.00037 2.2 0.00247 -1.8 ↕

Chr 9chr9:113509748-

113509689Pdcd6ip Mus musculus mRNA for mKIAA1375 protein [AK129340] 0.00942 1.7 0.00513 -1.6 0.01167 1.5 ↕

Chr 9chr9:121426667-

121426608Lyzl4 Mus musculus lysozyme-like 4 (Lyzl4), mRNA [NM_026915] 0.00433 1.9 0.03306 1.7 0.00261 1.9 ↑

Chr 9chr9:122229563-

122229622Abhd5

Mus musculus abhydrolase domain containing 5 (Abhd5),

mRNA [NM_026179]0.00114 1.5 0.00023 1.7 0.00059 1.5 ↑

Chr 9chr9:49257408-

49257349Ncam1

Mouse mRNA for 3'-end of NCAM-140 and NCAM-180

isoforms [X15052]0.02378 1.8 0.00635 1.5 2.65E-05 -1.9 ↕

Chr 9chr9:50503395-

50503336Hspb2

Mus musculus heat shock protein 2 (Hspb2), mRNA

[NM_024441]0.0333 -1.5 0.01575 -2.2 0.02169 -1.5 ↓

Chr 9chr9:59189752-

59189693Arih1

Mus musculus ariadne ubiquitin-conjugating enzyme E2

binding protein homolog 1 (0.00754 1.5 0.00854 1.5 0.00097 1.9 ↑

Chr 9chr9:66645435-

66645376Rab8b

Mus musculus RAB8B, member RAS oncogene family

(Rab8b), mRNA [NM_173413]0.0102 1.5 0.00889 1.6 7.07E-05 1.7 ↑

Chr 9chr9:7085054-

7084995Dync2h1

Mus musculus dynein cytoplasmic 2 heavy chain 1

(Dync2h1), mRNA [NM_029851]0.00026 -1.5 0.00316 -3.9 0.00519 -1.7 ↓

Chr 9chr9:72493465-

72493524LOC639396

Mus musculus similar to neural precursor cell expressed,

developmentally down-re0.00551 -2.3 0.00014 -3.9 0.00051 -3.9 ↓

Chr 9chr9:78476986-

78477045Cd109

Mus musculus CD109 antigen (Cd109), mRNA

[NM_153098]0.00198 1.6 0.01675 1.6 0.05398 1.7 ↑

Chr 9chr9:88251017-

88250958Syncrip

Mus musculus synaptotagmin binding, cytoplasmic RNA

interacting protein (Syncrip0.00999 1.5 0.00502 1.6 0.0178 1.5 ↑

Chr 9chr9:8968284-

89682259030420J04Rik

Mus musculus adult male colon cDNA, RIKEN full-length

enriched library, clone:900.0004 1.8 0.00138 1.5 0.04475 1.5 ↑

Chr 9chr9:95666837-

956667781700065D16Rik

Mus musculus RIKEN cDNA 1700065D16 gene

(1700065D16Rik), mRNA [NM_028533]0.00053 -2.7 0.01254 -2.1 0.00011 -2.8 ↓

Chr 10chr10:107951264-

107951205A830054O04Rik

Mus musculus 10 days neonate cortex cDNA, RIKEN full-

length enriched library, cl0.02837 -2.2 0.03239 2.3 0.00179 -3.1 ↕

Chr 10chr10:21802648-

21802589E030030I06Rik

PREDICTED: Mus musculus RIKEN cDNA E030030I06 gene,

transcript variant 1 (E030030.0266 -1.8 0.01213 -2.1 0.03615 -1.7 ↓

Chr 10chr10:23630760-

23630819Taar2

Mus musculus trace amine-associated receptor 2 (Taar2),

mRNA [NM_001007266]0.04811 -1.8 0.00478 -2.6 0.04089 -1.7 ↓

Chr 10chr10:57194700-

57199900Hsf2

Mus musculus heat shock factor 2 (Hsf2), mRNA

[NM_008297]0.01465 1.7 0.0049 2.1 0.00095 2.4 ↑

Chr 10chr10:58619999-

58620058Ankrd57

Mus musculus adult male thymus cDNA, RIKEN full-length

enriched library, clone:50.00077 2.1 0.0149 1.7 0.02384 1.7 ↑

Chr 10chr10:62641277-

62641336Herc4

Mus musculus hect domain and RLD 4 (Herc4), mRNA

[NM_026101]0.02183 1.8 0.00044 1.5 0.01128 1.8 ↑

Chr 10chr10:74489788-

74489847Rab36

Mus musculus RAB36, member RAS oncogene family

(Rab36), mRNA [NM_029781]0.00071 -1.9 0.02371 -1.6 0.05012 1.6 ↕

Chr 10chr10:75381333-

75381392Ndg2

Mus musculus Nur77 downstream gene 2 (Ndg2), mRNA

[NM_175329]0.00079 -2.0 0.00063 -2.2 0.02397 -1.6 ↓

Chr 10chr10:79806207-

79806148Uqcr

Mus musculus ubiquinol-cytochrome c reductase (6.4kD)

subunit (Uqcr), mRNA [NM_00.01409 -1.6 0.01473 -1.7 0.02244 -1.6 ↓

Chr 10chr10:90420963-

90420904Apaf1

Mus musculus apoptotic peptidase activating factor 1

(Apaf1), transcript variant0.0003 2.1 0.0293 1.6 0.02322 1.6 ↑

Chr 10chr10:92658506-

92658565Pctk2

Mus musculus 0 day neonate thymus cDNA, RIKEN full-

length enriched library, clon0.03008 1.5 0.01651 -2.0 0.02634 1.5 ↕

Chr 10chr10:93580081-

93580140Nr2c1

Mus musculus 0 day neonate eyeball cDNA, RIKEN full-

length enriched library, clo0.05334 -2.0 0.00421 -3.2 0.0273 -2.0 ↓

Chr 11chr11:101006492-

101006433BE980346

BE980346 UI-M-BG2-bch-f-04-0-UI.s1 NIH_BMAP_MSC_S1

Mus musculus cDNA clone UI-M-0.01329 -2.0 0.02836 -1.9 0.00078 -2.6 ↓

Chr 11chr11:120462273-

1204622141110012N22Rik

Mus musculus 18-day embryo whole body cDNA, RIKEN

full-length enriched library, 0.00028 -2.0 0.0367 -1.5 0.00487 -1.5 ↓

Chr 11chr11:29641510-

29641717Rtn4

Mus musculus reticulon 4 (Rtn4), transcript variant 1,

mRNA [NM_194054]5.17E-06 1.9 0.01747 -1.9 0.00848 1.9 ↕

Chr 11chr11:50915370-

50915429Zfp354a

Mus musculus zinc finger protein 354A (Zfp354a), mRNA

[NM_009329]0.05397 -1.7 0.04502 -1.8 0.03889 -1.6 ↓

243

Chr 11chr11:5160083-

5160024Kremen1

Mus musculus 16 days embryo head cDNA, RIKEN full-

length enriched library, clone0.03086 -1.5 0.03863 -1.6 0.00013 -2.5 ↓

Chr 11chr11:61955256-

61955315TC1644694

Q5DTQ5_MOUSE (Q5DTQ5) MKIAA4061 protein

(Fragment), partial (20%) [TC1644694]0.02513 1.7 0.04407 1.7 0.00142 2.0 ↑

Chr 11chr11:66917550-

66917609Myh3

PREDICTED: Mus musculus myosin, heavy polypeptide 3,

skeletal muscle, embryonic 0.01264 -1.6 0.03535 -1.5 0.01021 -1.6 ↓

Chr 11chr11:68790423-

68790482Slc25a35

Mus musculus solute carrier family 25, member 35

(Slc25a35), mRNA [NM_028048]0.02179 -1.6 0.00729 -1.7 0.03635 -1.6 ↓

Chr 11chr11:80680725-

80680784Spaca3

Mus musculus sperm acrosome associated 3 (Spaca3),

mRNA [NM_029367]0.0028 -2.0 0.02331 -1.8 0.00019 -2.2 ↓

Chr 12chr11:94295255-

94295196Spata20

Mus musculus spermatogenesis associated 20 (Spata20),

mRNA [NM_144827]0.0017 -1.5 0.00058 -1.6 0.04658 -1.5 ↓

Chr 12chr12:101445617-

101445558Smek1

Mus musculus SMEK homolog 1, suppressor of mek1

(Dictyostelium) (Smek1), mRNA [N0.00828 1.7 0.02801 1.6 0.00649 1.7 ↑

Chr 12chr12:108776768-

1087768271600002O04Rik

PREDICTED: Mus musculus RIKEN cDNA 1600002O04 gene

(1600002O04Rik), mRNA [XM_1474.06E-05 2.3 0.00346 1.9 0.04326 -1.8 ↕

Chr 12chr12:113637224-

113637283Tmem121

Mus musculus transmembrane protein 121 (Tmem121),

mRNA [NM_153776]0.00026 -1.6 0.00028 -1.7 0.00101 -1.5 ↓

Chr 12chr12:119632407-

119632348Itgb8

PREDICTED: Mus musculus integrin beta 8, transcript

variant 1 (Itgb8), mRNA [XM_0.01292 -1.6 0.00827 -1.7 0.02509 -2.2 ↓

Chr 12chr12:32105719-

32105660Slc26a4

Mus musculus solute carrier family 26, member 4

(Slc26a4), mRNA [NM_011867]3.98E-08 10.9 2.06E-07 11.3 9.57E-05 5.2 ↑

Chr 12chr12:53886191-

53886250AK048748

Mus musculus 0 day neonate cerebellum cDNA, RIKEN full-

length enriched library, 0.00185 2.1 0.00072 2.5 0.01676 1.8 ↑

Chr 12chr12:55812459-

55812400Baz1a

PREDICTED: Mus musculus bromodomain adjacent to zinc

finger domain 1A (Baz1a), m0.00056 1.9 0.0186 -1.8 0.01755 1.5 ↕

Chr 12chr12:71377444-

71377503Txndc1

Mus musculus thioredoxin domain containing 1 (Txndc1),

mRNA [NM_028339]0.017 1.6 0.01316 1.8 0.00082 2.0 ↑

Chr 12chr12:88338961-

883390201810035L17Rik

Mus musculus partial mRNA for hypothetical protein,

clone Telethon(Italy_B41)_St0.05006 -1.5 0.01728 -1.7 0.05196 -1.6 ↓

Chr 12chr12:90476912-

90476971AK081115

Mus musculus 10 days neonate cerebellum cDNA, RIKEN

full-length enriched library0.00263 -1.7 0.00622 -1.7 5.61E-05 -2.1 ↓

Chr 12chr12:99183734-

99183793NAP046120-1 Unknown 0.02329 -1.5 0.01087 -1.6 0.00124 -1.8 ↓

Chr 13chr13:102837027-

102836968AK085102

Mus musculus 13 days embryo lung cDNA, RIKEN full-

length enriched library, clone0.03004 1.5 0.04945 1.5 0.02859 -2.3 ↓

Chr 13chr13:32850377-

32850318Serpinb1a

Mus musculus serine (or cysteine) peptidase inhibitor,

clade B, member 1a (Serpi2.99E-08 2.6 0.00106 1.6 8.18E-07 2.0 ↑

Chr 13chr13:61205022-

61204963Cts6 Mus musculus cathepsin 6 (Cts6), mRNA [NM_021445] 0.04363 -1.7 0.01404 -2.1 0.01565 -2.0 ↓

Chr 13chr13:64149966-

64149907Zfp367

Mus musculus zinc finger protein 367 (Zfp367), mRNA

[NM_175494]0.00028 1.8 0.00023 2.0 0.04006 1.9 ↑

Chr 13chr13:74132349-

74131679Slc6a18

Mus musculus solute carrier family 6 (neurotransmitter

transporter), member 18 (0.00587 -1.5 0.00317 -1.6 0.04035 1.6 ↕

Chr 13chr13:74815274-

748152161500032O14Rik

Mus musculus adult male cerebellum cDNA, RIKEN full-

length enriched library, clo0.015 -1.8 4.34E-05 -3.5 0.01143 -1.8 ↓

Chr 13chr13:84141092-

84141151Mef2c

Mus musculus myocyte enhancer factor 2C (Mef2c), mRNA

[NM_025282]0.00767 -2.0 0.01344 -2.0 0.00122 -2.2 ↓

Chr 13chr13:94406010-

94442522Homer1

homer homolog 1 (Drosophila)

[Source:MarkerSymbol;Acc:MGI:1347345] [ENSMUST000000.00759 1.5 0.00131 1.7 0.01926 1.5 ↑

Chr 14chr14:100335001-

100334942Tbc1d4

Mus musculus activated spleen cDNA, RIKEN full-length

enriched library, clone:F80.02166 1.9 0.0214 1.5 0.00183 -2.1 ↕

Chr 14chr14:100814710-

100814769Lmo7

CG02_13_B03.x1 FH CG02 Mus musculus cDNA clone

CG02_13_B03 similar to LIM domain8.03E-10 3.4 4.55E-05 2.0 0.04728 1.5 ↑

Chr 14chr14:118120528-

118120469Ugcgl2

PREDICTED: Mus musculus UDP-glucose ceramide

glucosyltransferase-like 2, transcr0.0062 1.7 0.00674 -2.2 0.01131 1.7 ↕

Chr 14chr14:31838178-

31838237Lrrc18

Mus musculus leucine rich repeat containing 18 (Lrrc18),

mRNA [NM_026253]0.03136 -1.5 0.00137 -2.0 0.0069 -2.3 ↓

Chr 14chr14:32207646-

32207587Mapk8

Mus musculus mitogen activated protein kinase 8, mRNA

(cDNA clone MGC:62245 IMAG0.03124 1.7 0.001 2.5 0.02655 1.6 ↑

Chr 14chr14:33358618-

33358559Ldb3

Mus musculus LIM domain binding 3 (Ldb3), transcript

variant 1, mRNA [NM_011918]0.00194 -1.9 0.01198 -1.7 0.00362 -1.7 ↓

Chr 14chr14:54836480-

54836421Cbln3

Mus musculus cerebellin 3 precursor protein (Cbln3),

mRNA [NM_019820]0.04601 1.6 0.00809 2.1 0.00091 3.4 ↑

Chr 14chr14:66295961-

662960209430085L16Rik

Mus musculus 12 days embryo embryonic body between

diaphragm region and neck cDN0.00151 -3.4 0.02438 -2.5 0.00037 -4.2 ↓

Chr 14chr14:68198527-

68198468Slc25a37

Mus musculus 12 days embryo embryonic body between

diaphragm region and neck cDN0.01073 2.1 0.02783 2.0 0.00601 1.8 ↑

Chr 14chr14:87313420-

87313361Pcdh20

Mus musculus protocadherin 20 (Pcdh20), mRNA

[NM_178685]5.08E-05 -3.0 4.20E-05 -3.5 0.00153 -3.1 ↓

Chr 14chr14:8988272-

8962068NAP064811-1 Unknown 0.00626 -2.2 0.05147 -1.8 0.00292 -2.4 ↓

Chr 15chr15:10634379-

10634320Rai14

Mus musculus adult male corpora quadrigemina cDNA,

RIKEN full-length enriched li0.01661 -1.7 0.03005 -1.7 0.00745 -2.0 ↓

Chr 15chr15:26733928-

26733869Fbxl7

Mus musculus 12 days embryo eyeball cDNA, RIKEN full-

length enriched library, cl0.00091 -2.4 0.00299 -2.4 0.00048 -3.2 ↓

Chr 15chr15:44502535-

445024765730410E15Rik

Mus musculus adult male hippocampus cDNA, RIKEN full-

length enriched library, cl0.04512 -1.7 0.00304 2.9 0.00549 1.8 ↕

Chr 15chr15:54864258-

54861192Taf2

PREDICTED: Mus musculus TAF2 RNA polymerase II, TATA

box binding protein (TBP)-a0.03539 1.5 0.01457 1.8 0.00921 1.8 ↑

Chr 15chr15:6371412-

6371855Dab2

Mus musculus disabled homolog 2 (Drosophila) (Dab2),

transcript variant 3, mRNA 0.00126 1.7 0.0057 1.9 0.00365 1.8 ↑

244

Chr 15chr15:26733928-

26733869Fbxl7

Mus musculus 12 days embryo eyeball cDNA, RIKEN full-

length enriched library, cl0.00091 -2.4 0.00299 -2.4 0.00048 -3.2 ↓

Chr 15chr15:44502535-

445024765730410E15Rik

Mus musculus adult male hippocampus cDNA, RIKEN full-

length enriched library, cl0.04512 -1.7 0.00304 2.9 0.00549 1.8 ↕

Chr 15chr15:54864258-

54861192Taf2

PREDICTED: Mus musculus TAF2 RNA polymerase II, TATA

box binding protein (TBP)-a0.03539 1.5 0.01457 1.8 0.00921 1.8 ↑

Chr 15chr15:6371412-

6371855Dab2

Mus musculus disabled homolog 2 (Drosophila) (Dab2),

transcript variant 3, mRNA 0.00126 1.7 0.0057 1.9 0.00365 1.8 ↑

Chr 15chr15:6706780-

67068394921505C17Rik

Mus musculus RIKEN cDNA 4921505C17 gene

(4921505C17Rik), mRNA [NM_030168]0.02965 2.0 0.02166 1.7 0.00587 2.4 ↑

Chr 15chr15:74651369-

74651428Hemt1 Mus musculus mRNA for HemT-3 protein [AJ242831] 2.91E-05 5.8 0.00915 3.0 0.0291 -2.0 ↕

Chr 15chr15:82203312-

82203253Cyp2d22

Mus musculus 15 days embryo head cDNA, RIKEN full-

length enriched library, clone0.02451 -2.1 0.02195 -2.3 0.00561 -2.7 ↓

Chr 15chr15:9012640-

90180671110020G09Rik

Mus musculus 4 days neonate male adipose cDNA, RIKEN

full-length enriched librar0.01933 1.5 0.00181 1.7 0.01149 1.4 ↑

Chr 15chr15:90847007-

90846948AK078857

Mus musculus adult male colon cDNA, RIKEN full-length

enriched library, clone:900.02065 -2.9 0.05386 -2.6 0.00052 -4.7 ↓

Chr 15chr15:98497981-

98497922Rnd1

Mus musculus Rho family GTPase 1 (Rnd1), mRNA

[NM_172612]0.00047 1.8 0.02526 1.5 0.03859 -1.5 ↕

Chr 15chr15:98862343-

98862534Tuba6

Mus musculus tubulin, alpha 6 (Tuba6), mRNA

[NM_009448]0.00117 2.1 0.01048 1.9 0.01968 1.8 ↑

Chr 16chr16:032698506-

032698565Muc4 Mus musculus mucin 4 (Muc4), mRNA [NM_080457] 0.02166 -1.6 0.04541 -1.8 8.66E-05 -2.5 ↓

Chr 16chr16:20573640-

20573939Psmd2

Mus musculus proteasome (prosome, macropain) 26S

subunit, non-ATPase, 2 (Psmd2),0.01744 1.5 0.02008 1.6 0.00983 1.5 ↑

Chr 16chr16:49831819-

49831878Cd47

Mus musculus adult male hippocampus cDNA, RIKEN full-

length enriched library, cl0.02871 -1.7 0.02111 -1.9 0.03457 -1.7 ↓

Chr 16chr16:76234512-

76234453Nrip1

Mus musculus 16 days embryo lung cDNA, RIKEN full-

length enriched library, clone1.05E-06 1.7 1.05E-05 1.8 0.00012 -2.3 ↕

Chr 16chr16:77384095-

773841532810055G20Rik

Mus musculus 15 days embryo male testis cDNA, RIKEN

full-length enriched library0.03231 -1.8 0.03414 -2.1 0.03653 -1.8 ↓

Chr 16chr16:8561558-

8564000Pmm2

Mus musculus phosphomannomutase 2 (Pmm2), mRNA

[NM_016881]4.45E-05 1.8 0.01082 1.5 1.04E-05 1.7 ↑

Chr 17chr17:13627718-

13627777Mllt4 PREDICTED: Mus musculus myeloid [XM_890447] 0.05372 -1.6 0.00445 -2.9 0.00404 -2.2 ↓

Chr 17chr17:15104504-

15104563AK044799

Mus musculus 9.5 days embryo parthenogenote cDNA,

RIKEN full-length enriched lib0.04372 -1.6 0.01864 -1.9 0.00722 -2.0 ↓

Chr 17chr17:21356011-

213560703110052M02Rik

Mus musculus 12 days embryo eyeball cDNA, RIKEN full-

length enriched library, cl0.01608 -1.6 0.00017 -2.4 0.01389 -1.9 ↓

Chr 17chr17:21524657-

21524598MGC29393

Mus musculus hypothetical gene MGC29393 (MGC29393),

mRNA [NM_029281]0.00407 -1.9 0.00036 -2.5 0.0152 -1.6 ↓

Chr 17chr17:28565376-

28565435Brpf3

Mus musculus cDNA, RIKEN full-length enriched library,

clone:M5C1075N18 product:0.01111 1.9 0.03633 1.8 0.04881 1.6 ↑

Chr 17chr17:32280266-

32280325Cyp4f16

Mus musculus cytochrome P450, family 4, subfamily f,

polypeptide 16 (Cyp4f16), m0.00112 1.8 0.04189 1.5 0.00248 1.6 ↑

Chr 17chr17:63608687-

63609305Fert2

Mus musculus fer (fms/fps related) protein kinase, testis

specific 2 (Fert2), tr0.04928 1.5 0.00581 1.9 0.02297 1.5 ↑

Chr 17chr17:74130419-

741220320610016J10Rik

Mus musculus 15 days embryo head cDNA, RIKEN full-

length enriched library, clone0.00281 1.5 0.03551 -1.5 0.00533 1.6 ↕

Chr 17chr17:84607731-

84607790Abcg8

Mus musculus ATP-binding cassette, sub-family G

(WHITE), member 8 (Abcg8), mRNA 0.00173 -2.1 0.00011 -2.9 1.89E-08 -3.5 ↓

Chr 18chr18:12286520-

12286579Riok3

Mus musculus RIO kinase 3 (yeast) (Riok3), mRNA

[NM_024182]9.21E-09 1.9 1.24E-05 1.6 0.00016 1.6 ↑

Chr 18chr18:34761926-

34761867Cdc23

Mus musculus CDC23 (cell division cycle 23, yeast,

homolog) (Cdc23), mRNA [NM_177.70E-07 1.5 0.03039 1.6 0.00056 1.9 ↑

Chr 18chr18:61421963-

61421904Ppargc1b

Mus musculus 3 days neonate thymus cDNA, RIKEN full-

length enriched library, clo0.00185 -1.7 0.00013 -2.2 0.00353 -1.6 ↓

Chr 18chr18:66984818-

66984759Mc4r

Mus musculus melanocortin 4 receptor (Mc4r), mRNA

[NM_016977]0.00035 2.2 0.02516 1.7 0.00568 1.7 ↑

Chr 18chr18:78263922-

78263863Slc14a1

Mus musculus solute carrier family 14 (urea transporter),

member 1 (Slc14a1), mR0.00086 2.2 0.01359 1.7 0.00112 2.0 ↑

Chr 19chr19:17740656-

17740597Pcsk5

PREDICTED: Mus musculus proprotein convertase

subtil isin [XM_129214]0.02247 -1.9 0.00442 -1.9 0.0288 -2.0 ↓

Chr 19chr19:3407031-

3407090Mtl5

Mus musculus metallothionein-like 5, testis-specific

(tesmin) (Mtl5), transcript0.00028 2.0 0.02309 1.5 0.00124 1.7 ↑

Chr 19chr19:42653405-

42653346Loxl4

Mus musculus lysyl oxidase-like 4 (Loxl4), mRNA

[NM_053083]0.03309 1.5 0.05063 1.5 0.00041 1.9 ↑

Chr 19chr19:47673498-

47673557Slk

Mus musculus STE20-like kinase (yeast) (Slk), mRNA

[NM_009289]0.00201 1.6 0.00066 1.8 0.00563 1.5 ↑

Chr XchrX:102487244-

102487185EG436227

PREDICTED: Mus musculus similar to RAN binding protein

5 (LOC436227), mRNA [XM_60.00547 2.5 0.01508 2.4 0.00166 2.8 ↑

Chr XchrX:118513543-

118513484Nap1l3

Mus musculus nucleosome assembly protein 1-like 3

(Nap1l3), mRNA [NM_138742]0.0169 -1.5 0.0493 -1.5 0.0275 -2.7 ↓

Chr XchrX:12718478-

12716303Cask

Mus musculus calcium/calmodulin-dependent serine

protein kinase (MAGUK family) (0.02564 1.7 0.05268 1.7 0.03362 1.6 ↑

Chr XchrX:131066050-

1310650791700129I15Rik

Mus musculus RIKEN cDNA 1700129I15 gene

(1700129I15Rik), mRNA [NM_030106]0.04256 -1.8 0.01327 -2.2 0.02143 -1.8 ↓

Chr XchrX:147262104-

147263076Huwe1

Mus musculus HECT, UBA and WWE domain containing 1

(Huwe1), mRNA [NM_021523]0.05109 1.9 0.01007 2.7 0.03339 2.0 ↑

Chr XchrX:155969795-

155968708Ppef1

PREDICTED: Mus musculus protein phosphatase with EF

hand calcium-binding domain 0.01617 1.8 0.02792 1.8 0.00166 2.2 ↑

Chr XchrX:32601604-

32601663Il13ra1

Mus musculus interleukin 13 receptor, alpha 1 (Il13ra1),

mRNA [NM_133990]5.03E-08 2.6 0.00076 1.7 0.00363 1.7 ↑

245

Chr XchrX:147262104-

147263076Huwe1

Mus musculus HECT, UBA and WWE domain containing 1

(Huwe1), mRNA [NM_021523]0.05109 1.9 0.01007 2.7 0.03339 2.0 ↑

Chr XchrX:155969795-

155968708Ppef1

PREDICTED: Mus musculus protein phosphatase with EF

hand calcium-binding domain 0.01617 1.8 0.02792 1.8 0.00166 2.2 ↑

Chr XchrX:32601604-

32601663Il13ra1

Mus musculus interleukin 13 receptor, alpha 1 (Il13ra1),

mRNA [NM_133990]5.03E-08 2.6 0.00076 1.7 0.00363 1.7 ↑

246

Appendix II

Genome wide candidate genes associated with autotomy in the spinal cord. The expression of

these genes differentially contrasted between A high vs. A sham, A low, and B low autotomy

mice at a p-value<0.05 and a -1.5>fold change>1.5

247

p-value FC p-value FC p-value FC

A_51_P144143 Unknown 0.00311 2.2 0.02768 1.8 0.004705 2.0 ↑

A_52_P756294 Unknown 0.028155 -1.8 0.006459 -2.2 0.002368 -2.5 ↓

1

chr1:173419775-

173419834 Cd244

Mus musculus CD244 natural kil ler cell

receptor 2B4 (Cd244), mRNA [NM_018729] 0.000589 4.7 0.004608 3.5 0.001678 4.5 ↑

1 chr1:93204297-93204356 BC056923

Mus musculus cDNA sequence BC056923

(BC056923), mRNA [NM_173395] 0.000575 -2.3 0.052241 -1.6 0.000315 4.9 ↕

2

chr2:152840517-

152842444 Hck

Mus musculus hemopoietic cell kinase (Hck),

mRNA [NM_010407] 0.000439 1.8 0.01733 1.5 0.002317 1.5 ↑

2

chr2:158194053-

158194111 BC054059

Mus musculus cDNA sequence BC054059

(BC054059), mRNA [NM_145635] 0.010497 1.5 0.010769 1.5 0.000432 -2.6 ↕

2 chr2:85380612-85380671 Olfr996

Mus musculus olfactory receptor 996

(Olfr996), mRNA [NM_146437] 0.05319 1.5 0.038565 1.6 0.020682 1.7 ↑

3

chr3:015348529-

015348470 Sirpb1

Mus musculus 2 days pregnant adult female

ovary cDNA, RIKEN full-length enriched 0.008169 2.7 0.027844 2.3 0.000311 3.4 ↑

3

chr3:096369943-

096369880 Fcgr1

Mus musculus strain AB/H (Biozzi) high

affinity immunoglobulin gamma Fc receptor 0.006367 2.1 0.035451 1.6 0.030384 1.7 ↑

4

chr4:133365725-

133365666 Cd52

Mus musculus CD52 antigen (Cd52), mRNA

[NM_013706] 0.012086 1.9 0.033703 1.7 0.027094 1.6 ↑

4 chr4:44546223-44546164 Pax5

Mus musculus adult male aorta and vein

cDNA, RIKEN full-length enriched library, 0.031117 -1.5 0.004985 -1.7 0.00291 -1.9 ↓

4 chr4:57804030-57804089 Palm2

Mus musculus paralemmin 2 (Palm2), mRNA

[NM_172868] 0.006506 1.5 0.012424 1.8 0.008827 1.5 ↑

5

chr5:115709119-

115709178 Msi1

Mus musculus 12 days embryo spinal

ganglion cDNA, RIKEN full-length enriched lib 0.039337 -2.1 0.049721 -2.1 0.017942 -2.2 ↓

5

chr5:137960881-

137960822 6430598A04Rik

Mus musculus RIKEN cDNA 6430598A04 gene

(6430598A04Rik), mRNA [NM_175521] 0.013192 -1.6 0.040411 -1.5 0.002022 -1.6 ↓

5

chr5:138082142-

138082083 Pilrb1

Mus musculus paired immunoglobin-like type

2 receptor beta 1 (Pilrb1), mRNA [NM_ 0.031787 2.0 0.021466 2.2 0.000677 2.7 ↑

5

chr5:151255290-

151255349 Kl

Mus musculus mRNA for secreted isoform of

Klotho protein, complete cds [AB010088 0.043075 -1.5 0.019356 -1.6 0.042877 -1.5 ↓

5 chr5:26334255-26332802 NAP030421-1 Unknown 0.000382 2.9 0.045309 1.8 0.000272 2.4 ↑

6

chr6:119738783-

119738724 Erc1

Mus musculus ELKS/RAB6-interacting/CAST

family member 1 (Erc1), transcript varia 0.042747 1.7 0.007141 1.8 0.008765 1.6 ↑

6

chr6:125652152-

125652211 Vwf

Mus musculus Von Willebrand factor

homolog (Vwf), mRNA [NM_011708] 0.000629 2.1 0.007385 1.8 0.011167 1.7 ↑

6

chr6:128336896-

128336955 TC1608296

U74612 forkhead box M1A (Homo sapiens)

(exp=-1; wgp=0; cg=0), partial (7%) [TC16 0.001126 -2.1 0.030692 -1.6 0.000403 -2.2 ↓

6 chr6:48676977-48677036 Gimap5

Mus musculus 0 day neonate lung cDNA,

RIKEN full-length enriched library, clone: 0.041022 1.7 0.052663 1.7 0.000418 2.2 ↑

6 chr6:70633716-70652162 L18942

Mus musculus immunoglobulin l ight chain

AbH130 VJ region mRNA, partial cds. [L18 0.006933 -1.8 0.016065 -1.7 0.002701 -2.3 ↓

7

chr7:100058635-

100058694 Kcne3

Mus musculus potassium voltage-gated

channel, Isk-related subfamily, gene 3 (Kcn 0.000857 4.7 0.024391 2.7 0.00055 4.2 ↑

7

chr7:100811781-

100811722 P2ry6

Mus musculus pyrimidinergic receptor P2Y, G-

protein coupled, 6 (P2ry6), mRNA [NM 0.000279 2.0 0.014567 1.6 0.011099 1.5 ↑

7

chr7:102452451-

102452510 Olfr550

Mus musculus olfactory receptor 550

(Olfr550), mRNA [NM_147104] 0.018138 -1.6 0.001984 -2.0 0.007857 -1.8 ↓

7

chr7:115332904-

115332845 Sox6

Mus musculus SRY-box containing gene 6

(Sox6), transcript variant 1, mRNA [NM_01 0.046212 -1.9 0.016585 2.3 0.006843 -2.5 ↕

7 chr7:29288473-29288532 6330444E15Rik

Mus musculus RIKEN cDNA 6330444E15 gene

(6330444E15Rik), mRNA [NM_001002767] 0.017829 -3.1 0.023119 -3.1 0.004761 -3.4 ↓

7 chr7:3315702-3315643 Lilrb3

Mus musculus leukocyte immunoglobulin-like

receptor, subfamily B (with TM and IT 0.020983 2.2 0.021992 2.1 0.053998 1.7 ↑

7 chr7:34829102-34829161 Cebpa

Mus musculus CCAAT/enhancer binding

protein (C/EBP), alpha (Cebpa), mRNA

[NM_007 0.023195 1.5 0.027609 1.5 0.027393 1.5 ↑

8 chr8:19023541-19023482 Defb38

Mus musculus defensin beta 38 (Defb38),

mRNA [NM_183036] 0.039239 -1.8 0.049692 -1.8 0.000759 -3.2 ↓

8 chr8:69447128-69447187 Tktl2

Mus musculus adult male testis cDNA, RIKEN

full-length enriched library, clone:4 0.022256 -1.7 0.035506 1.5 0.000427 1.8 ↕

8 chr8:87987554-87987613 Man2b1

Mus musculus mannosidase 2, alpha B1

(Man2b1), mRNA [NM_010764] 0.000316 1.5 0.001743 1.5 6.23E-05 1.5 ↑

9 chr9:20779144-20779203 Icam1

Mus musculus intercellular adhesion

molecule, mRNA (cDNA clone MGC:6195

IMAGE:35 0.003055 2.0 0.041917 1.6 0.002198 1.9 ↑

9 chr9:51676988-51677047 4933407I05Rik

Mus musculus adult male testis cDNA, RIKEN

full-length enriched library, clone:4 0.02292 1.6 0.029478 1.6 0.004362 1.6 ↑

9 chr9:88997625-89005486 Bcl2a1b

Mus musculus B-cell leukemia/lymphoma 2

related protein A1b (Bcl2a1b), mRNA [NM_ 0.000314 2.7 0.015572 1.9 0.012493 2.4 ↑

10

chr10:74401286-

74401227 Rtdr1

Mus musculus adult male testis cDNA, RIKEN

full-length enriched library, clone:4 0.016465 -1.8 0.041637 -1.7 0.000609 -2.2 ↓

10

chr10:78696506-

78696565 AJ543404

Mus musculus cDNA sequence AJ543404

(AJ543404), mRNA [NM_175936] 0.00223 -2.0 0.022038 -1.7 0.004851 -2.6 ↓

10

chr10:78988523-

78987832 Theg

Mus musculus testicular haploid expressed

gene (Theg), transcript variant 1, mRN 0.042957 -1.5 0.003788 -1.9 8.84E-05 -2.2 ↓

10

chr10:79295284-

79295509 NAP025923-1 Unknown 0.020349 -2.3 0.047093 -2.1 0.000255 -3.9 ↓

10

chr10:97043284-

97043343 Kera

Mus musculus keratocan (Kera), mRNA

[NM_008438] 0.014682 2.9 0.048062 2.4 0.000293 3.9 ↑

A high vs. B lowGene RegulationChrom Genomic Coordinates Gene Name Description

A high vs. A sham A high vs. A low

248

11

chr11:031082433-

031082386 Mszf78

Mus musculus mRNA for mszf78, partial cds.

[AB010323] 0.039002 1.5 0.030728 1.6 0.037899 1.5 ↑

11

chr11:21273723-

21273782 EG666013

Mus musculus adult male hypothalamus

cDNA, RIKEN full-length enriched library, c 0.001124 -1.8 0.021117 -1.5 0.000242 -2.0 ↓

11 chr11:7106653-7106594 Igfbp3

Mus musculus insulin-like growth factor

binding protein 3 (Igfbp3), mRNA [NM_008 0.00819 2.0 0.047011 2.0 0.025895 1.7 ↑

11

chr11:81919403-

81919462 Ccl12

Mus musculus chemokine (C-C motif) l igand

12 (Ccl12), mRNA [NM_011331] 1.47E-05 5.6 7.71E-05 4.8 3.66E-05 3.4 ↑

12

chr12:104621405-

104621464 Serpina3f

Mus musculus serine (or cysteine) peptidase

inhibitor, clade A, member 3F (Serpi 0.009704 -2.4 0.026822 -2.2 0.013644 -2.4 ↓

12

chr12:76320159-

76320218 Rhoj

Mus musculus ras homolog gene family,

member J (Rhoj), mRNA [NM_023275] 0.000744 1.8 0.012332 1.5 0.00012 1.8 ↑

12

chr12:84999757-

84999816 Acot6

Mus musculus adult male spinal cord cDNA,

RIKEN full-length enriched library, cl 0.000452 -2.4 0.008729 -1.9 7.18E-06 -5.1 ↓

12

chr12:88879335-

88879276 3200001D21Rik

Mus musculus 14, 17 days embryo head

cDNA, RIKEN full-length enriched library, c 0.00865 -1.6 0.000695 -2.0 0.016517 -1.5 ↓

13

chr13:60905568-

60905509 TC1637884

CTL2B_MOUSE (P12400) CTLA-2-beta protein

precursor (Fragment), complete [TC16378 0.033006 1.9 0.034612 1.9 0.002748 2.2 ↑

13

chr13:95328260-

95328201 Scamp1

Mus musculus 0 day neonate cerebellum

cDNA, RIKEN full-length enriched library, 0.012249 -2.5 0.05019 -2.1 4.83E-05 -4.7 ↓

14

chr14:61706698-

61706639 AI316844

Mus musculus 3 days neonate thymus cDNA,

RIKEN full-length enriched library, clo 0.032242 1.6 0.009622 1.8 0.031679 1.5 ↑

15

chr15:66611336-

66611277 Sla

Mus musculus src-l ike adaptor (Sla),

transcript variant 1, mRNA [NM_001029841] 0.000947 2.2 0.013993 1.8 0.004177 1.8 ↑

15

chr15:78177993-

78178052 Csf2rb1

Mus musculus NOD-derived CD11c +ve

dendritic cells cDNA, RIKEN full-length enric 0.000133 1.8 8.68E-05 1.9 3.04E-05 1.7 ↑

15

chr15:81471273-

81471332 Ep300

Mus musculus 0 day neonate thymus cDNA,

RIKEN full-length enriched library, clon 0.040741 1.6 0.041864 2.1 0.014688 1.7 ↑

15

chr15:83694888-

83694829 4931407K02Rik

Mus musculus RIKEN cDNA 4931407K02 gene

(4931407K02Rik), mRNA [NM_029946] 0.042672 1.6 0.041624 1.6 0.04649 -1.7 ↕

15

chr15:99106905-

99106846 EG545136

PREDICTED: Mus musculus hypothetical

LOC545136 (LOC545136), mRNA [XM_619361] 0.009199 -1.6 0.028115 -1.5 0.000907 -1.8 ↓

16

chr16:23348323-

23348382 Rtp1

Mus musculus receptor transporter protein 1

(Rtp1), mRNA [NM_001004151] 0.048165 -1.6 0.02608 -1.8 7.48E-05 -2.6 ↓

16

chr16:36107196-

36107255 EG433016

Mus musculus NOD-derived CD11c +ve

dendritic cells cDNA, RIKEN full-length enric 0.018383 2.1 0.00012 4.2 0.006976 2.5 ↑

16

chr16:36241019-

36240960 Stfa1

Mus musculus stefin A1 (Stfa1), mRNA

[NM_001001332] 0.010563 2.5 0.035392 3.2 0.001007 2.6 ↑

16

chr16:77519972-

77520031 8030498J20Rik

Mus musculus 15 days embryo male testis

cDNA, RIKEN full-length enriched library 0.048618 1.4 0.013988 1.6 0.017843 1.5 ↑

17 chr17:4001295-4001236 4930470H14Rik

Mus musculus adult male testis cDNA, RIKEN

full-length enriched library, clone:4 0.03256 1.5 0.034325 1.5 0.007483 1.6 ↑

18

chr18:52457867-

52457926 Ftmt

Mus musculus ferritin mitochondrial (Ftmt),

mRNA [NM_026286] 0.002504 -1.7 0.002061 -1.7 0.003922 -1.6 ↓

19

chr19:011657146-

011657087 Ms4a6d

Mus musculus mRNA for MS4A11, complete

cds. [AB026047] 0.000849 2.8 0.034682 1.9 0.000887 2.3 ↑

19

chr19:11538135-

11538194 Ms4a6c

Mus musculus membrane-spanning 4-

domains, subfamily A, member 6C (Ms4a6c),

mRNA 0.002279 2.7 0.002194 2.8 0.007212 2.0 ↑

19

chr19:11596938-

11596997 Ms4a6b

Mus musculus membrane-spanning 4-

domains, subfamily A, member 6B (Ms4a6b),

mRNA 0.004275 2.8 0.018882 2.3 0.026919 1.9 ↑

19

chr19:11619895-

11621871 Ms4a4d

Mus musculus membrane-spanning 4-

domains, subfamily A, member 4D (Ms4a4d),

mRNA 0.000629 3.7 0.032947 2.2 0.000817 5.9 ↑

19

chr19:17396465-

17396524 NAP123465-1 Unknown 0.029378 -1.5 0.003171 -1.9 0.006154 -2.1 ↓

19

chr19:32729219-

32729278 Papss2

Mus musculus 3'-phosphoadenosine 5'-

phosphosulfate synthase 2 (Papss2), mRNA

[NM 0.006712 1.5 0.029386 1.6 0.008858 1.5 ↑

19

chr19:34189048-

34188989 Ankrd22

Mus musculus ankyrin repeat domain 22

(Ankrd22), mRNA [NM_024204] 0.03128 1.5 0.041699 1.5 0.006589 1.5 ↑

19

chr19:46721922-

46721981 TC1683850

Q6P200_MOUSE (Q6P200) Cytochrome P450,

family 17, subfamily a, polypeptide 1, pa 0.013156 -1.8 0.032376 -1.7 0.023005 -1.7 ↓

X

chrX:113826670-

113826729 4933434C23Rik

Mus musculus adult male testis cDNA, RIKEN

full-length enriched library, clone:4 0.042171 -1.7 0.000622 -2.7 0.004489 -2.0 ↓

X chrX:39142125-39142066 ENSMUST00000084357

odd Oz/ten-m homolog 1 (Drosophila)

[Source:MarkerSymbol;Acc:MGI:1345185]

[ENSMU 0.006081 -1.9 0.020282 -1.7 0.011016 -1.9 ↓

X chrX:92733954-92734013 Heph

Mus musculus hephaestin (Heph), transcript

variant 2, mRNA [NM_181273] 0.010036 2.1 0.025144 1.9 0.00745 2.0 ↑

249

Appendix III

SNP variations in A vs. B mice, and other high- (C3H/HeJ, BALBc/cByJ), low- (C57BL/10J, AKR/J,

C58/J) and intermediate-autotomy strains (129S1/SvImJ, 129x1/SvJ, DBA/2J)

Gene : dbSNP Function Class

Assays

SNP A/J C3H/HeJ BALB/cByJ 129S1/SvImJ 129X1/SvJ DBA/2J C57BL/6J C57BL/10J AKR/J C58/J

Allele

(ss) Summary

Dennd3 : Intron 2 1 C C C G G C C/G

Dennd3 : Intron 2 2 C

T C C

C/T

Dennd3 : Intron 1 3 C

T C

C/T

Dennd3 : Intron 1 4 C

T C

C/T

Dennd3 : Intron 1 5 T

C T

C/T

Dennd3 : Intron 1 6 G

A

A/G

Dennd3 : Intron 1 7 T

A

A/T

Dennd3 : Intron 1 8 C

T

C/T

Dennd3 : Intron 1 9 C

A

A/C

Dennd3 : Intron 2 10 A

G A

A/G

Dennd3 : Intron 2 11 A

C A

A/C

Dennd3 : Intron 2 12 C

T C

C/T

Dennd3 : Intron 2 13 A

G A

A/G

Dennd3 : Intron 2 14 C

T

C/T

Dennd3 : Intron 2 15 T

C T T

C/T

Dennd3 : Intron 2 16 A

T A A

A/T

Dennd3 : Intron 1 17 T

C T

C/T

Dennd3 : Intron 1 18 T

G T

G/T

Dennd3 : Intron 2 19 G

G A G

A/G

Dennd3 : Intron 1 20 T

G T

G/T

Dennd3 : Intron 2 21 T

G G

G/T

Dennd3 : Intron 2 22 T

A A

A/T

Dennd3 : Intron 3 23 C C C G G C C

C

C/G

Dennd3 : Intron 3 24 A A A G G A A

A

A/G

Dennd3 : Intron 3 25 C C C T T C C

C

C/T

Dennd3 : Intron 2 26 C C C T T C

C

C/T

Dennd3 : Intron 2 27 G G G T

T G

G

G/T

Dennd3 : Intron 2 28 A A A G G A

A

A/G

Dennd3 : Intron 2 29 A A A G

G

A

A/G

Dennd3 : Intron 1 30 T T T A

T

T

A/T

Dennd3 : Intron 3 31 T T T

C C

T

C/T

Dennd3 : Intron 1 32

A G

A/G

Dennd3 : Intron 2 33 A

T A

A/T

Dennd3 : Intron 2 34 C C C T T C

C

C/T

250

Dennd3 : Intron 2 35 G

A G G

A/G

Dennd3 : Intron 1 36 G

A G

A/G

Dennd3 : Intron 3 37 C C C T T C C

C

C/T

Dennd3 : Intron 2 38 G

T G G

G/T

Dennd3 : Intron 1 39 A

T A

A/T

Dennd3 : Intron 1 40 G

A G

A/G

Dennd3 : Intron 1 41 T

C T

C/T

Dennd3 : Intron 1 42 T

C T

C/T

Dennd3 : Intron 1 43 G

A G

A/G

Dennd3 : Intron 1 44 T

G T

G/T

Dennd3 : Intron 1 45 T

C T

C/T

Dennd3 : Intron 1 46 T

C T

C/T

Dennd3 : Intron 2 47 A

G G

A/G

Dennd3 : Intron 1 48 C C C T

C

C

C/T

Dennd3 : Intron 3 49 A A A G

G

A

A/G

Dennd3 : Intron 3 50 C C C C

T C

C

C/T

Dennd3 : Intron 3 51 C C C T

C C

C

C/T

Dennd3 : Intron 3 52 C C C T

T C

C

C/T

Dennd3 : Intron 3 53 C C C T

C C

C

C/T

Dennd3 : Intron 1 54 T T T T

T

T

C/T

Dennd3 : Intron 3 55 C C C T

C C

C

C/T

Dennd3 : Intron 1 56 C

T

C C

C/T

Dennd3 : Intron 1 57 C

T

C C

C/T

Dennd3 : Intron 1 58 G G G G

G

G

A/G

Dennd3 : Intron 2 59 G G G T

G G

G

G/T

Dennd3 : Intron 3 60 T T T G

G T

T

G/T

Dennd3 : Intron 2 61 C

C G C

C/G

Dennd3 : Intron 1 62 A

G A

A/G

Dennd3 : Intron 1 63 G

A G

A/G

Dennd3 : Intron 2 64 T T T C C T

T

C/T

Dennd3 : Coding-Synonymous

2 65 C C C C C G

C

C/G

Dennd3 : Coding-Synonymous

3 66 T T T C C T T

T

C/T

Dennd3 : Intron 2 67 G G G C C G

G

C/G

Dennd3 : Intron 1 68 T

G T

G/T

Dennd3 : Intron 1 69 A

G

A/G

Dennd3 : Intron 1 70 A

G

A/G

Dennd3 : Intron 1 71

A G

A/G

Dennd3 : Intron 1 72

G A

A/G

Dennd3 : Intron 1 73 C

C T

C/T

Dennd3 : Intron 2 74 A A A T T T

A

A/T

Dennd3 : Intron 1 75 G

A A

A/G

251

Dennd3 : Intron 1 76 T

C C

C/T

Dennd3 : Intron 2 77 C C C T T T

C

C/T

Dennd3 : Intron 1 78 G

C G

C/G

Dennd3 : Intron 2 79 A A A G G G

A

A/G

Dennd3 : Intron 2 80 A A A G G G

A

A/G

Dennd3 : Intron 1 81 T

C T

C/T

Dennd3 : Intron 2 82 C C C T T T

C

C/T

Dennd3 : Intron 1 83 T

T C

C/T

Dennd3 : Intron 1 84 G G G G

G

G

A/G

Dennd3 : Intron 2 85 T T T C C C

T

C/T

Dennd3 : Intron 1 86 T T T C

C

T

C/T

Dennd3 : Intron 3 87 G G G A A G

G

A/G

Dennd3 : Intron 3 88 A A A G G G

A

A/G

Dennd3 : Coding-Synonymous

1 89 C C C C

C

C/G

Dennd3 : Intron 3 90 A A A G G G A

A

A/G

Dennd3 : Intron 1 91 G G G G

G

G

A/G

Dennd3 : Intron 2 92 G

A A G

A/G

Dennd3 : Intron 2 93 A

A G A

A/G

Dennd3 : Intron 2 94 G

T T G G

G/T

Dennd3 : Intron 2 95 T T T C C C

T

C/T

Dennd3 : Intron 1 96 A

G G

A/G

Dennd3 : Intron 1 97 G G G C

G

G

C/G

Dennd3 : Intron 1 98 T

G G

G/T

Dennd3 : Intron 1 99 G

G A

A/G

Dennd3 : Intron 1 100 C

T T

C/T

Dennd3 : Intron 1 101 A

G G

A/G

Dennd3 : Intron 2 102 A A A

G G

A

A/G

Dennd3 : Intron 1 103 T

C C

C/T

Dennd3 : Intron 2 104 A A A G G G

A

A/G

Dennd3 : Intron 2 105 G G G A A A

G

A/G

Dennd3 : Intron 1 106 A A A

A

A

A/G

Dennd3 : Intron 2 107 G G G

C

G

C/G

Dennd3 : Intron 1 108 A

T

A/T

Dennd3 : Intron 1 109 G

A

A/G

Dennd3 : Intron 1 110 T

C

C/T

Dennd3 : Intron 1 111 G

A

A/G

Dennd3 : Intron 1 112 G

A

A/G

Dennd3 : Intron 1 113 T

C

C/T

Dennd3 : Intron 1 114 T

C

C/T

Dennd3 : Intron 1 115 C

T

C/T

Dennd3 : Intron 1 116 T T T C

T

T

C/T

Dennd3 : Intron 1 117 T T T C

T

T

C/T

252

Dennd3 : Intron 1 118 C C C T

C

C

C/T

Dennd3 : Intron 2 119 C C C T

C

C

C/T

Dennd3 : Intron 2 120 C C C T T C

C

C/T

Dennd3 : Intron 1 121 G

A G

A/G

Dennd3 : Intron 1 122 C

T C

C/T

Dennd3 : Intron 1 123 T

C T

C/T

Dennd3 : Intron 2 124 G G G A A G

G

A/G

Dennd3 : Intron 2 125 T T T C C C

T

C/T

Dennd3 : Intron 2 126 A A A G G A

A

A/G

Dennd3 : Intron 2 127 C C C A A C

C

A/C

Dennd3 : Intron 2 128 G G G C C C

G

C/G

Dennd3 : Intron 2 129 T T T C

C

T

C/T

Dennd3 : Intron 1 130 A

C

A/C

Dennd3 : Intron 1 131 A

C

A/C

Dennd3 : Intron 1 132 G

A

A/G

Dennd3 : Intron 1 133 A

G

A/G

Dennd3 : Intron 1 134 G

T

G/T

Dennd3 : Intron 1 135

T T

T

Dennd3 : Intron 1 136 C C C T

T

C

C/T

Dennd3 : Intron 2 137 G G G G G A

G

A/G

Dennd3 : Intron 1 138 T

T C

C/T

Dennd3 : Intron 1 139 G

G A

A/G

Dennd3 : Intron 1 140 A

A T

A/T

Dennd3 : Intron 2 141 A A A

G G

A

A/G

Dennd3 : Intron 2 142 A A A G G G

A

A/G

Dennd3 : Intron 2 143 C C C T T C

C

C/T

Dennd3 : Intron 2 144 A A A G G G

A

A/G

Dennd3 : Intron 1 145 C

C T

C/T

Dennd3 : Intron 1 146 A A A A

T

A

A/T

Dennd3 : Intron 2 147 T T T C C C

T

C/T

Dennd3 : Intron 1 148 A

T

A/T

Dennd3 : Intron 1 149 T

A

A/T

Dennd3 : Intron 1 150 G

C

C/G

Dennd3 : Intron 1 151 G

T

G/T

Dennd3 : Intron 1 152 T

C

C/T

Dennd3 : Intron 2 153 T T T

C C

T

C/T

Dennd3 : Intron 2 154 G G G C C

G

C/G

Dennd3 : Intron 1 155 C

T

C/T

Dennd3 : Intron 1 156 C

A

A/C

Dennd3 : Intron 1 157 T

C

C/T

Dennd3 : Intron 2 158 A A A

G

A

A/G

Dennd3 : Intron 2 159 G G G

A

G

A/G

Dennd3 : Intron 1 160 A A A G

G

A

A/G

253

Dennd3 : Intron 1 161 G

A

A/G

Dennd3 : Intron 1 162 C

T

C/T

Dennd3 : Intron 1 163 T

G

G/T

Dennd3 : Intron 2 164 C

A C

A/C

Dennd3 : Intron 2 165 G

A G

A/G

Dennd3 : Intron 3 166 T T T C

C T

T

C/T

Dennd3 : Intron 1 167 C C C

C

C

A/C

Dennd3 : Intron 1 168 T T T C

T

T

C/T

Dennd3 : Intron 1 169 T T T C

T

T

C/T

Dennd3 : Intron 1 170 C C C T

C

C

C/T

Dennd3 : Intron 1 171 T T T C

T

T

C/T

Dennd3 : Intron 1 172 C C C A

C

C

A/C

Dennd3 : Intron 1 173 C C C T

C

C

C/T

Dennd3 : Intron 1 174 G

A G

A/G

Dennd3 : Intron 2 175 T

C C T

C/T

Dennd3 : Intron 1 176 G

G T

G/T

Dennd3 : Intron 1 177 C

G C

C/G

Dennd3 : Intron 2 178 C C C T T C

C

C/T

Dennd3 : Intron 1 179 G G G C

G

G

C/G

Dennd3 : Intron 2 180 G

T G

G/T

Dennd3 : Intron 1 181 G G G A

G

G

A/G

Dennd3 : Intron 3 182 G G G A

A G

G

A/G

Dennd3 : Intron 3 183 G G G T

T G

G

G/T

Dennd3 : Intron 2 184 A A A G G A

A

A/G

Dennd3 : Intron 1 185 C

T

C/T

Dennd3 : Intron 2 186 T T T G G G

T

G/T

Dennd3 : Intron 1 187 C C C T

C

C

C/T

Dennd3 : Intron 1 188 C

G

C/G

Dennd3 : Intron 1 189 C

T

C/T

Dennd3 : Intron 2 190 G G G A A G

G

A/G

Dennd3 : Intron 1 191 C

A

A/C

Dennd3 : Intron 2 192 T T T C C T

T

C/T

Dennd3 : Intron 1 193 T

A

A/T

Dennd3 : Intron 2 194 C

T C C

C/T

Dennd3 : Intron 3 195 C C C

T C C

C

C/T

Dennd3 : Intron 3 196 G G G C C G G

G

C/G

Dennd3 : Intron 2 197 A A A A A G

A

A/G

Dennd3 : Intron 3 198 T T T C C C

T

C/T

Dennd3 : Intron 1 199 G G G A

G

G

A/G

Dennd3 : Intron 1 200 A A A C

A

A

A/C

Dennd3 : Intron 1 201 G

A G

A/G

Dennd3 : Intron 1 202 G

A G

A/G

Dennd3 : Intron 1 203 G

A G

A/G

254

Dennd3 : Intron 1 204 T

C C

C/T

Dennd3 : Intron 1 205 T

C T

C/T

Dennd3 : Intron 1 206 C

T

C/T

Dennd3 : Intron 1 207

G A

A/G

Dennd3 : Intron 1 208

C T

C/T

Dennd3 : Intron 1 209

T C

C/T

Dennd3 : Intron 1 210 T T T C

T

T

C/T

Dennd3 : Intron 1 211 G

T

G

G/T

Dennd3 : Intron 3 212 ? C C C C T C

C

C/T

Dennd3 : Intron 1 213 A

G

A/G

Dennd3 : Intron 1 214 C

T C

C/T

Dennd3 : Intron 2 215 T

C T C

C/T

Dennd3 : Intron 2 216 C C C A A C

C

A/C

Dennd3 : Intron 3 217 A A A G G A A

A

A/G

Dennd3 : Intron 3 218 A A A

G A A

A

A/G

Dennd3 : Intron 2 219 G

A G G

A/G

Dennd3 : Intron 2 220 G

A G G

A/G

Dennd3 : Intron 3 221 C C C T T C C

C

C/T

Dennd3 : Intron 2 222 C C C T T C

C

C/T

Dennd3 : Intron 1 223 G

G A

A/G

Dennd3 : Intron 1 224 C

C T

C/T

Dennd3 : Intron 1 225 G

G A

A/G

Dennd3 : Intron 2 226 T T T

C C

T

C/T

Dennd3 : Intron 2 227 A A A T T A

A

A/T

Dennd3 : Intron 1 228 G G G

G

A/G

Dennd3 : Intron 2 229 C C C T T C

C

C/T

Dennd3 : Intron 1 230 C

A C

A/C

Dennd3 : Intron 2 231 G G G A A G

G

A/G

Dennd3 : Intron 2 232 C C C A A C

C

A/C

Dennd3 : Intron 2 233 T T T C C

T

C/T

Dennd3 : Intron 2 234 G G G A A

G

A/G

Dennd3 : Intron 1 235 A

G

A/G

Dennd3 : Intron 1 236 G

A

A/G

Dennd3 : Intron 1 237 G

T

G/T

Dennd3 : Intron 1 238 T

C T

C/T

Dennd3 : Intron 1 239 A

C A

A/C

Dennd3 : Intron 2 240 C C C T T C

C

C/T

Dennd3 : Intron 1 241 G

T G

G/T

Dennd3 : Intron 1 242 C

T C

C/T

Dennd3 : Intron 2 243 A

G G

A/G

Dennd3 : Intron 2 244 G G G A A G

G

A/G

Dennd3 : Intron 1 245 G

G A

A/G

Dennd3 : Intron 1 246 G

A A

A/G

255

Dennd3 : Intron 1 247 C

T C

C/T

Dennd3 : Intron 2 248 A A A G G G

A

A/G

Dennd3 : Intron 2 249 C C C T T T

C

C/T

Dennd3 : Intron 1 250 G

G A

A/G

Dennd3 : Intron 2 251 A A A G G

A

A/G

Dennd3 : Intron 2 252 T

C

T

C/T

Dennd3 : Intron 2 253 G

A

G

A/G

Dennd3 : Intron 3 254 G G

T

G

G

G/T

Dennd3 : Intron 2 255 C

G

C C

C/G

Dennd3 : Intron 2 256 A

G

A A

A/G

Dennd3 : Intron 2 257

A

G G

A/G

Dennd3 : Intron 2 258 C

C C T C

C/T

Dennd3 : Intron 3 259 C C C C C T C

C

C/T

Dennd3 : Intron 2 260 T T T

C

T

C/T

Dennd3 : Intron 3 261 G G G A A A G

G

A/G

Dennd3 : Intron 3 262 T T T

C T T

T

C/T

Dennd3 : Intron 3 263 T T T

C C T

T

C/T

Dennd3 : Intron 1 264 T T T C

T

C/T

Dennd3 : Intron 1 265 G G G A

G

A/G

Dennd3 : Intron 2 266 A A A T

A

A

A/T

Dennd3 : Intron 1 267 A A A A

A

A/G

Dennd3 : Intron 2 268 T

G

T T

G/T

Dennd3 : Intron 1 269 G G G G

G

A/G

Dennd3 : Intron 3 270 A A A G

A A

A

A/G

Dennd3 : Intron 1 271 T

T T G T

G/T

Dennd3 : Intron 2 272 T

G G T T

G/T

Dennd3 : Intron 2 273 C

G G

C

C/G

Dennd3 : Intron 1 274 G

A G G

A/G

Dennd3 : Intron 2 275 T T T C C C T

T

C/T

Dennd3 : Intron 1 276 G G G A

G

A/G

Dennd3 : Intron 1 277 A A A C

A

A/C

Dennd3 : Coding-Synonymous

2 278 C

T C

C/T

Dennd3 : Intron 2 279 G G G A A A

G

A/G

Dennd3 : Intron 1 280 A

T

A/T

Dennd3 : Intron 1 281 G

A G

A/G

Dennd3 : Intron 1 282 T

T

T

Dennd3 : Intron 1 283 C

G G

C/G

Dennd3 : Intron 1 284 G

A G

A/G

Dennd3 : Intron 1 285 T

C T

C/T

Dennd3 : Intron 2 286 T T T G

T

T

G/T

Dennd3 : Intron 1 287 C

C

C

Dennd3 : Intron 1 288 C

C

C

256

Dennd3 : Intron 2 289 T T T

A

T

A/T

Dennd3 : Intron 2 290 G G G T

G

G

G/T

Dennd3 : Intron 2 291 A A A G

G

A

A/G

Dennd3 : Intron 1 292 T

T C

C/T

Dennd3 : Intron 1 293 A

G A

A/G

Dennd3 : Intron 2 294 T T T C C C

T

C/T

Dennd3 : Intron 1 295 T

T C

C/T

Dennd3 : Intron 2 296 A A A G G A

A

A/G

Dennd3 : Intron 2 297 A A A C C A

A

A/C

Dennd3 : Intron 1 298 A

G A

A/G

Dennd3 : Intron 2 299 G G G A A G

G

A/G

Dennd3 : Intron 2 300 A

G A A

A/G

Dennd3 : Intron 2 301 C

T C C

C/T

Dennd3 : Intron 1 302 T

A T T

A/T

Dennd3 : Intron 2 303 T

C T T

C/T

Dennd3 : Intron 2 304 G

A G G

A/G

Dennd3 : Intron 2 305 C

T C C

C/T

Dennd3 : Intron 3 306 A A A G G G A

A

A/G

Dennd3 : Coding-Synonymous

1 307 A A A A

G

A

A/G

Dennd3 : Coding-Synonymous

1 308 G G G A

G

G

A/G

Dennd3 : Coding-NonSynonymous

1 309 G G G G

G

G

A/G

Dennd3 : Intron 2 310 A

G A A

A/G

Dennd3 : Intron 1 311 C

T

C/T

Dennd3 : Intron 1 312 A

T

A/T

Dennd3 : Intron 1 313 G

A

A/G

Dennd3 : Intron 1 314 A

G

A/G

Dennd3 : Intron 1 315 T

C

C/T

Dennd3 : Intron 1 316 T

C

C/T

Dennd3 : Intron 2 317 T

C

T

C/T

Dennd3 : Intron 1 318 A

G

A/G

Dennd3 : Intron 1 319 C

T

C/T

Dennd3 : Intron 1 320 T

A

A/T

Dennd3 : Intron 1 321 G

G A

A/G

Dennd3 : Intron 1 322 T

C T

C/T

Dennd3 : Intron 2 323 C

T C C

C/T

Dennd3 : Intron 1 324 A

G G

A/G

Dennd3 : Intron 1 325

A T

A/T

Dennd3 : Intron 1 326

G G

G

Dennd3 : Intron 2 327 A A A A A G

A

A/G

Dennd3 : Intron 2 328 G G G A A G

G

A/G

257

Dennd3 : Intron 2 329 C C C C C T

C

C/T

Dennd3 : Intron 1 330

A G

A/G

Dennd3 : Intron 1 331

G A

A/G

Dennd3 : Intron 1 332

T C

C/T

Dennd3 : Intron 2 333 G G G

G T

G

G/T

Dennd3 : Intron 1 334

G A

A/G

Dennd3 : Intron 2 335 T T T

C T

T

C/T

Dennd3 : Intron 2 336 G G G A A G

G

A/G

Dennd3 : Intron 1 337 C

G G

C/G

Dennd3 : Intron 3 338 A A A G G A A

A

A/G

Dennd3 : Intron 3 339 A A A G G A A

A

A/G

Dennd3 : Intron 2 340 C C C T T C

C

C/T

Dennd3 : Intron 1 341 T

C T

C/T

Dennd3 : Intron 3 342 T T T C C C T

T

C/T

Dennd3 : Intron 3 343 C C C G

G C

C

C/G

Dennd3 : Intron 2 344 A

G A

A/G

Dennd3 : Intron 2 345 T

G T

G/T

Dennd3 : Intron 1 346 A

A

A

Dennd3 : Intron 1 347 G

G

G

Dennd3 : Intron 2 348 C

A A A C

A/C

Dennd3 : Intron 2 349 T

C C T T

C/T

Dennd3 : Intron 2 350 A

G G G A

A/G

Dennd3 : Intron 2 351 G G G A A G

G

A/G

Dennd3 : Intron 1 352 A

G A

A/G

Dennd3 : Intron 1 353 T

C T

C/T

Dennd3 : Intron 1 354 G

A G

A/G

Dennd3 : Intron 1 355 G G G G

A

G

A/G

Dennd3 : Intron 2 356 A A A G

A

A

A/G

Dennd3 : Intron 2 357 T T T C

C

T

C/T

Dennd3 : Intron 1 358 A A A C

A

A

A/C

Dennd3 : Intron 1 359 T T T C

T

T

C/T

Dennd3 : Intron 2 360 T T T C

T T

T

C/T

Dennd3 : Intron 2 361 T T T C

T T

T

C/T

Dennd3 : Intron 2 362 C

T

C

C/T

Dennd3 : Intron 2 363 T

C

T

C/T

Dennd3 : Intron 2 364 C

T C C

C/T

Dennd3 : Intron 2 365 A A A T T A

A

A/T

Dennd3 : Intron 2 366 A A A G G A

A

A/G

Dennd3 : Intron 1 367 A

T A

A/T

Dennd3 : Intron 1 368 T

C T

C/T

Dennd3 : Intron 1 369 T

T C

C/T

Dennd3 : Intron 1 370 G G G C

G

G

C/G

Dennd3 : Intron 2 371 T T T C C T

T

C/T

258

Dennd3 : Intron 2 372 T T T C C T

T

C/T

Dennd3 : Intron 2 373 G G G G G A

G

A/G

Dennd3 : Intron 1 374 G G G A

G

G

A/G

Dennd3 : Intron 1 375 G G G A

G

G

A/G

Dennd3 : Intron 1 376 A A A C

A

A

A/C

Dennd3 : Intron 2 377 G G G A A G

G

A/G

Dennd3 : Intron 1 378 C

G

C/G

Dennd3 : Intron 2 379 A A A G G A

A

A/G

Dennd3 : Intron 2 380 A A A

G A

A

A/G

Dennd3 : Intron 1 381

G T

G/T

Dennd3 : Intron 1 382 T

C T

C/T

Dennd3 : Intron 1 383 G

A G

A/G

Dennd3 : Intron 2 384 T T T C C C

T

C/T

Dennd3 : Intron 2 385 C C C T T C

C

C/T

Dennd3 : Intron 2 386 C C C T T C

C

C/T

Dennd3 : Coding-Synonymous

2 387 G G G C C G

G

C/G

Dennd3 : Coding-Synonymous

2 388 C C C T T C

C

C/T

Dennd3 : Coding-Synonymous

2 389 C C C T T C

C

C/T

Dennd3 : Coding-Synonymous

2 390 C C C T T C

C

C/T

Dennd3 : Intron 1 391 C C C T

C

C

C/T

Dennd3 : Intron 1 392 A A A G

A

A

A/G

Dennd3 : Intron 1 393 C

T C

C/T

Dennd3 : Intron 1 394 T

A

A/T

Dennd3 : Intron 1 395 T

A

A/T

Dennd3 : Intron 1 396 C

T

C/T

Dennd3 : Intron 1 397 C

A

A/C

Dennd3 : Intron 1 398 C

A

A/C

Dennd3 : Intron 1 399 A

G

A/G

Dennd3 : Intron 1 400 C

T

C/T

Dennd3 : Intron 1 401 A

G

A/G

Dennd3 : Intron 1 402 T

G

G/T

Dennd3 : Intron 1 403 T

C C

C/T

Dennd3 : Intron 1 404 A A A C

A

A

A/C

Dennd3 : Intron 1 405 C C C G

C

C

C/G

Dennd3 : Intron 1 406 T T T C

T

T

C/T

Dennd3 : Coding-Synonymous

2 407 T T T C C C

T

C/T

Dennd3 : Coding-Synonymous

1 408 A A A G

A

A

A/G

259

Dennd3 : Intron 1 409 A A A

G

A

A/G

Dennd3 : Intron 1 410 T T T C

T

T

C/T

Dennd3 : Intron 2 411 C C C T T C

C

C/T

Dennd3 : Intron 2 412 G G G A A A

G

A/G

Dennd3 : Intron 2 413 G G G A A A

G

A/G

Dennd3 : Intron 2 414 G G G A A G

G

A/G

Dennd3 : Intron 1 415 A

G A

A/G

Dennd3 : Intron 1 416 G G G A

G

G

A/G

Dennd3 : Intron 1 417 A

G A

A/G

Dennd3 : Intron 1 418 T

C C

C/T

Dennd3 : Intron 1 419 A

C C

A/C

Dennd3 : Intron 1 420 G G G C

C

G

C/G

Dennd3 : Intron 1 421 A A A G

A

A

A/G

Dennd3 : Intron 1 422 T

C

C/T

Dennd3 : Intron 1 423 C

C T

C/T

Dennd3 : Intron 1 424

A A

A

Dennd3 : Intron 1 425 C

G C

C/G

Dennd3 : Intron 1 426

G G

G

Dennd3 : Intron 1 427

G G

G

Dennd3 : Intron 1 428 C

T C

C/T

Dennd3 : Intron 1 429 C

T C

C/T

Dennd3 : Intron 1 430

G A

A/G

Dennd3 : Intron 1 431 T

C

C/T

Dennd3 : Intron 2 432 C

T C

C/T

Dennd3 : Intron 1 433 G

G A

A/G

Dennd3 : Intron 2 434 A

G G

A/G

Dennd3 : Intron 1 435 A

G A

A/G

Dennd3 : Intron 2 436 T

C C C T

C/T

Dennd3 : Intron 2 437 T

G G T T

G/T

Dennd3 : Intron 1 438 G

A A

A/G

Dennd3 : Intron 2 439 C

T T C C

C/T

Dennd3 : Intron 2 440 T

G T T

G/T

Dennd3 : Intron 1 441 T

G G

G/T

Dennd3 : Intron 1 442

G

G

A/G

Dennd3 : Intron 2 443 T

G

G/T

Dennd3 : Intron 2 444 C

C

T

C/T

Dennd3 : Intron 1 445 T

C T

C/T

Dennd3 : Intron 1 446 G

A G

A/G

Dennd3 : Intron 1 447 T

C T

C/T

Dennd3 : Intron 1 448 C

T C

C/T

Dennd3 : Intron 1 449 A

T A

A/T

Dennd3 : Intron 1 450 T

C T

C/T

Dennd3 : Intron 1 451 A

G G

A/G

260

Dennd3 : Intron 1 452 C

T C

C/T

Dennd3 : Intron 1 453 C

T C

C/T

Dennd3 : Intron 1 454

A G

A/G

Dennd3 : Intron 1 455 G

A G

A/G

Dennd3 : Intron 2 456 A

C A

A/C

Dennd3 : Intron 1 457

C C

C

C/T

Dennd3 : Intron 1 458 G

C

C/G

Dennd3 : Intron 1 459 A

T

A/T

Dennd3 : Intron 1 460 A

G

A/G

Dennd3 : Intron 1 461 T

A T

A/T

Dennd3 : Intron 1 462 A

G

A/G

Dennd3 : Intron 2 463 C

C C G

C/G

Dennd3 : Intron 1 464 T

C T

C/T

Dennd3 : Intron 1 465 A

G A

A/G

Dennd3 : Intron 1 466 A

G

A/G

Dennd3 : Intron 1 467 T

C T

C/T

Dennd3 : Intron 1 468 C

T C

C/T

Dennd3 : Intron 1 469 T

G T

G/T

Dennd3 : Intron 2 470 T

C T

C/T

Dennd3 : Intron 1 471 G

C G

C/G

Dennd3 : Intron 1 472 C

T C

C/T

Dennd3 : Intron 1 473 C

A C

A/C

Dennd3 : Intron 1 474 G

T G

G/T

Dennd3 : Intron 1 475 C

T C

C/T

Dennd3 : Intron 2 476 ?

C C T

C/T

Dennd3 : Coding-Synonymous

3 477 A

G

A A

A/G

Dennd3 : Intron 3 478 C

C

T C

C/T

Dennd3 : Intron 2 479 T

C

T T

C/T

Dennd3 : Intron 1 480 A

G A

A/G

Dennd3 : Intron 1 481 A

A

A

Dennd3 : Intron 1 482 A

G

A/G

Dennd3 : Intron 2 483 C

G G

C/G

Dennd3 : Intron 2 484 C

C A

A/C

Dennd3 : Intron 1 485 G

T

G/T

Dennd3 : Intron 1 486 C

T

C/T

Dennd3 : Intron 1 487 G

A

A/G

Dennd3 : Intron 1 488 C

T

C/T

Dennd3 : Intron 1 489 T T T C

T

T

C/T

Dennd3 : Intron 1 490

T

A

A/T

Dennd3 : Intron 2 491 G

A G G

A/G

Dennd3 : Intron 3 492 T T T

C T T

T

C/T

261

Dennd3 : Coding-Synonymous

3 493 G G G A A G G

G

A/G

Dennd3 : Coding-NonSynonymous

2 494 A

G G

A

A/G

Dennd3 : Intron 2 495 T

C C

T

C/T

Dennd3 : Intron 2 496 A A A G G A

A

A/G

Dennd3 : Intron 2 497 A A A G

G

A

A/G

Dennd3 : Intron 1 498 C C C A

C

C

A/C

Dennd3 : Intron 1 499 C C C T

C

C

C/T

Dennd3 : Intron 1 500 C C C T

C

C

C/T

Dennd3 : Intron 1 501 T T T C

T

T

C/T

Dennd3 : Intron 1 502 A A A

T

A

A/T

Dennd3 : Intron 1 503 G G G A

G

G

A/G

Dennd3 : Intron 1 504 G G G A

G

G

A/G

Dennd3 : Intron 2 505 G

A G G

A/G

Dennd3 : Intron 3 506 G G G A A G G

G

A/G

Dennd3 : Intron 2 507 A

G G A A

A/G

Dennd3 : Intron 2 508 T

C C T T

C/T

Dennd3 : Intron 3 509 C C C T T C C

C

C/T

Dennd3 : Intron 3 510 T T T C

T T

T

C/T

Dennd3 : Intron 3 511 C C C T T C C

C

C/T

Dennd3 : Intron 3 512 G G G A A

G

G

A/G

Dennd3 : Intron 1 513 G G G G

T

G

G/T

Dennd3 : Intron 2 514 A

G G

A

A/G

Dennd3 : Intron 2 515 C

T T

C

C/T

Dennd3 : Intron 2 516 T

C C

T

C/T

Dennd3 : Intron 2 517 T

C C

T

C/T

Dennd3 : Intron 2 518 A

G G

A

A/G

Dennd3 : Intron 2 519 C

G C

C/G

Dennd3 : Intron 2 520 C

T C C

C/T

Dennd3 : Intron 2 521 A

G A A

A/G

Dennd3 : Intron 1 522 A

G A

A/G

Dennd3 : Intron 3 523 A A A G G A A

A

A/G

Dennd3 : Intron 3 524 G G G C C G

G

C/G

Dennd3 : Intron 2 525 C

T T C C

C/T

Dennd3 : Intron 3 526 G G G A A G G

G

A/G

Dennd3 : Coding-NonSynonymous

3 527 G G G A A G

G

A/G

Dennd3 : mRNA-UTR 3 528 G G G T T G G

G

G/T

Dennd3 : mRNA-UTR 1 529 A A A A

A

A

A/G

Dennd3 : mRNA-UTR 1 530 G G G A

G

G

A/G

Dennd3 : mRNA-UTR 1 531 G G G A

G

G

A/G

Dennd3 : mRNA-UTR 3 532 C C C T T C C

C

C/T

262

Dennd3 : mRNA-UTR 1 533 G G G G

G

G

A/G

Dennd3 : mRNA-UTR 3 534 C C C T T C C

C

C/T

Slc45a4 : Intron 1 1 A A A A G A A/G

Slc45a4 : mRNA-UTR 2 2 C

T T C C

C/T

Slc45a4 : mRNA-UTR 2 3 G G G A A G G

G

A/G

Slc45a4 : mRNA-UTR 2 4 C

A A C C

A/C

Slc45a4 : mRNA-UTR 3 5 A A A T T A A

A

A/T

Slc45a4 : mRNA-UTR 3 6 A A A G G A A

A

A/G

Slc45a4 : mRNA-UTR 1 7 T T T C

T

T

C/T

Slc45a4 : Coding-NonSynonymous

2 8 T

A

T

A/T

Slc45a4 : Coding-Synonymous

1 9 G G G G

A

G

A/G

Slc45a4 : Coding-Synonymous

3 10 G G G A A A G

G

A/G

Slc45a4 : Intron 3 11 G G G A A G G

G

A/G

Slc45a4 : Intron 3 12 T T T C C C T

T

C/T

Slc45a4 : Intron 1 13 A

G G

A/G

Slc45a4 : Intron 2 14 T

G G T T

G/T

Slc45a4 : Intron 1 15 T

A A A T

A/T

Slc45a4 : Intron 2 16

A A C C

A/C

Slc45a4 : Intron 3 17 A A A G G G A

A

A/G

Slc45a4 : Intron 3 18 A A A G

G A

A

A/G

Slc45a4 : Intron 3 19 G G G A

A G

G

A/G

Slc45a4 : Intron 2 20 A

G

G A

A/G

Slc45a4 : Intron 3 21 A A A G G A A

A

A/G

Slc45a4 : Intron 1 22 G

G G A G

A/G

Slc45a4 : Intron 3 23 G

C C C G

C/G

Slc45a4 : Intron 4 24 A A A G G A A

A

A/G

Slc45a4 : Intron 4 25 A A A G G A A

A

A/G

Slc45a4 : Coding-Synonymous

3 26 A A A G G A

A

A/G

Slc45a4 : Intron 2 27 C

T T

C

C/T

Slc45a4 : Intron 1 28 T T T C

C

T

C/T

Slc45a4 : Intron 1 29 C C C T

C

C

C/T

Slc45a4 : Intron 1 30 C C C T

C

C

C/T

Slc45a4 : Intron 2 31 C

A C

A/C

Slc45a4 : Intron 3 32 A A A G

G A

A

A/G

Slc45a4 : Intron 2 33 C

T T C

C/T

Slc45a4 : Intron 3 34 T T T G G T

T

G/T

Slc45a4 : Intron 3 35 C C C T T C C

C

C/T

Slc45a4 : Intron 3 36 G G G A A G G

G

A/G

263

Slc45a4 : Intron 3 37 A A A C C A A

A

A/C

Slc45a4 : Intron 3 38 G G G C C G G

G

C/G

Slc45a4 : Coding-Synonymous

3 39 G G G A A G

G

A/G

Slc45a4 : Coding-Synonymous

3 40 C C C T T C

C

C/T

Slc45a4 : Coding-Synonymous

3 41 T T T G G T

T

G/T

Slc45a4 : Coding-Synonymous

1 42 A

G A

A/G

Slc45a4 : Intron 3 43 C C C T T C C C C C C/T

Slc45a4 : Intron 4 44 G G G A A G G

G

A/G

Slc45a4 : Intron 2 45 G

C G G

C/G

Slc45a4 : Intron 2 46 G

G A G

A/G

Slc45a4 : Intron 3 47 C C C T T C C

C

C/T

Slc45a4 : Intron 2 48 T

C C T T

C/T

Slc45a4 : Intron 2 49 C

T T C C

C/T

Slc45a4 : Intron 3 50 T T T C C T T

T

C/T

Slc45a4 : Intron 2 51 G G G A A G

G

A/G

Slc45a4 : Intron 3 52 A

G G G A

A/G

Slc45a4 : Intron 3 53 A

C C A A

A/C

Slc45a4 : Intron 3 54 T T T C C T T

T

C/T

Slc45a4 : Intron 3 55 A A A G G A A

A

A/G

Slc45a4 : Intron 3 56 C C C T T C C

C

C/T

Slc45a4 : Intron 2 57 C

T C C

C/T

Slc45a4 : mRNA-UTR 3 58 G G G A A G G

G

A/G

Slc45a4 : Intron 2 59 C

T T C C

C/T

Slc45a4 : Intron 2 60 C

G G C C

C/G

Slc45a4 : Intron 1 61 A

G A

A/G

Slc45a4 : Intron 3 62 T T T C C T T

T

C/T

Slc45a4 : Intron 2 63 G G G A A G

G

A/G

Slc45a4 : Intron 2 64 T T T C C C

T

C/T

Slc45a4 : Intron 1 65 G

A G

A/G

Slc45a4 : Intron 2 66 A A A C C A

A

A/C

Slc45a4 : Intron 1 67

T G

G/T

Slc45a4 : Intron 1 68

T C

C/T

Slc45a4 : Intron 1 69 A

G A

A/G

Slc45a4 : Intron 1 70 C

T C

C/T

Slc45a4 : Intron 1 71 C

T C

C/T

Slc45a4 : Intron 1 72 T

G T

G/T

Slc45a4 : Intron 1 73 C

T C

C/T

Slc45a4 : Intron 1 74 T T T C

T

T

C/T

Slc45a4 : Intron 1 75 A

C

A/C

264

Slc45a4 : Intron 1 76 C

T C

C/T

Slc45a4 : Intron 2 77 T T T C C C

T

C/T

Slc45a4 : Intron 1 78 A A A C

A

A

A/C

Slc45a4 : Intron 2 79 G G G A A G

G

A/G

Slc45a4 : Intron 2 80 A A A G G A

A

A/G

Slc45a4 : Intron 1 81

T A

A/T

Slc45a4 : Intron 2 82 G G G

C G

G

C/G

Slc45a4 : Intron 2 83 A A A A A G

A

A/G

Slc45a4 : Intron 2 84 A A A C C A

A

A/C

Slc45a4 : Intron 1 85 T

T G

G/T

Slc45a4 : Intron 1 86 A

G

A/G

Slc45a4 : Intron 1 87 A

C A

A/C

Slc45a4 : Intron 3 88 C C C T T C C

C

C/T

Slc45a4 : Intron 2 89 C C C G G C

C

C/G

Slc45a4 : Intron 3 90 T T T G

T T

T

G/T

Slc45a4 : Intron 3 91 G G G A A G G

G

A/G

Slc45a4 : Intron 3 92 C C C T T C C

C

C/T

Slc45a4 : Intron 3 93 T T T C C T T

T

C/T

Slc45a4 : Intron 3 94 C C C T T C C

C

C/T

Slc45a4 : Intron 1 95 G

A G

A/G

Slc45a4 : Intron 2 96 A A A G G A

A

A/G

Slc45a4 : Intron 1 97 T

A T

A/T

Slc45a4 : Intron 3 98 C C C

G G C

C

C/G

Slc45a4 : Intron 1 99 T

C T T

C/T

Slc45a4 : Intron 2 100 G

A G G

A/G

Slc45a4 : Intron 2 101 T T T G G T

T

G/T

Slc45a4 : Intron 3 102

T

C

C/T

Slc45a4 : Intron 4 103 A A A

C A A

A

A/C

Slc45a4 : Intron 1 104 A A A A

G

A

A/G

Slc45a4 : Intron 4 105 C C C A A C C

C

A/C

Slc45a4 : Intron 3 106 C C C T T C C

C

C/T

Slc45a4 : Intron 3 107 C C C T T T C

C

C/T

Slc45a4 : Intron 2 108 A A A A A G G

A

A/G

Slc45a4 : Intron 1 109 T T T G

T

T

G/T

Slc45a4 : Intron 2 110 G

C G

C/G

Slc45a4 : Intron 3 111 A A A G

G A

A

A/G

Slc45a4 : Intron 3 112 G G G G

A G

G

A/G

Slc45a4 : Intron 3 113 A A A G

G A

A

A/G

Slc45a4 : Intron 4 114 T T T C

T T

T

C/T

Slc45a4 : Intron 4 115 T T T C

T T

T

C/T

Slc45a4 : Intron 2 116

A

G G

A/G

Slc45a4 : Intron 4 117 C C C T

C C

C

C/T

Slc45a4 : Intron 1 118 C C C C

C

C

C/T

265

Slc45a4 : Intron 3 119 C C C T T C C

C

C/T

Slc45a4 : Intron 3 120 G G G A A G G

G

A/G

Slc45a4 : Intron 3 121 T

G G T T

G/T

Slc45a4 : Intron 2 122 T T T T T G

T

G/T

Slc45a4 : Intron 3 123 A

G G G A

A/G

Slc45a4 : Intron 3 124 C C C C C T C

C

C/T

Slc45a4 : Intron 2 125 G G G G G A

G

A/G

Slc45a4 : Intron 2 126 G G G A A G

G

A/G

Slc45a4 : Intron 2 127 C C C T T T

C

C/T

Slc45a4 : Intron 2 128 G

A

G G

A/G

Slc45a4 : Intron 3 129 T T T C C T T

T

C/T

Slc45a4 : Intron 4 130 G G G A A G G

G

A/G

Slc45a4 : Intron 2 131 T

C T T

C/T

Slc45a4 : Intron 1 132 C C C A

C

C

A/C

Slc45a4 : Intron 3 133 T T T C

C

T

C/T

Slc45a4 : Intron 1 134 A A A G

A

A/G

Slc45a4 : Intron 3 135 T T T C

C

T

C/T

Slc45a4 : Intron 1 136 A A A T

A

A

A/T

Slc45a4 : Intron 2 137 T

C T T

C/T

Slc45a4 : Intron 2 138 C

G C C

C/G

Slc45a4 : Intron 1 139 T T T C

T

T

C/T

Slc45a4 : Intron 1 140 C

T C

C/T

Slc45a4 : Intron 1 141 C

A C

A/C

Slc45a4 : Intron 3 142 A A A T T A A

A

A/T

Slc45a4 : Intron 2 143 C C C T T C

C

C/T

Slc45a4 : Intron 2 144 A A A

G A

A

A/G

Slc45a4 : Intron 2 145 G

A G G

A/G

Slc45a4 : Intron 3 146 T T T G G G T

T

G/T

Slc45a4 : Intron 1 147 C

C T

C/T

Slc45a4 : Intron 3 148 C C C A A C C

C

A/C

Slc45a4 : Intron 3 149 G G G A A G G

G

A/G

Slc45a4 : Coding-Synonymous

3 150 T T T G G T T

T

G/T

Slc45a4 : Coding-Synonymous

1 151 G G G G

G

G

A/G

Slc45a4 : mRNA-UTR 3 152 C C C T T C

C

C/T

Slc45a4 : mRNA-UTR 3 153 G G G A A G G

G

A/G

Slc45a4 : Intron 2 154 T T T G G T T

T

G/T

Slc45a4 : Intron 2 155 C C C T T C C

C

C/T

Slc45a4 : Intron 1 156 T T T

T

G/T

Slc45a4 : Intron 1 157 A A A

A

A/C

Slc45a4 : Intron 2 158 G G G A A G G

G

A/G

Slc45a4 : Intron 3 159 G G G A A A G

G

A/G

266

Slc45a4 : Intron 2 160 G

C G

C/G

Slc45a4 : Intron 2 161 G

C G

C/G

Slc45a4 : Intron 3 162 G G G A A G G

G

A/G

Slc45a4 : Intron 3 163 C C C T T C C

C

C/T

Slc45a4 : Intron 3 164 C C C T T C C

C

C/T

Slc45a4 : Intron 2 165 T

A A T T

A/T

Slc45a4 : Intron 2 166 G

G C G

C/G

Slc45a4 : Intron 1 167 A A A G

A

A

A/G

Slc45a4 : Intron 2 168 C

T C

C/T

Slc45a4 : Intron 3 169 C C C G G C

C

C/G

Slc45a4 : Intron 1 170 C C C C

C

C

C/T

Slc45a4 : Intron 2 171 G G G A A G G

G

A/G

Slc45a4 : Intron 1 172 A A A G

A

A

A/G

Slc45a4 : Intron 3 173 T T T C C C T

T

C/T

Slc45a4 : Intron 3 174 C C C C C T C

C

C/T

Slc45a4 : Intron 1 175 C C C C

C

C/T

Slc45a4 : Intron 2 176 A A A T T A A

A

A/T

Slc45a4 : Intron 1 177 G G G G

T

G

G/T

Slc45a4 : Intron 1 178 C C C C

T

C

C/T

Slc45a4 : Intron 2 179 A

G G A

A/G

Slc45a4 : Intron 3 180 A A A G G A A

A

A/G

Slc45a4 : Intron 3 181 T T T G G T T

T

G/T

Slc45a4 : Intron 2 182 A

G G A

A/G

Slc45a4 : Intron 1 183 T T T G

T

T

G/T

Slc45a4 : Intron 2 184 C

T C C

C/T

Slc45a4 : Intron 3 185 T T T C C T T

T

C/T

Slc45a4 : Intron 3 186 G G G A A G G

G

A/G

Slc45a4 : Intron 3 187 T T T C C C T

T

C/T

Slc45a4 : Intron 3 188 G G G G G A G

G

A/G

Slc45a4 : Intron 3 189 A A A G G A A

A

A/G

Slc45a4 : Intron 2 190 T

C T

C/T

Slc45a4 : Intron 2 191 A

G A A

A/G

Slc45a4 : Intron 2 192 A

G A A

A/G

Slc45a4 : Intron 2 193 C

A C C

A/C

Slc45a4 : Intron 2 194 C

G C C

C/G

Slc45a4 : Intron 2 195 T T T C C T T

T

C/T

Slc45a4 : Intron 1 196 C C C T

C

C

C/T

Slc45a4 : Intron 1 197 G G G C

G

G

C/G

Slc45a4 : Intron 1 198 G

A G

A/G

Slc45a4 : Intron 2 199 A A A

G A A

A

A/G

Slc45a4 : Intron 2 200 T

C C T

C/T

Slc45a4 : Intron 2 201 G

G T G

G/T

Slc45a4 : Intron 2 202 C C C C

T C

C

C/T

267

Slc45a4 : Intron 1 203 A

G A A

A/G

Slc45a4 : Intron 1 204 T T T A

T

T

A/T

Slc45a4 : Intron 3 205 C C C T T C C

C

C/T

Slc45a4 : Intron 3 206 T T T C C T T

T

C/T

Slc45a4 : Intron 3 207 A A A G G A A

A

A/G

Slc45a4 : Intron 3 208 T T T C C T T

T

C/T

Slc45a4 : Intron 2 209 T T T T T C

T

C/T

Slc45a4 : Intron 2 210 T

A T

A/T

Slc45a4 : Intron 1 211 C C C A

C

C

A/C

Slc45a4 : Intron 1 212 A A A A

G

A

A/G

Slc45a4 : Intron 2 213 G

A

G

A/G

Slc45a4 : Intron 2 214 T

G

T

G/T

Slc45a4 : Intron 3 215 A A A G G A A

A

A/G

Slc45a4 : Intron 1 216 A

G

A/G

Slc45a4 : Intron 2 217 A

G

A

A/G

Slc45a4 : Intron 2 218 T

A T T

A/T

Slc45a4 : Intron 2 219 T T T C

T

T

C/T

Slc45a4 : Intron 2 220 T T T G G T

T

G/T

Slc45a4 : Intron 1 221 T T T C

T

T

C/T

Slc45a4 : Intron 2 222 G G G T T G

G

G/T

Slc45a4 : Intron 1 223 C

T

C/T

Slc45a4 : Intron 2 224 T T T C C C

T

C/T

Slc45a4 : Intron 2 225 G G G A A G

G

A/G

Slc45a4 : Intron 2 226 G G G A A G

G

A/G

Slc45a4 : mRNA-UTR 2 227 A A A G G A

A

A/G

Slc45a4 : mRNA-UTR 2 228 G G G A A G

G

A/G

Slc45a4 : mRNA-UTR 2 229 A A A

C A

A

A/C

Slc45a4 : mRNA-UTR 2 230 A A A

C A

A

A/C

Slc45a4 : mRNA-UTR 1 231 T

G T

G/T

Slc45a4 : mRNA-UTR 2 232 T T T C C T

T

C/T

Slc45a4 : mRNA-UTR 2 233 T T T A A T

T

A/T

Slc45a4 : mRNA-UTR 1 234 A

G

A/G

Slurp1 : Intron 1 1

G

A

A/G

Slurp1 : Intron 2 2 G

A

G

A/G

Slurp1 : Intron 2 3 T

C

T

C/T

Ly6d : Intron 3 1 G G G A A G G

G

A/G

Ly6d : Intron 2 2

T T G G

G/T

Ly6d : mRNA-UTR 3 3 A A A G G A A

A

A/G

Ly6d : mRNA-UTR 2 4 A

G G A A

A/G

Ly6d : Intron 2 5 A

T T A

A/T

Ly6d : Intron 2 6 G

A A G

A/G

268

Ly6d : Intron 3 7 C C C G G C

C

C/G

Ly6d : Intron 3 8 A A A G G A

A

A/G

Ly6d : Intron 3 9 G G G A A G

G

A/G

Ly6d : Intron 2 10 C

T T C C

C/T

Ly6d : Intron 2 11 C

T T C C

C/T

Ly6k : Intron 1 1 A A A G

A

A

A/G

Ly6k : Intron 1 2 A A A G

A

A

A/G

Ly6k : Coding-NonSynonymous

3 3 C C C T T C C

C

C/T

Hemt1 : mRNA-UTR 1 1 G

A

A/G

Hemt1 : Intron 1 2 G

A

A/G

Hemt1 : Intron 1 3 G

C

C/G

Hemt1 : Intron 1 4 G

A

A/G

Hemt1 : Intron 1 5 C

T C

C/T

Hemt1 : Coding-Synonymous 2 6 G

T T G

G/T

Hemt1 : Coding-NonSynonymous

2 7 A

G G A

A/G

Hemt1 : Intron 3 8 A

C C A

A/C

Hemt1 : Intron 1 9 T

C

C/T

Hemt1 : Intron 1 10 G G G A

G

A/G

Hemt1 : Intron 1 11 C

T

C/T

Hemt1 : Intron 2 12 C

T T C C

C/T

Hemt1 : Intron 1 13 T T T C

T

T

C/T

Hemt1 : Intron 1 14 T

C

C

C/T

Hemt1 : Intron 1 15 T T T A

T

T

A/T

Hemt1 : Intron 1 16 C C C G

C

C

C/G

Hemt1 : Coding-NonSynonymous

1 17 A A A G

A

A

A/G

Hemt1 : Coding-Synonymous 2 18 C

T T

C

C/T

2010109I03Rik : mRNA-UTR 2 1 C C C T T C

C

C/T

2010109I03Rik : mRNA-UTR 3 2 G G G G G A

A

A/G

2010109I03Rik : mRNA-UTR 2 3 G G G A A G

G

A/G

2010109I03Rik : mRNA-UTR 2 4 G G G A A G

G

A/G

2010109I03Rik : mRNA-UTR 3 5 C C C C C T T

T

C/T

2010109I03Rik : mRNA-UTR 2 6 A

G G G

A/G

2010109I03Rik : mRNA-UTR 1 7 G

G A A

A/G

2010109I03Rik : mRNA-UTR 2 8 T

C C C C

C/T

2010109I03Rik : Coding-Synonymous

3 9 A A A A A G

G

A/G

269

2010109I03Rik : Coding-Synonymous

3 10 T T T C C C C

C

C/T

2010109I03Rik : Intron 2 11 G

G A A

A/G

2010109I03Rik : Intron 1 12 C C C T

C

C

C/T

2010109I03Rik : Intron 1 13 T T T C

T

T

C/T

2010109I03Rik : mRNA-UTR 2 14 A A A G G G

G

A/G

Ly6i : mRNA-UTR 2 1 T T T C

C

C

C/T

Ly6i : mRNA-UTR 1 2 G

A

A/G

Ly6i : mRNA-UTR 1 3 T

C

C/T

Ly6i : mRNA-UTR 1 4 G

T T

G/T

Ly6i : mRNA-UTR 1 5 A

G

A/G

Ly6i : mRNA-UTR 1 6 A

C

A/C

Ly6i : mRNA-UTR 1 7 T

G

G/T

Ly6i : mRNA-UTR 1 8 A

G

A/G

Ly6i : Coding-NonSynonymous

1 9 C

T T

C/T

Ly6i : Intron 2 10 C C C G G G

G

C/G

Ly6i : Intron 1 11 C

A A

A/C

Ly6i : Intron 1 12 C

A A

A/C

Ly6i : Intron 1 13 T

C C

C/T

Ly6i : Intron 1 14 G

A A

A/G

Ly6i : Intron 1 15 A

C C

A/C

Ly6i : Intron 1 16 T

C C

C/T

Ly6i : Intron 1 17 T

G G

G/T

Ly6i : Intron 1 18 T

C C

C/T

Ly6i : Intron 1 19 G

A A

A/G

Ly6i : Intron 1 20 A

G G

A/G

Ly6i : Intron 1 21 G

T T

G/T

Ly6i : Intron 1 22 G

A A

A/G

Ly6i : Intron 1 23 A

C C

A/C

Ly6i : Intron 1 24 T

C C

C/T

Ly6i : Intron 1 25 A

G G

A/G

Ly6i : Intron 1 26 A A A T

T

T

A/T

Ly6i : Intron 1 27 G

T T

G/T

Ly6i : Intron 1 28 A A A G

G

G

A/G

Ly6i : Intron 1 29 G

A

A/G

Ly6i : Intron 1 30 T

C C

C/T

Ly6i : Intron 1 31 G

A

A/G

Ly6i : Intron 1 32 T

G

G/T

Ly6i : Intron 1 33 C

T T

C/T

Ly6i : Intron 1 34 G

A A

A/G

Ly6i : Intron 1 35 C

G G

C/G

Ly6i : Intron 1 36 T

A A

A/T

270

Ly6i : Intron 1 37 T T T C

C

C

C/T

Ly6i : Intron 1 38 C

A A

A/C

Ly6i : Intron 1 39 T

C C

C/T

Ly6i : Intron 1 40 G

C C

C/G

Ly6i : Intron 2 41 C C C T T T

T

C/T

Ly6i : Intron 3 42 T T T G G G G

G

G/T

Ly6i : Intron 1 43 G

C C

C/G

Ly6i : Intron 2 44 T

G

G G

G/T

Ly6i : Intron 3 45 C C C G

G G

G

C/G

Ly6i : Intron 3 46 G G G T T T T

T

G/T

Ly6i : Coding-Synonymous 2 47 G

C C C C

C/G

Ly6i : Coding-NonSynonymous

3 48 T T T G G G G

G

G/T

Ly6i : mRNA-UTR 3 49 T

C C C C C C C C/T

Ly6i : mRNA-UTR 1 50 G G G

C

C/G

Ly6c1 : Intron 2 1 G G G A A A

A

A/G

Ly6c1 : Intron 2 2 C C C T T T

T

C/T

Ly6c1 : Intron 2 3 A A A G G G

G

A/G

Ly6c1 : Intron 2 4 A A A G G G

G

A/G

Ly6c1 : Intron 1 5 T

A A

A/T

Ly6c1 : Intron 1 6 G

C C

C/G

Ly6c1 : Intron 1 7

C

A

A

A

A/C

Ly6c1 : Intron 2 8 G

G C C C

C

C/G

Ly6c1 : Intron 2 9 C

C T T T

T

C/T

Ly6c1 : Intron 1 10 G

T T

G/T

Ly6c1 : mRNA-UTR 1 11

C C

C

Ly6c1 : Coding-NonSynonymous

1 12 A

G G

A/G

Ly6c1 : Intron 2 13 G

G T G

G/T

Ly6c1 : Intron 2 14 G

C C

C/G

Ly6c1 : Intron 1 15 G

C C

C/G

Ly6c1 : Intron 1 16 G G G

A

A

A/G

Ly6c1 : Intron 2 17 C C C T

T

T

C/T

Ly6c1 : Intron 1 18

C C T

T

T

C/T

Ly6c1 : Intron 1 19 G

C C

C/G

Ly6c1 : Intron 1 20 G

T T

G/T

Ly6c1 : Intron 1 21 G

C

C/G

Ly6c1 : Intron 1 22 T

C

C/T

Ly6c1 : Intron 1 23 T

C

C/T

Ly6c1 : Intron 1 24 C

T

C/T

Ly6c1 : Intron 1 25 A

T T

A/T

271

Ly6c1 : Intron 1 26 T

C C

C/T

Ly6c1 : Intron 2 27 T T T C

C

C

C/T

Ly6c1 : Intron 2 28 A A A G G G

G

A/G

Ly6c1 : Intron 1 29 T

A A

A/T

Ly6c1 : Intron 1 30 T

C

C/T

BC025446 : Intron 2 1 G

A

A

A/G

BC025446 : Intron 1 2 G G G A

A

A/G

BC025446 : Intron 3 3 T T T C C C C

C

C/T

BC025446 : Intron 1 4 T T T C

C

C

C/T

BC025446 : Intron 2 5 G

A A A

A/G

BC025446 : Intron 3 6 C C C T T T T

T

C/T

BC025446 : Intron 2 7 C

T T T

C/T

BC025446 : Intron 1 8 T

T

C

C

C/T

BC025446 : Intron 2 9 T

C C C

C/T

BC025446 : Intron 2 10 T

C C C

C/T

BC025446 : Intron 3 11 G G G C C C C

C

C/G

BC025446 : Intron 2 12 A A A G G G

G

A/G

BC025446 : Intron 2 13 A A A C C C

C

A/C

BC025446 : Intron 1 14 G G G G

G

G

A/G

BC025446 : Intron 1 15 A A A G

G

G

A/G

BC025446 : Intron 1 16 T T T C

T

C/T

BC025446 : Intron 1 17

A T

T

A/T

BC025446 : Intron 2 18 A A A T T T

T

A/T

BC025446 : Intron 2 19 T T T G G G

G

G/T

BC025446 : Intron 1 20 C

T T

C/T

BC025446 : Intron 1 21 C

T T

C/T

BC025446 : Intron 1 22 C

A

A/C

BC025446 : Intron 1 23 C

T T

C/T

BC025446 : Intron 1 24 C

T T

C/T

BC025446 : Intron 1 25 C

T T

C/T

BC025446 : Intron 2 26 T T T C C C

C

C/T

BC025446 : Intron 1 27 C

T T

C/T

BC025446 : Intron 1 28 A

G

A/G

BC025446 : Intron 1 29 T

C C

C/T

BC025446 : Intron 1 30 G

A A

A/G

BC025446 : Intron 1 31 T

A A

A/T

BC025446 : Intron 1 32 G

A A

A/G

BC025446 : Intron 1 33 T

G G

G/T

BC025446 : Intron 1 34 C

A A

A/C

BC025446 : Intron 1 35 A

G G

A/G

BC025446 : Intron 1 36 A

G G

A/G

BC025446 : Intron 1 37 G

A A

A/G

272

BC025446 : Intron 1 38 T

A A

A/T

BC025446 : Intron 1 39 C

T

C/T

Scrt1 : mRNA-UTR 2 1 G

A

A/G

C030006K11Rik : Intron 2 15 A A

C

C

C

A/C

C030006K11Rik : Intron 3 16 A A A C C C

C

A/C

C030006K11Rik : Intron 3 17 A A A G G G

G

A/G

C030006K11Rik : mRNA UTR 4 18 C C C T T T T

T

C/T

Recql4 : Coding-NonSynonymous

3 1 T T T G G G G

G

G/T

Recql4 : Intron 2 2 A A A G

G

G

A/G

Recql4 : Intron 3 3 T

C C C

C/T

Recql4 : Intron 4 4 G G G A A A A

A

A/G

Zfp7 : Intron 1 1 A A A

G

A/G

Zfp7 : Intron 2 2 T

C C

C/T

Zfp7 : Intron 1 3 C

T T

C/T

Zfp7 : Intron 3 4 A

C C C

A/C

Zfp7 : Intron 3 5 C

T T T

C/T

Zfp7 : Intron 3 6 T

C C

C/T

Zfp7 : Coding-Synonymous 3 7 T

C

C/T

Zfp7 : Intron 1 8 C

A

A/C

Zfp7 : Intron 1 9 C

T T

C/T

Zfp7 : Intron 3 10 T

C C C

C/T

Zfp7 : Intron 4 11 C

T T

T

C/T

Zfp7 : Intron 1 12 A A A T

A

A

A/T

Zfp7 : Intron 3 13 C

T T T

C/T

Zfp7 : Intron 3 14 G G G T

T T

T

G/T

Zfp7 : Intron 4 15 G G G T

T T

T

G/T

Zfp7 : Coding-NonSynonymous

3 16 T

G G

G/T

Zfp7 : Intron 4 17 A A A G

G G

G

A/G

Zfp7 : Intron 3 18 C

T T T

C/T

Zfp7 : Intron 4 19 A A A G G G G

G

A/G

Zfp7 : Intron 4 20 C C C G G G G

G

C/G

Rbfox2 : mRNA-UTR 3 1 A G G A/G

Rbfox2 : mRNA-UTR 3 2 A

G G

A/G

Rbfox2 : Intron 4 3 A

A G G G G

G

A/G

273

Rbfox2 : Intron 2 4 C

T T

C/T

Rbfox2 : Intron 2 5 T T T C

C

C

C/T

Rbfox2 : Intron 3 6 C

T T T

C/T

Rbfox2 : Intron 3 7 A A A C

C

C

A/C

Rbfox2 : Intron 3 8 G G G T

T T

G/T

Rbfox2 : Intron 3 9 A A A G G G

G

A/G

Rbfox2 : Intron 3 10 A

G G G

A/G

Rbfox2 : Intron 1 11 T

A A

A/T

Rbfox2 : Intron 3 12 C

A A

A/C

Rbfox2 : Intron 4 13 T T T C

C C

C

C/T

Rbfox2 : Intron 4 14 A A A G

G G

G

A/G

Rbfox2 : Intron 4 15 T

T G

G

G

G/T

Rbfox2 : Intron 3 16 T

C C

C/T

Rbfox2 : Intron 3 17 T

C C

C/T

Rbfox2 : Intron 3 18 T

C C C

C/T

Rbfox2 : Intron 3 19 T

G

G

G/T

Rbfox2 : Intron 4 20 C C C A A A A

A

A/C

Rbfox2 : Intron 3 21 C

T T T

C/T

Rbfox2 : Intron 1 22 C C C C

C

C

C/T

Rbfox2 : Intron 4 23 A A A T

T

T

A/T

Rbfox2 : Intron 3 24 A

G G G

A/G

Rbfox2 : Intron 4 25 C C C G G G G

G

C/G

Rbfox2 : Intron 4 26 T T T C

C C

C

C/T

Rbfox2 : Intron 4 27 C C C T

T T

T

C/T

Rbfox2 : Intron 1 28 C C C C

C

C

C/T

Rbfox2 : Intron 3 29 G

A A

A

A/G

Rbfox2 : Intron 2 30 T

C C C C

C/T

Rbfox2 : Intron 2 31 A A A G

G

G

A/G

Rbfox2 : Intron 4 32 T T T C C C C

C

C/T

Rbfox2 : Intron 3 33 A

G G G

A/G

Rbfox2 : Intron 1 34 G G G G

G

G

A/G

Rbfox2 : Intron 2 35 A

G G G

A/G

Rbfox2 : Intron 4 36 C C C T T T T

T

C/T

Rbfox2 : Intron 1 37 G G G G

G

G

C/G

Rbfox2 : Intron 3 38 C C C A A A

A

A/C

Rbfox2 : Intron 3 39 T T T C C C

C

C/T

Rbfox2 : Intron 2 40 C C C T

T

T

C/T

Rbfox2 : Intron 3 41 T T T A A A

A

A/T

Rbfox2 : Intron 4 42 T T T A A A A

A

A/T

Rbfox2 : Intron 4 43 C C C T T T T

T

C/T

Rbfox2 : Intron 3 44 G G G T T T T

T

G/T

Rbfox2 : Intron 4 45 G G G A A A A

A

A/G

Rbfox2 : Intron 4 46 T

T A A A A

A

A/T

274

Rbfox2 : Intron 1 47 G G G G

G

G

A/G

Rbfox2 : Intron 4 48 C C C T T T T

T

C/T

Rbfox2 : Intron 3 49 A

G

G

A/G

Rbfox2 : Intron 4 50 G G G T

T T

T

G/T

Rbfox2 : Intron 3 51 T

C

C

C/T

Rbfox2 : Intron 3 52 G

A

A

A/G

Rbfox2 : Intron 3 53 A A A G

G

G

A/G

Rbfox2 : Intron 3 54 A

C C C

A/C

Rbfox2 : Intron 3 55 T T T C

C C

C

C/T

Rbfox2 : Intron 3 56 T

A A A

A/T

Rbfox2 : Intron 3 57 G

A

A

A/G

Rbfox2 : Intron 1 58 T

A A

A/T

Rbfox2 : Intron 4 59 G G G A A A A

A

A/G

Rbfox2 : Intron 3 60 G

T T T

G/T

Rbfox2 : Intron 4 61 T

T C C C C

C

C/T

Rbfox2 : Intron 4 62 C C C T

T T

T

C/T

Rbfox2 : Intron 3 63 T

C

C

C/T

Rbfox2 : Intron 4 64 A A A C C C C

C

A/C

Rbfox2 : Intron 1 65 A

A A

A

Rbfox2 : Intron 1 66 C C C C

C

C

C/T

Rbfox2 : Intron 1 67 A A A A

A

A

A/G

Rbfox2 : Intron 2 68 A

C C

A/C

Rbfox2 : Intron 4 69 A A A G G G G

G

A/G

Rbfox2 : Intron 3 70 G G G C

C

C

C/G

Rbfox2 : Intron 1 71

G

T

T

T

G/T

Rbfox2 : Intron 2 72 C C C T

T

T

C/T

Rbfox2 : Intron 1 73 T

A

A

A/T

Rbfox2 : Intron 3 74 G

A A A

A/G

Rbfox2 : Intron 3 75 C

G G G

C/G

Rbfox2 : Intron 3 76 C

T T T

C/T

Rbfox2 : Intron 3 77 C

C G

G G

G

C/G

Rbfox2 : Intron 3 78 A

G G

A/G

Rbfox2 : Intron 2 79 T

T G T T

G/T

Rbfox2 : Intron 2 80 A

A C A

A/C

Rbfox2 : Intron 2 81 G

A

A

A/G

Rbfox2 : Intron 3 82 T

C C C

C/T

Rbfox2 : Intron 3 83 A

C C C

A/C

Rbfox2 : Intron 4 84 G G G A A A A

A

A/G

Rbfox2 : Intron 3 85 A

G G

A/G

Rbfox2 : Intron 4 86 T

C C C C

C/T

Rbfox2 : Intron 4 87 T

T C C C C

C

C/T

Rbfox2 : Intron 4 88 C C C T T T T T T T C/T

Rbfox2 : Intron 1 89 C C C C

C

C

C/T

275

Rbfox2 : Intron 1 90 G G G G

G

G

A/G

Rbfox2 : Intron 1 91 A A A A

A

A

A/C

Rbfox2 : Intron 4 92 G

G A A A A

A

A/G

Rbfox2 : Intron 4 93 G

G A A A

A

A/G

Rbfox2 : Intron 4 94 G

G A A A A

A

A/G

Rbfox2 : Intron 3 95 T T T G G G G

G

G/T

Rbfox2 : Intron 1 96 A

G G G

A/G

Rbfox2 : Intron 2 97 A

T

A/T

Rbfox2 : Intron 2 98 A

G G

A/G

Rbfox2 : Intron 4 99 A A A C C C C

C

A/C

Rbfox2 : Intron 3 100 A

G

G

A/G

Rbfox2 : Intron 1 101 C

G

C/G

Rbfox2 : Intron 2 102 G

A

A/G

Rbfox2 : Intron 3 103 A

G G G

A/G

Rbfox2 : Intron 4 104 C C C T T T T

T

C/T

Rbfox2 : Intron 3 105 C

A A A

A/C

Rbfox2 : Intron 3 106 G

A A A

A/G

Rbfox2 : Intron 3 107 C

A A

A/C

Rbfox2 : Intron 4 108 A A A G G G

G

A/G

Rbfox2 : Intron 2 109 A

T T

A/T

Rbfox2 : Intron 3 110 G

A A A

A/G

Rbfox2 : Intron 3 111 T T T C

C

C

C/T

Rbfox2 : Intron 4 112 T T T C

C

C

C/T

Rbfox2 : Intron 2 113 T

C C

C/T

Rbfox2 : Intron 4 114 C C C T

T T

T

C/T

Rbfox2 : Intron 2 115 T T T A

A

A

A/T

Rbfox2 : Intron 4 116 G G G A

A A

A

A/G

Rbfox2 : Intron 4 117 A A A G

G G

G

A/G

Rbfox2 : Intron 3 118 C

T

T

C/T

Rbfox2 : Intron 3 119 T

C C C C

C/T

Rbfox2 : Intron 3 120 C

T T T

C/T

Rbfox2 : Intron 4 121 C

T T T T

C/T

Rbfox2 : Intron 2 122 A

G G G

A/G

Rbfox2 : Intron 3 123 G G G A A A

A

A/G

Rbfox2 : Intron 2 124 G

A A A

A/G

Rbfox2 : Intron 1 125 G

G A

A/G

Rbfox2 : Intron 1 126 A

G

G

A/G

Rbfox2 : Intron 1 127 A

G

G

A/G

Rbfox2 : Intron 3 128 A

A

C

A/C

Rbfox2 : Intron 2 129 G

A A A

A/G

Rbfox2 : Intron 2 130 G

T T T

G/T

Rbfox2 : Intron 3 131 G

A A

A/G

Rbfox2 : Intron 1 132 G

A

A/G

276

Rbfox2 : Intron 3 133 T

G G

G/T

Rbfox2 : Intron 3 134 G G G T T T

T

G/T

Rbfox2 : Intron 2 135 C

T T T

C/T

Rbfox2 : Intron 4 136 C C C T

T T

T

C/T

Rbfox2 : Intron 1 137 G

A

A/G

Rbfox2 : Intron 2 138 A

G G

A/G

Rbfox2 : Intron 2 139 A

G G

A/G

Rbfox2 : Intron 4 140 G G G A A A A

A

A/G

Rbfox2 : Intron 2 141 T T T C

C

C

C/T

Rbfox2 : Intron 3 142 A A A G G G G

A

A/G

Rbfox2 : Intron 2 143 C

G G

C/G

Rbfox2 : Intron 3 144 G

A A A

A/G

Rbfox2 : Intron 3 145 A

G G

A/G

Rbfox2 : Intron 4 146 A A A G G G G G G G A/G

Rbfox2 : Intron 2 147 T

G G G

G/T

Apol10b : mRNA-UTR 2 1 G

A

A/G

Apol10b : mRNA-UTR 2 2 G

A

A/G

Apol10b : Coding-Synonymous

2 3 C

G

C/G

Apol10b : Coding-Synonymous

2 4 T

C C

C/T

Apol10b : Coding-NonSynonymous

2 5 A

T T

A/T

Apol10b : Coding-NonSynonymous

2 6 G

A A

A/G

Apol10b : Coding-NonSynonymous

2 7 G

A A

A/G

Apol10b : Coding-Synonymous

1 8 G

A

A/G

Apol10b : Intron 1 9 A

C C

A/C

Apol10b : Intron 1 10 G

A

A/G

Apol10b : Intron 1 11 C

G

C/G

Apol10b : Intron 1 12 G

A

A/G

Apol10b : Intron 1 13 C

A

A/C

Apol10b : Intron 2 14 A

T

A/T

Apol10b : Intron 1 15 T

A

A/T

Apol10b : Intron 1 16 T

C

C/T

Apol10b : Intron 2 17 T

C

C/T

Apol10b : Intron 1 18 T

G

G/T

Apol10b : Intron 2 19 C

T T

C/T

Apol10b : Intron 2 20 A

G

A/G

Apol10b : Intron 3 21 A

G G G

A/G

Apol10b : Intron 1 22 A

G G

A/G

Apol10b : Intron 2 23 G

A A

A/G

Apol10b : Intron 2 24 T

C C

C/T

277

Apol10b : Intron 2 25 A

G G

A/G

Apol10b : Intron 2 26 T

C C

C/T

Apol10b : Intron 2 27 A

G G

A/G

Apol10b : Intron 2 28 G

A

A/G

Apol10b : Intron 2 29 G

T

G/T

Apol10b : Intron 2 30 C

T

C/T

Apol10b : Intron 2 31 A

G

A/G

Apol10b : Intron 1 32 T

A

A/T

Apol10b : Intron 1 33 A

G G

A/G

Apol10b : Intron 1 34 G

A A

A/G

Apol10b : Intron 2 35 A

T

A/T

Apol10b : Intron 3 36 C T

T T T

T

C/T

Apol10b : Intron 2 37 G

A A

A/G

Apol10b : Intron 2 38 A

C C

A/C

Apol10b : Intron 2 39 A

T T

A/T

Apol10b : Intron 1 40 C

T

T

C/T

Apol10b : Intron 3 41 T

C C

C/T

Apol10b : Intron 3 42 C

T T

C/T

Apol10b : Intron 3 43 T

C C C C

C/T

Apol10b : Intron 1 44

A

T

A/T

Apol10b : Intron 4 45 T

C

C C

C

C/T

Apol10b : Intron 2 46 T T T C

C

C

C/T

Apol10b : Intron 4 47 T T T C

C

C

C/T

Apol10b : Intron 3 48 G

A A A A

A/G

Apol10b : Intron 1 49 T T T T

T

T

C/T

Apol10b : Intron 1 50 A

G G

A/G

Apol10b : Intron 1 51 C

T T

C/T

Apol10b : Intron 1 52 T

G

G/T

Apol10b : Intron 1 53 T

C

C/T

Apol10b : Intron 1 54 A

G

A/G

Apol10b : Intron 1 55

C C

T

C/T

Cacng2 : Intron 3 1 T A A A/T

Cacng2 : Intron 3 2 C

A A

A/C

Cacng2 : Intron 4 3 G G

A A A A

A

A/G

Cacng2 : Intron 3 4 T

C

C

C

C/T

Cacng2 : Intron 3 5 G G

A

A

A

A/G

Cacng2 : Intron 3 6 C

T

C/T

Cacng2 : Intron 4 7 C C

T

T

C/T

Cacng2 : Intron 4 8 C C

T

T

T

C/T

Cacng2 : Intron 4 9 C C

T T

T

C/T

Cacng2 : Intron 2 10 G

A

A/G

Cacng2 : Intron 4 11 C C

T

T T

T

C/T

278

Cacng2 : Intron 3 12 C

T T

T

C/T

Cacng2 : Intron 2 13 A

G G

G

A/G

Cacng2 : Intron 3 14 A

G

G G

A/G

Cacng2 : Intron 3 15 G G G A A A A

A/G

Cacng2 : Intron 2 16 C C C A

A

A

A/C

Cacng2 : Intron 4 17 C C C T T T T

T

C/T

Cacng2 : Intron 2 18 T

C C C

C/T

Cacng2 : Intron 1 19 C

T T T

C/T

Cacng2 : Intron 1 20 G G G T

T

T

G/T

Cacng2 : Intron 3 21 T

C C C

C/T

Cacng2 : Intron 3 22 G

T

T

G/T

Cacng2 : Intron 3 23 T

C

C

C/T

Cacng2 : Intron 2 24 C

T

T

C/T

Cacng2 : Intron 3 25 C

T

T

C/T

Cacng2 : Intron 1 26 T T T T

T

T

G/T

Cacng2 : Intron 3 27 C

T

T

C/T

Cacng2 : Intron 3 28 T

G

G

G/T

Cacng2 : Intron 2 29 C

T

C/T

Cacng2 : Intron 3 30 C

T T T

C/T

Cacng2 : Intron 3 31 T

C C C

C/T

Cacng2 : Intron 2 32

T C

C/T

Cacng2 : Intron 3 33 G

A

A

A/G

Cacng2 : Intron 2 34 T T T C

C

C

C/T

Cacng2 : Intron 3 35 C

T T T C

C/T

Cacng2 : Intron 3 36 A

G G

A/G

Cacng2 : Intron 4 37 C C C T

T T

T

C/T

Cacng2 : Intron 3 38 C

C C T

C/T

Cacng2 : Intron 2 39 G

A A G

A/G

Cacng2 : Intron 3 40 A

A A C

A/C

Cacng2 : Intron 1 41 A

T T

A/T

Cacng2 : Intron 3 42 A

A C

A/C

Cacng2 : Intron 1 43 C

C C T

C/T

Cacng2 : Intron 4 44 G G G G G G A

G

A/G

Cacng2 : Intron 3 45 T

T

T C

C/T

Cacng2 : Intron 3 46 G G G A

A

A

A/G

Cacng2 : Intron 4 47 A A A A A A G

A

A/G

Cacng2 : Intron 3 48 C

T

C/T

Cacng2 : Intron 3 49 T

C

C/T

Cacng2 : Intron

3 50

C

C

T

C/T

Cacng2 : Intron 2 51 G

G C

C/G

Cacng2 : Intron 3 52 A

A A G

A/G

Cacng2 : Intron 3 53 G

G C

C/G

279

Cacng2 : Intron 3 54 T

T T A

A/T

Cacng2 : Intron 5 55 A

A A A A G

A/G

Cacng2 : Intron 4 56 A

A A A G

A/G

Cacng2 : Intron 4 57 T

T G

G/T

Cacng2 : Intron 3 58 C

C T

C/T

Cacng2 : Intron 4 59 G G G G G G C

G

C/G

Cacng2 : Intron 4 60 A A A A A A C

A

A/C

Cacng2 : Intron 3 61 C

C C C T

C/T

Cacng2 : Intron 2 62 T

T

C

C/T

Cacng2 : Intron 3 63 C

C C C T

C/T

Cacng2 : Intron 3 64 A

A A A G

A/G

Cacng2 : Intron 3 65 A

A A A G

A/G

Cacng2 : Intron 3 66 G

G G G A

A/G

Cacng2 : Intron 4 67 G G G G G G A

G

A/G

Cacng2 : Intron 3 68 A

A G

A/G

Cacng2 : Intron 3 69 T

T G

G/T

Cacng2 : Intron 4 70 G G G G G G C

G

C/G

Cacng2 : Intron 1 71 G

C

C/G

Cacng2 : Intron 3 72 G

G G G A

A/G

Cacng2 : Intron 3 73 C

C C C T

C/T

Cacng2 : Intron 3 74 G

G G G C

C/G

Cacng2 : Intron 3 75 T

C C C T

C/T

Cacng2 : Intron 4 76 A A A A A A G

A

A/G

Cacng2 : Intron 3 77 G

G

A

A/G

Cacng2 : Intron 3 78 C

C C T

C/T

Cacng2 : Intron 3 79 C C C A

A

A

A/C

Cacng2 : Intron 3 80 T

T T C

C/T

Cacng2 : Intron 3 81 C

C C T

C/T

Cacng2 : Intron 4 82 T T T T

T C

T

C/T

Cacng2 : Intron 3 83 C

C C T

C/T

Cacng2 : Intron 3 84 T

T T C

C/T

Cacng2 : Intron 3 85 C

C C T

C/T

Cacng2 : Intron 3 86 T

T T C

C/T

Cacng2 : Intron 3 87 G

G G A

A/G

Cacng2 : Intron 3 88 A

A A C

A/C

Cacng2 : Intron 2 89 G

G G A

A/G

Cacng2 : Intron 2 90 C

C C T

C/T

Cacng2 : Intron 3 91 G

G G C

C/G

Cacng2 : Intron 3 92 A

A A G

A/G

Cacng2 : Intron 3 93 C

C C T

C/T

Cacng2 : Intron 4 94 T T T T T T A

T

A/T

Cacng2 : Intron 3 95 C

C

T

C/T

Cacng2 : Intron 4 96 A A A A A A G

A

A/G

280

Cacng2 : Intron 3 97 G

G G A

A/G

Cacng2 : Intron 3 98 G

G G A

A/G

Cacng2 : Intron 3 99 G

G G G A

A/G

Cacng2 : Intron 3 100 C

C C C T

C/T

Cacng2 : Intron 3 101 C

C C C T

C/T

Cacng2 : Intron 3 102 G

G G G A

A/G

Cacng2 : Intron 4 103 C C C C C C G

C

C/G

Cacng2 : Intron 4 104 T T T T T T C

T

C/T

Cacng2 : Intron 4 105 A A A G G G

G

A/G

Cacng2 : Intron 3 106 G

G

G A

A/G

Cacng2 : Intron 3 107 C

C T

C/T

Cacng2 : Intron 3 108 A

A

A G

A/G

Cacng2 : Intron 3 109 G

G

G A

A/G

Pvalb : Intron 2 1 T

T C

C/T

Pvalb : Intron 3 2 T T T T T C

C/T

Pvalb : Intron 3 3 G G G G G A

A/G

Pvalb : Intron 2 4 G

G A

A/G

Pvalb : Intron 2 5 C

C T

C/T

Pvalb : Intron 4 6 T T T T T C C

C/T

Pvalb : Intron 4 7 A A A A A G G

A

A/G

Pvalb : Intron 3 8 A

A G G

A/G

Pvalb : Intron 3 9 T

T A A

A/T

Pvalb : Intron 3 10 G

G T T

G/T

Pvalb : Intron 3 11 C

C T T

C/T

Pvalb : Intron 1 12 G

G G

T

G/T

Pvalb : Intron 3 13 C

C T

C/T

Pvalb : Intron 2 14 C C C C

T

C/T

Pvalb : Intron 2 15 C C C C

T

C/T

Pvalb : Intron 4 16 G G G

A A

G

A/G

Pvalb : Intron 2 17 G

A A

A/G

Pvalb : Intron 4 18 A A A A A G G

A

A/G

Pvalb : Intron 2 19 C C C C C T C

C

C/T

Pvalb : Intron 3 20 T

T T C C

C/T

Pvalb : Intron 4 21 T T

T

C

T

C/T

Pvalb : Intron 2 22 G

G A G

A/G

Pvalb : Intron 4 23 T T T T

C C

T

C/T

Pvalb : Intron 2 24 G

G A G

A/G

Csf2rb2 : mRNA-UTR 3 1 C

C T T

C/T

Csf2rb2 : mRNA-UTR 4 2 A A A A A T T

A

A/T

Csf2rb2 : mRNA-UTR 4 3 T T T T T G G

T

G/T

Csf2rb2 : mRNA-UTR 4 4 C C C C C T T

C

C/T

281

Csf2rb2 : mRNA-UTR 4 5 T T T T T C C

T

C/T

Csf2rb2 : mRNA-UTR 4 6 C C C C C G G

C

C/G

Csf2rb2 : mRNA-UTR 3 7 T

T T C

C/T

Csf2rb2 : mRNA-UTR 3 8 G

G G A

A/G

Csf2rb2 : mRNA-UTR 3 9 G

G G A

A/G

Csf2rb2 : mRNA-UTR 2 10 T

T

T

Csf2rb2 : mRNA-UTR 3 11 C C C C C T

C

C/T

Csf2rb2 : mRNA-UTR 3 12 G G G G

T

G

G/T

Csf2rb2 : mRNA-UTR 2 13 A

G

A/G

Csf2rb2 : mRNA-UTR 3 14 A

G G

A/G

Csf2rb2 : mRNA-UTR 2 15 A

G

A/G

Csf2rb2 : Coding-Synonymous

3 16 C

G G

C/G

Csf2rb2 : Coding-Synonymous

4 17 T

T

C C

C/T

Csf2rb2 : Coding-Synonymous

1 18 C

A A

A/C

Csf2rb2 : Coding-NonSynonymous

2 19 T

C C

C/T

Csf2rb2 : Coding-Synonymous

4 20 C

C

A A

A/C

Csf2rb2 : Coding-NonSynonymous

3 21 C

C C

T

C/T

Csf2rb2 : Coding-Synonymous

2 22 C

C T

C/T

Csf2rb2 : Coding-NonSynonymous

2 23 T

T

C/T

Csf2rb2 : Coding-Synonymous

2 24 G

G A

A/G

Csf2rb2 : Coding-NonSynonymous

3 25 G G G G G A

G

A/G

Csf2rb2 : Coding-NonSynonymous

2 26 C C C C C T

C

C/T

Csf2rb2 : Intron 3 27 C C C C C T

C

C/T

Csf2rb2 : Intron 3 28 G G G G G C

G

C/G

Csf2rb2 : Intron 2 29 T T T T T C

T

C/T

Csf2rb2 : Intron 3 30 A A A A A G

A

A/G

Csf2rb2 : Intron 3 31 A A A A A G

A

A/G

Csf2rb2 : Intron 3 32

C T

T

C/T

Csf2rb2 : Intron 2 33 A

A A A T

A/T

Csf2rb2 : Intron 2 34 T T

T T C

T

C/T

Csf2rb2 : Intron 1 35 C

C

C

Csf2rb2 : Intron 1 36 G

G

G

282

Csf2rb2 : Intron 3 37 C C

C C A

A/C

Csf2rb2 : Intron 1 38

G

G

G

A/G

Csf2rb2 : Intron 3 39 T T T T T C

T

C/T

Csf2rb2 : Intron 3 40 A A A A

G

A

A/G

Csf2rb2 : Intron 2 41 T

C

C/T

Csf2rb2 : Intron 1 42 G

A

A/G

Csf2rb2 : Intron 1 43 G

A

A/G

Csf2rb2 : Intron 1 44 A

C

A/C

Csf2rb2 : Intron 1 45 G

A

A/G

Csf2rb2 : Intron 2 46 C C C C

T

C

C/T

Csf2rb2 : Intron 1 47 A

G

A/G

Csf2rb2 : Intron 1 48 G

T

G/T

Csf2rb2 : Intron 1 49 G

A

A/G

Csf2rb2 : Intron 1 50 C

T

C/T

Csf2rb2 : Intron 1 51 G

A

A/G

Csf2rb2 : Coding-NonSynonymous

2 52 G

A

A/G

Csf2rb2 : Intron 1 53 C

G

C/G

Csf2rb2 : Intron 1 54 T

C

C/T

Csf2rb2 : Intron 1 55 A

A G

A/G

Csf2rb2 : Intron 1 56 C

C T

C/T

Csf2rb2 : Intron 1 57 A

A T

A/T

Csf2rb2 : Intron 1 58 T

T C

C/T

Csf2rb2 : Intron 1 59 C

C T

C/T

Csf2rb2 : Intron 1 60 T

T A

A/T

Csf2rb2 : Intron 1 61 C

C T

C/T

Csf2rb2 : Intron 1 62 A

A G

A/G

Csf2rb2 : Intron 1 63 A

A G

A/G

Csf2rb2 : Intron 1 64 A

A T

A/T

Csf2rb2 : Intron 1 65 G

G C

C/G

Csf2rb2 : Intron 1 66 C

C T

C/T

Csf2rb2 : Intron 1 67 T

T G

G/T

Csf2rb2 : Intron 1 68 G

G C

C/G

Csf2rb2 : Intron 2 69 A

A A G G

A/G

Csf2rb2 : Intron 3 70 A

A A G G

A/G

Csf2rb2 : Intron 3 71 C

C

T T

C/T

Csf2rb2 : Intron 3 72 C

C

T T

C/T

Csf2rb2 : Intron 3 73 A

A

G G

A/G

Csf2rb2 : Intron 3 74 T T T T

A

A/T

Csf2rb2 : Intron 3 75 A

A A

G

A/G

Csf2rb2 : Intron 1 76 T T T T

T

T

G/T

Csf2rb2 : Intron 3 77 C C C C

A

C

A/C

283

Csf2rb2 : Coding-NonSynonymous

4 78 A A A A

T T

A/T

Csf2rb2 : Intron 3 79 T

G G

G/T

Csf2rb2 : Intron 3 80 C

T T

C/T

Csf2rb2 : Intron 3 81 T

C C

C/T

Csf2rb2 : Intron 4 82 T T T T

C C

T

C/T

Csf2rb2 : Intron 4 83 G G G G

A A

G

A/G

Csf2rb2 : Intron 5 84 C C C C C G G

C

C/G

Csf2rb2 : Intron 1 85

C C

C

C/T

Csf2rb2 : Intron 3 86 C

C C T T

C/T

Csf2rb2 : Intron 3 87 T

T T C C

C/T

Csf2rb2 : Intron 1 88

A G

A/G

Csf2rb2 : Intron 2 89 T T T T T C

T

C/T

Csf2rb2 : Intron 1 90 C C C C

T

C

C/T

Csf2rb2 : Intron 2 91 T T T T T C

T

C/T

Csf2rb2 : Intron 1 92 A

A

A

Csf2rb2 : Intron 1 93 T

G

G/T

Csf2rb2 : Intron 1 94 T

G

G/T

Csf2rb2 : Coding-Synonymous

2 95 T

T A

A/T

Csf2rb2 : Coding-NonSynonymous

3 96 A A A A A G

A

A/G

Csf2rb2 : Coding-NonSynonymous

2 97 G G G G G T

G

G/T

Csf2rb2 : Intron 3 98 G G G G G A

G

A/G

Csf2rb2 : Coding-Synonymous

2 99 A

A G

A/G

Csf2rb2 : Coding-NonSynonymous

2 100 A

A G

A/G

Csf2rb2 : Coding-Synonymous

2 101 T

T G

G/T

Csf2rb2 : Intron 3 102 G

G G G A

G

A/G

Csf2rb2 : Intron 1 103 A

A C

A/C

Csf2rb2 : Intron 1 104

C

C

C

C/G

Csf2rb2 : Intron 2 105 T T T T T C

T

C/T

Csf2rb2 : Intron 2 106 C

A

A/C

Csf2rb2 : Intron 2 107 T

T A

A/T

Csf2rb2 : Intron 2 108 T

C C

C/T

Csf2rb2 : Intron 2 109 C C C C C T

C

C/T

Csf2rb2 : Intron 2 110 A

A A G G

A/G

Csf2rb2 : Intron 3 111 C C C C C A

C

A/C

Csf2rb2 : Intron 3 112 C C C C C A

C

A/C

Csf2rb2 : Intron 2 113 C C C C

C

C

C/T

284

Csf2rb2 : Intron 2 114 C

C A

A/C

Csf2rb2 : Intron 3 115 A A A A A G

A

A/G

Csf2rb2 : Intron 4 116 G G G G G A

G

A/G

Csf2rb2 : Intron 4 117 T T T T

A A

T

A/T

Csf2rb2 : Intron 3 118 T T T T T G

T

G/T

Csf2rb2 : Intron 2 119 A A A A A G

A

A/G

Csf2rb2 : Intron 1 120 G G G G

A

G

A/G

Csf2rb2 : Intron 2 121 G

G A

A/G

Csf2rb2 : Intron 1 122 C

T

C/T

Csf2rb2 : Intron 4 123 T T T T T C C

T

C/T

Csf2rb2 : Coding-Synonymous

3 124 A A A A

G G

A

A/G

Csf2rb2 : Coding-NonSynonymous

2 125 G G G G

C

G

C/G

Csf2rb2 : Intron 4 126 T T T T

G G

G/T

Csf2rb2 : Intron 3 127 G G G G

A A

G

A/G

Csf2rb2 : Intron 3 128 G G G G

A

G

A/G

Csf2rb2 : Intron 2 129 G

A

A/G

Csf2rb2 : Intron 1 130 C

C G

C/G

Csf2rb2 : Intron 3 131 C

C C C A

C

A/C

Csf2rb2 : Intron 4 132 A A A A

C

A

A/C

Csf2rb2 : Intron 2 133 G

G

T

G/T

Csf2rb2 : Intron 2 134 A

A A T

A/T

Csf2rb2 : Intron 2 135 T

T A

A/T

Csf2rb2 : Intron 2 136

A C

A/C

Csf2rb2 : Intron 2 137 A

A C

A/C

Csf2rb2 : Intron 2 138 C

C G

C/G

Csf2rb2 : Intron 2 139 G

G T

G/T

Csf2rb2 : Intron 1 140 C

T

C/T

Csf2rb2 : Intron 2 141 G

G

A/G

Csf2rb2 : Intron 2 142 A

A T

A/T

Csf2rb2 : Intron 2 143 G

G A

A/G

Csf2rb2 : Intron 2 144 T

T

G G

G/T

Csf2rb2 : Intron 2 145 A

A

C C

A/C

Csf2rb2 : Intron 3 146 A

A

G G

A/G

Csf2rb2 : Intron 2 147 A

A

C C

A/C

Csf2rb2 : Intron 4 148 G C

? G C C

C

C/G

Csf2rb2 : Intron 3 149 A

A A C C

A/C

Csf2rb2 : Intron 2 150 T

T T G G

G/T

Csf2rb2 : Intron 3 151 C

C C C A A

C

A/C

Csf2rb2 : Intron 1 152 T

T C

C/T

Csf2rb2 : Intron 3 153 C

C T T

C/T

Csf2rb2 : Intron 3 154 T

T T C C

C/T

285

Csf2rb2 : Intron 3 155 A

A A G G

A/G

Csf2rb2 : Intron 3 156 A

A A G

A/G

Csf2rb2 : Intron 3 157 T

T A

A/T

Csf2rb2 : Intron 4 158 G G G G G A A

G

A/G

Csf2rb2 : Intron 4 159 T T T T T G G

T

G/T

Csf2rb2 : Intron 3 160

G A A

A/G

Csf2rb2 : Intron 3 161 G

G A A

A/G

Csf2rb : Intron 2 1 A A C A/C

Csf2rb : Intron 2 2 G

G A

A/G

Csf2rb : Intron 4 3 G G G G G A A

G

A/G

Csf2rb : Intron 1 4 A

A T

A/T

Csf2rb : Intron 1 5 T

T G

G/T

Csf2rb : Intron 4 6 A A A A A G

A

A/G

Csf2rb : Intron 4 7 T T T T T A A

T

A/T

Csf2rb : Intron 3 8 A

A G

A/G

Csf2rb : Intron 3 9 T

T C C

C/T

Csf2rb : Intron 2 10 G

G

T

G/T

Csf2rb : Intron 3 11 C

C

T T

C/T

Csf2rb : Coding-NonSynonymous

1 12 C C C C

G

C

C/G

Csf2rb : Intron 3 13 A

A A C C

A/C

Csf2rb : Intron 1 14 A A A A

G

A

A/G

Csf2rb : Intron 4 15 G

G G G A A

A/G

Csf2rb : Intron 3 16 C

C T

C/T

Csf2rb : Intron 1 17 T T T T

C

T

C/T

Csf2rb : Intron 1 18 A A A A

C

A

A/C

Csf2rb : Intron 1 19 G G G G

G

G

G/T

Csf2rb : Intron 1 20 A A A A

T

A

A/T

Csf2rb : Intron 1 21 C C C C

T

C

C/T

Csf2rb : Intron 1 22 T T T T

C

T

C/T

Csf2rb : Intron 1 23 G G G G

T

G

G/T

Csf2rb : Coding-NonSynonymous

1 24 C C C C

A

C

A/C

Csf2rb : Coding-NonSynonymous

1 25

T T T

C

T

C/T

Csf2rb : Intron 2 26

T T T

G

G/T

Csf2rb : Intron 2 27 T T T T T ?

T

C/T

Csf2rb : Intron 2 28 G G G G G A

G

A/G

Csf2rb : Intron 1 29 C

C T

C/T

Csf2rb : Intron 1 30

T

T

C/T

Csf2rb : Intron 3 31 A

A G G

A/G

Csf2rb : Intron 1 32 G

G

A/G

286

Csf2rb : Intron 4 33 C C C C C G G

C

C/G

Csf2rb : Intron 1 34 T

C

C/T

Csf2rb : Intron 1 35 G

A

A/G

Csf2rb : Intron 1 36 A

G

A/G

Csf2rb : Intron 1 37 C

T

C/T

Csf2rb : Intron 1 38 T

A T

A/T

Csf2rb : Intron 1 39 G

A G

A/G

Csf2rb : Intron 1 40 C

A C

A/C

Csf2rb : Intron 2 41 A

? G G

A/G

Csf2rb : Intron 1 42 T

A T

A/T

Csf2rb : Intron 1 43 C

C

C

Csf2rb : Intron 3 44 C

C G

C/G

Csf2rb : Intron 1 45 G G G G

G

G

G/T

Csf2rb : Intron 4 46 A

A

G G

A/G

Csf2rb : Intron 3 47 T

T

A

A/T

Csf2rb : Intron 2 48 G G G G

C

G

C/G

Csf2rb : Intron 3 49 G G G G G A A

G

A/G

Csf2rb : Intron 2 50 A

A A G G

A/G

Csf2rb : Coding-Synonymous 4 51 C

C

C T T

C/T

Csf2rb : Coding-NonSynonymous

1 52 A

A

G G

A/G

Csf2rb : Coding-NonSynonymous

2 53 ? A

C C A A

A/C

Csf2rb : mRNA-UTR 2 54 C

C G G

C/G

Csf2rb : mRNA-UTR 1 55 T

G T

G/T

Csf2rb : mRNA-UTR 3 56 T

T T G G

G/T

Csf2rb : mRNA-UTR 2 57 A

A A G G

A/G

Csf2rb : mRNA-UTR 1 58 G

G G C C

C/G

Csf2rb : mRNA-UTR 2 59 A

A G

A/G

Elfn2 : mRNA-UTR 3 1 T

T C

C/T

Elfn2 : Intron 3 2 G

G A

A/G

Elfn2 : Intron 3 3 T

T T T C

C/T

Elfn2 : Intron 4 4 T T T T

T C

T

C/T

Elfn2 : Intron 3 5 G

G G T

G/T

Elfn2 : Intron 3 6 G

G A

A/G

Elfn2 : Intron 4 7 G

G G

G A

G

A/G

Elfn2 : Intron 3 8 G

G C

C/G

Elfn2 : Intron 1 9

T G

G/T

Card10 : Intron 3 1 C C C T C/T

Card10 : mRNA-UTR 2 2 C

? T

C/T

287

Card10 : mRNA-UTR 1 3 G

A A

A/G

Nol12 : mRNA-UTR 3 1 C C C T T C

C

C/T

Nol12 : Intron 3 2 T

T G G T

T

G/T

Nol12 : Intron 3 3 T T T C

T

T

C/T

Nol12 : Intron 3 4 A A A C

A

A

A/C

Nol12 : Coding-Synonymous 3 5 A A A G

A

A

A/G

Nol12 : Intron 3 6 G G G A A G

G

A/G

Nol12 : Intron 3 7 T T

C C T

C/T

Nol12 : Intron 3 8 A A

G G A

A/G

Nol12 : Intron 3 9 T T

C C T

C/T

Nol12 : Intron 3 10 G G G C C G

G

C/G

Nol12 : Intron 3 11 A A A G G A

A

A/G

Nol12 : Intron 2 12 A

G A

A/G

Nol12 : Intron 2 13 G

A G

A/G

Nol12 : Intron 3 14 C

C T T C

C/T

Nol12 : Intron 2 15 T

C T

C/T

Nol12 : Intron 2 16 C

C

C

Nol12 : Intron 3 17 A A A G

A

A

A/G

Nol12 : Intron 2 18 C

A C

A/C

Nol12 : Intron 2 19 A

G A

A/G

Nol12 : Intron 3 20 A A A C C A

A

A/C

Nol12 : Intron 2 21

C T

C/T

Nol12 : Intron 2 22

C T

C/T

Nol12 : Intron 2 23

A C

A/C

Nol12 : Coding-NonSynonymous

3 24 T T T C C T

T

C/T

Nol12 : Intron 2 25 A

G A

A/G

Nol12 : Intron 2 26 A

G A

A/G

Nol12 : Intron 2 27 T

C T

C/T

Nol12 : Intron 2 28 C

T C

C/T

Nol12 : Intron 2 29 A

C A

A/C

Nol12 : Intron 3 30 G

G C C G

G

C/G

Nol12 : Intron 3 31 A

A G G A

A

A/G

Nol12 : Intron 2 32 T

G T

G/T

Nol12 : Intron 3 33 G

T

G

G/T

Nol12 : Intron 2 34 A

A

A

Nol12 : Intron 2 35 T

T

T

Nol12 : Intron 3 36 A A A G

A

A

A/G

Nol12 : Intron 2 37 T

T

T

Nol12 : Intron 3 38 G G G A A G

G

A/G

Nol12 : Intron 3 39 C

C T T C

C

C/T

288

Nol12 : Intron 2 40 G

T G

G/T

Nol12 : Intron 2 41 A

G A

A/G

Nol12 : Intron 2 42 T

C T

C/T

Nol12 : Intron 2 43 G

C G

C/G

Nol12 : Intron 2 44 G

A G

A/G

Nol12 : Intron 2 45 A

T A

A/T

Nol12 : Intron 2 46 C

T C

C/T

Nol12 : Intron 2 47 A

G A

A/G

Nol12 : mRNA-UTR 3 48 G G G T T G

G

G/T

Nol12 : mRNA-UTR 3 49 G G G A A G

G

A/G

Nol12 : mRNA-UTR 3 50 C

C G G C

C

C/G

Nol12 : mRNA-UTR 3 51 G G G A

G

G

A/G

Nol12 : mRNA-UTR 3 52 A A A G G A

A

A/G

Nol12 : mRNA-UTR 3 53 A A A G G A

A

A/G

1700088E04Rik : Intron 2 1 G

T G G

G/T

1700088E04Rik : Intron 3 2 G

A A G A

A/G

1700088E04Rik : Intron 3 3 A

G G A G

A/G

1700088E04Rik : Intron 3 4 G

C G C

C/G

1700088E04Rik : Intron 3 5 T

G T G

G/T

1700088E04Rik : Intron 4 6 T T T C C T C

T

C/T

1700088E04Rik : Intron 4 7 A A A G G A G

A

A/G

1700088E04Rik : Intron 2 8

G T T

G/T

1700088E04Rik : Intron 3 9

T C

C/T

1700088E04Rik : Intron 2 10

A G

A/G

1700088E04Rik : Intron 2 11 G

A G

A/G

1700088E04Rik : Intron 2 12 A

T A

A/T

1700088E04Rik : Intron 4 13 G G G A A G A

G

A/G

1700088E04Rik : mRNA-UTR 4 14 G G G A A G A

G

A/G

Pick1 : Intron 4 1 A A A

G A

A

A/G

Pick1 : Coding-Synonymous 4 2 G G G A A G A

G

A/G

Pick1 : Intron 3 3

G A G

A/G

Pick1 : Intron 1 4 G G G C

G

G

C/G

Pick1 : Intron 3 5 T

T C

T C

T

C/T

Pick1 : Intron 4 6 A A A A

A T

A

A/T

Pick1 : Intron 3 7 T T T C

T

T

C/T

Pick1 : Intron 2 8 T T T C C T

T

C/T

Pick1 : Intron 2 9 T

C T

C/T

Pick1 : Intron 3 10

C C A

C

C

A/C

Pick1 : Intron 3 11 T

T C

T

T

C/T

289

Pick1 : Intron 3 12 A A A G

A

A

A/G

Pick1 : Intron 3 13 G

G T

G

G

G/T

Pick1 : Intron 3 14 G G G C

G

G

C/G

Pick1 : Intron 3 15 T T T C

T

T

C/T

Pick1 : Intron 3 16 C C C A

C

C

A/C

Pick1 : Intron 3 17 A A A C

A

A

A/C

Pick1 : Intron 2 18 G

G

G

Pick1 : Intron 3 19 T

A T A

A/T

Pick1 : Intron 4 20 T T T C C T C

T

C/T

Pick1 : Intron 3 21 A

C A C

A/C

Pick1 : Intron 4 22 C

C G G C G

C

C/G

Pick1 : Intron 1 23 C

C

C

Pick1 : Intron 4 24 T T T C C T C

T

C/T

Pick1 : Intron 3 25 G

C G C

C/G

Pick1 : Intron 3 26 T

G T G

G/T

Pick1 : Intron 3 27 G

G G A

A/G

Pick1 : Intron 4 28 G G G G G G A

G

A/G

Pick1 : Intron 3 29 G G G A A G

G

A/G

Pick1 : Intron 2 30 C C C T T C

C

C/T

Pick1 : Intron 3 31 T

T C

T

C/T

Pick1 : Intron 1 32 G

A

A

A/G

Pick1 : Intron 2 33 A A A G

A

A

A/G

Pick1 : Intron 2 34 T

T

T

Pick1 : Intron 2 35 T

C

C/T

Pick1 : Intron 2 36 G G G T T G

G

G/T

Pick1 : Intron 2 37 C

T

C/T

Pick1 : Intron 3 38 A A A G G A

A

A/G

Pick1 : Intron 2 39 C

A C

A/C

Pick1 : Intron 1 40 G

A G

A/G

Pick1 : Intron 2 41 G

A G

A/G

Pick1 : Intron 2 42

C T

C/T

Pick1 : Intron 2 43 G G G A A G

G

A/G

Pick1 : Intron 2 44 G G G A

G

G

A/G

Pick1 : Intron 2 45 T

C T

C/T

Pick1 : Intron 2 46 A

T A

A/T

Pick1 : Intron 2 47 G

A G

A/G

Pick1 : Intron 3 48 T

T C

C/T

Pick1 : Intron 4 49 A A A G G A G

A

A/G

Pick1 : Intron 4 50 T T T C C T C

T

C/T

Pick1 : Intron 3 51 C

A C A

A/C

Pick1 : Intron 3 52 C

T T C T

C/T

Pick1 : Intron 3 53 C

T T C T

C/T

Pick1 : Intron 3 54 T

C

T C

C/T

290

Pick1 : Intron 4 55 A

T T A T

A/T

Pick1 : Intron 3 56 A

G G A

A

A/G

Pick1 : Intron 3 57 T

C C T T

T

C/T

Pick1 : Intron 2 58 A

C C A C

A/C

Pick1 : Intron 4 59 C

C T T C T

C

C/T

Pick1 : Coding-Synonymous 1 60 G

G

G

G

A/G

Pick1 : Intron 3 61 T

C C T C

C/T

Pick1 : Intron 3 62

G

T G

G/T

Pick1 : Intron 1 63

T

C

C/T

Pick1 : Intron 2 64 C

T

C C

C/T

Pick1 : Intron 2 65 G

A

G G

A/G

Pick1 : Intron 1 66 C

C

C

Pick1 : Intron 1 67 A

T

A T

A/T

Pick1 : Intron 4 68 C

T

C T

C

C/T

Pick1 : Intron 2 69 G

T

G

G/T

Pick1 : Intron 2 70 C

G

C

C/G

Pick1 : Intron 2 71 C

T

C T

C/T

Pick1 : Intron 2 72 G

G

G A

A/G

Pick1 : Intron 2 73 C

T

C

C/T

Pick1 : Intron 3 74 C

C T

C/T

Pick1 : Intron 3 75 C

C T

C/T

Pick1 : Intron 3 76 G

G G G A

A/G

Pick1 : Intron 4 77 G

A A G A

A/G

Pick1 : Intron 3 78 G

G A

A/G

Pick1 : Intron 1 79 G

G

G

Pick1 : Intron 2 80 T

G G T

G/T

Pick1 : Intron 1 81 T

G G T

G/T

Pick1 : Intron 1 82 G

G G

G

G

A/G

Pick1 : Intron 2 83 G

A G

A/G

Pick1 : Intron 1 84 A

A G

A

A

A/G

Pick1 : Coding-NonSynonymous

3 85 G

G

G A

A/G

Pick1 : Intron 4 86 G

G

G A

A/G

291

Appendix IV

mRNA levels of the reference gene Hprt across treatment groups and strains following real-time

PCR. (A) Hprt remained insignificantly different in the naïve, sham, and denervated low groups

of A and B mice, and in denervated high autotomy A mice (P>0.05). (B) Hprt remained

insignificantly different in the naïve, sham, and denervated low groups of C3H/HeJ and AKR/J

mice, and in denervated high autotomy C3H/HeJ mice (P>0.05). (C) Hprt remained

insignificantly different in the naïve, sham, and denervated low- and high-autotomy groups of

C3H/HeN (Tlr4 +/+

) and C3H/HeJ (Tlr4 -/-

) mice (P>0.05). One-way ANOVA with post hoc

Tukey’s test

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

C3H/HeJ AKR/J

mRN

A le

vel o

f Hpr

t

Hprt gene expression in C3H and AKR mice

Naive

Sham

Low Autotomy

High Autotomy

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

A mice B mice

mR

NA

leve

l of H

prt

Hprt gene expression in A and B mice

Naive

Sham

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Naïve Sham LowAutotomy

HighAutotomy

mRN

A le

vel o

f Hpr

t

Hprt expression in wild-type vs. mutant Tlr4 mice

Tlr4 +/+

Tlr4 -/-

A

B

C

292

Appendix V

Number of CSF2RB1+ cell extensions around the central canal in naïve and sham-operated A

and B mice, and in denervated A high autotomy and low autotomy mice. P-values show

insignificant differences in the number of extensions between naïve and sham-operated A mice,

and between naïve and sham-operated B mice (paired T-test). Data for these 2 groups of mice,

per strain, were pooled together for further analysis. As well, p-values show insignificant

differences in the number of extensions between MHS-A and NLS-A mice (paired T-test). Data

for these 2 groups of mice were pooled together for further analysis.

Mice Number of CSF2RB1+ extensions in CC

Dorsal Ventral Lateral Total

Naïve A 4.11±1.45 4.43±1.26 13.20±4.76 20.58±6.54

Sham A 5.28±0.68 5.01±0.56 11.88±1.74 20.01±2.21

P-value 0.38 0.66 0.79 0.93

Naïve B 1.46±1.40 1.58±1.58 1.88±1.88 4.33±4.28

Sham B 0.04±0.04 0.13±0.13 0.96±0.96 1.08±1.08

P-value 0.35 0.39 0.68 0.49

MHS-A 6.60±0.26 7.58±0.55 18.19±1.02 30.23±1.51

NLS-A 6.21±0.17 6.33±0.35 20.29±2.90 30.54±2.87

P-value 0.34 0.16 0.41 0.19

293

Appendix VI

Number of CSF2RB1+ cell processes crossing the dentate gyrus per 200 μm unit length in naïve

and sham-operated A and B mice. P-values show insignificant differences in the number of

processes between naïve and sham-operated A mice, and between naïve and sham-operated B

mice (paired T-test). Data for these 2 groups of mice, per strain, were pooled together for further

analysis.

Mice Number of CSF2RB1+ processes crossing the DG per 200 μm

Naïve A 6.83±6.86

Sham A 6.50±4.04

P-value 0.68

Naïve B 5.11±2.73

Sham B 7.51±3.41

P-value 0.6

Appendix VII

Number of CSF2RB1+ cells in the polymorph dentate gyrus per ROI of 2500 μm2 of naïve and

sham-operated A and B mice. P-values show insignificant differences in the number of cells

between naïve and sham-operated A mice, and between naïve and sham-operated B mice (paired

T-test). Data for these 2 groups of mice, per strain, were pooled together for further analysis.

Mice Number of CSF2RB1+ cells in polymorph DG

Naïve A 3.37±0.45

Sham A 7.15±2.70

P-value 0.37

Naïve B 7.96±1.58

Sham B 8.24±1.39

P-value 0.9

294

Copyright Acknowledgements

RightsLink, permission to reproduce Scheme 1, License Number 3378941306268

RightsLink, permission to reproduce Scheme 2, License Number 3378941061859