Axon and muscle spindle hyperplasia in the myostatinnull mouseMohamed I. Elashry,1,2 Anthony Otto,1 Antonios Matsakas,1 Salah E. El-Morsy,2 Lisa Jones,1

Bethan Anderson1 and Ketan Patel1

1School of Biological Sciences, Hopkins Building, University of Reading, Reading, UK2Anatomy Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt

Abstract

Germline deletion of the myostatin gene results in hyperplasia and hypertrophy of the tension-generating

(extrafusal) fibres in skeletal muscle. As this gene is expressed predominantly in myogenic tissues it offers an

excellent model with which to investigate the quantitative relationship between muscle and axonal develop-

ment. Here we show that skeletal muscle hyperplasia in myostatin null mouse is accompanied by an increase in

nerve fibres in major nerves of both the fore- and hindlimbs. We show that axons within these nerves undergo

hypertrophy. Furthermore, we provide evidence that the age-related neural atrophic process is delayed in the

absence of myostatin. Finally, we show that skeletal muscle hyperplasia in the myostatin null mouse is accompa-

nied by an increase in the number of muscle spindles (also called stretch receptors or proprioceptors). However,

our work demonstrates that the mechanisms regulating intrafusal fibre hyperplasia and hypertrophy differ from

those that control the aetiology of extrafusal fibres.

Key words axon; muscle; myostatin; nerve fibre; proprioceptor; skeletal; spindle.

Introduction

The relationship between nerves and muscles has fascinated

biologists for almost 500 years, ever since the first detailed

description by Fallopius of the muscular innervation by the

cranial nerves (reviewed by Steinberg, 2002). Key work car-

ried out at the beginning of the last century by Shorey,

amongst others, demonstrated that nerves and muscles are

not only linked in terms of being part of a functional unit

(to initiate contraction) but also influence each other’s

development (Shorey, 1909). Studies performed by Ham-

burger (1934) developed these ideas further by demonstrat-

ing that the survival of motor neurons is regulated by

target tissues in the embryonic limb. A key breakthrough in

this field came from the study of Hollyday & Hamburger

(1976) who transplanted an additional limb field to the

flank of a developing chick and discovered that this opera-

tion resulted in an increase of the motor neuron popula-

tion. The most significant finding from this landmark study

was that the increase in cell number was not due to an

expansion of the progenitor pool but rather to a decrease

in the degree of cell death, which would normally eliminate

motor neurons that failed to form stable synaptic connec-

tions. The identity of the tissues that promoted motor neu-

ron survival was established through tissue ablation studies

in which the somites, the source of skeletal muscle, were

shown to have a major influence on motor neuron death

(Phelan & Hollyday, 1991).

Today it is generally believed that the vertebrate body

generates considerably more motor neurons than are

needed, and their survival is dependent on the limited pro-

duction of trophic factors from muscle and Schwann cells

(Sendtner et al., 2000; Strelau et al., 2009). The exact num-

ber of cells that are eliminated is thought to be in the

region of 50–60% compared to those that survive (Lance-

Jones, 1982). It is believed that trophic factors, expressed at

elevated levels at the neuromuscular junction, are delivered

to the cell body through the deployment of retrograde

transport mechanisms along the axon (Bartlett et al., 1998;

Pu et al., 1999; Reynolds et al., 2000). These suggestions

have been assimilated into the trophic factor access hypoth-

esis (reviewed by Banks & Chamberlain, 2005; Oppenheim

et al., 1999). Numerous trophic factors have been shown to

promote motor neuron survival, including insulin-like

growth factor (IGF)-1 and ciliary neurotrophic factor (CNTF),

often most efficacious when used in combination. Further-

more, there is evidence that certain motor neuron sub-types

may depend on specific combinations of trophic factors to

survive (Forger et al., 2003).

Correspondence

Ketan Patel, School of Biological Sciences, Hopkins Building, Whitek-

nights, Reading, RG6 6UB, Berkshire, UK. T: + 44 (0)118 378 8079;

E: ketan.patel@reading.ac.uk

Accepted for publication 19 November 2010

ªª 2011 The AuthorsJournal of Anatomy ªª 2011 Anatomical Society of Great Britain and Ireland

J. Anat. (2011) 218, pp173–184 doi: 10.1111/j.1469-7580.2010.01327.x

Journal of Anatomy

Many experiments have been performed in which the

number of muscle fibres was reduced and the outcome on

motor neuron number assessed. It was shown that the num-

ber of surviving motor neurons decreased when the number

of muscle fibres was reduced (Habgood et al., 1984; Tanaka

& Landmesser, 1986; Grieshammer et al., 1998). In contrast,

very few experiments have determined axon numbers in

nerves following manipulations that increased the number

of muscle fibres. Such studies would be valuable as they

would not only validate the trophic factor access hypothesis

but, importantly, would determine whether the extra sur-

viving cells can be maintained over extended periods.

We here exploit the phenotype of the myostatin null

mouse specifically to determine the effect of a genetic

manipulation that results in increased muscle fibre number

on nerve fibre development in two major limb nerves. The

radial and ischiatic nerves were chosen as representative

elements in the anterior and posterior, respectively, of the

mouse. The radial is the largest nerve in the brachial plexus.

It conducts action potentials that mediate the contraction

of extensor muscles of the upper arm and forearm, as well

as relaying cutaneous sensation from the dorsal autopod.

The ischiatic nerve is the broadest nerve in the body and is

a continuation of the sacral plexus. It innervates thigh and

shank muscles as well as relaying cutaneous sensation (Gray

et al., 1995). Myostatin (GDF-8) is a member of the trans-

forming growth factor-beta (TGF-b) super-family of proteins

that negatively regulate skeletal muscle mass (McPherron

et al., 1997). Genetic deletion of myostatin in mice results in

muscle enlargement, which is brought about by enlarge-

ment (hypertrophy) and increase in the number (hyperpla-

sia) of tension-generating fibres (McPherron et al., 1997;

Elashry et al., 2009). The mutant is particularly valuable for

this study as the gene is expressed predominantly in muscle,

not in connective tissue or in tissues of the central nervous

system.

Muscle fibres of skeletal muscle perform two major func-

tions. The vast majority generate tension through contrac-

tion. The fibres responsible for generating tension are

called extrafusal fibres. They are categorised in terms of

their myosin heavy chain (MHC) content and, in mice, are

classed as oxidative (type I and type IIa), intermediate (type

IIx) or glycolytic (type IIb). However, another population of

contractile protein-rich fibres are also present in skeletal

muscle. These muscle spindles (also called stretch receptors

or proprioceptors) are essential for the optimal working of

the tissues as they prevent overstretching by acting as mec-

hano-sensors (Maier, 1997). Muscle spindles are composed

of a connective tissue capsule that surrounds a varying

number of discrete muscle fibres called intrafusal fibres. In

contrast, extrafusal fibres contain only one muscle fibre,

surrounded by a connective tissue layer. Two forms of mus-

cle spindles have been identified based on the organisation

of the nuclei; nuclear bag fibres, where the nuclei are

grouped at the equatorial region, and nuclear chain fibres,

where the nuclei are centrally located and arranged in a

chain-like fashion. Typically, a type Ia sensory fibre projects

from the non-contracting portion of the muscle spindle to

the CNS, relaying information about the degree of contrac-

tion in extrafusal fibres.

Here we report that the deletion of myostatin, which

causes muscle fibre hyperplasia, results in an increase in the

number of axons present in both the radial and ischiatic

nerves. Previous work has shown that the diameter of a

motor neuron is a reflection of the size of the motor unit.

Fast (glycolytic) muscle fibres are part of large motor units

in which the axon tends to have a large diameter. In con-

trast, slow (oxidative) fibres are part of small motor units

and are innervated by small-diameter neurons (Carmeli &

Reznick, 1994). We report that the large glycolytic muscle

fibre type profile of myostatin null animals is reflected in an

increase in the average axon diameter in both the radial

and ischiatic nerves. Lastly, we show that the increase in

extrafusal fibre number is accompanied by an increase in

the number of muscle spindles.

Material and methods

Animals

C57 ⁄ BL6 and transgenic mstn) ⁄ ) male mice (minimum of n = 3)

were bred in the Biological Resource Unit at the University of

Reading. Mice were housed with food and water ad libitum

until they reached the required age.

Muscle tissue collection

All the mice were killed through schedule one procedures. Two

muscles, the flexor digitorum superficialis (FDS) and the flexor

digitorum brevis (FDB), from the forelimbs and hindlimbs,

respectively, were carefully dissected, snap frozen with liquid

nitrogen pre-cooled with isopentane and kept at )80 �C until

processing. These muscles were chosen as they have a high sen-

sory function and are rich in muscle spindles.

Nerve tissue collection

The radial nerve and ischiatic nerve from the forelimb and hind-

limbs, respectively, were dissected before their branching points.

From the anatomical point of view, the radial nerve is the largest

of the brachial plexus nerves. It passes behind the axillary artery

to the caudal surface of the humerus to traverse from the medial

to the lateral surface of the upper forelimb. The ischiatic nerve

runs under the biceps femoris and semitendinosus muscles.

Before the knee joint, the nerve divides into two main branches,

the tibial and the peroneal nerves (Gray’s Anatomy, 1995). Both

the radial and ischiatic nerves were identified, isolated and fixed

in situ by the addition of a few drops of 2.5% glutaraldehyde in

0.1 M phosphate buffer (pH. 7.3) prior to resection. Sections 2 mm

in length were fixed in 2.5% glutaraldehyde in 0.1 M phosphate

buffer (pH. 7.3) for 3 days, ethanol-dehydrated, washed in resin

(LR medium resin kit; TAAB, UK) and then equilibrated in resin

overnight at 4 �C. The samples were placed in moulds before the

ªª 2011 The AuthorsJournal of Anatomy ªª 2011 Anatomical Society of Great Britain and Ireland

Myostatin null nerve fibre phenotype, M. I. Elashry et al.174

addition of hardener (acrylic resin accelerator; TAAB). The resin

blocks were sectioned at 1 lm thickness using an ultra-microtome

with a disposable glass knife.

Histological analysis

Resin sections were left to dry on a hot surface (60 �C) over-

night. Subsequently, sections were covered with 1% toluidine

blue with 1% sodium borate for 1 min, washed in distilled

water, dried and mounted using DPX, as previously performed

(Illanes et al., 1990).

Immunohistochemistry

Frozen muscle samples were rapidly embedded in tissue tech

freezing medium (Jung) using dry ice-cooled ethanol. Whole

muscles were cryosectioned and placed on polylysine-coated

slides from the tendon of origin to the tendon of insertion to

get a single 10-lm section for every 50 lm of muscle length.

The sections were air-dried for at least 2 h before staining. The

sections were blocked in wash buffer [5% foetal calf serum

(v ⁄ v) in phosphate-buffered saline containing 0.05% Triton

X-100]. Basal lamina and myosin heavy chain isoforms (MHC)

type I-, IIa- and IIb-expressing fibres were identified using rabbit

anti-laminin (1 : 200) polyclonal antibody (Sigma) and

A4.840 IgM (1 : 1), A4.74 IgG (1 : 4) and BFF3 IgM (1 : 1) super-

natant monoclonal primary antibodies, respectively (Develop-

mental Studies Hybridoma Bank). Primary antibodies were

visualised using Alexa Fluor 488 goat anti-mouse IgG (Molecular

Probes A11029, 1 : 200) and Alexa Fluor 633 goat anti-mouse

IgM (Molecular Probes A21046, 1 : 200) secondary antibodies.

All antibodies were diluted in wash buffer 30 min prior to use.

Transmission electron microscopy

The nerve sections were stained with lead acetate. Transmission

electron microscopy was carried out using a Philips CM 20 with

an accelerating voltage of 80 kV.

Digital image analysis

Images of toluidine blue-stained sections were captured using a

light microscope under an oil immersion lens (· 100) with an

Axiocam digital camera. Quantification of nerve fibres was per-

formed manually as previously described (Illanes et al., 1990).

Measurement of the nerve fibre cross-sectional area (CSA) was

carried out via manual drawing around each fibre using Zeiss

AXIOVISION software version 4.7. First, the total CSA of the nerve

fibre was determined by measuring the area inside the outer limit

of the myelin sheath. Secondly, the axonal CSA was measured.

Finally, the myelin sheath CSA was calculated by subtracting the

nerve fibre value from the axon area (Fig. 2K). Images of stained

FDS and FDB muscle transverse sections were captured using a

Zeiss Axioscop2 fluorescent microscope. Images of whole cross-

sections were reconstructed using ADOBE PHOTOSHOP CS. Quantifica-

tion of proprioceptors and the number of intrafusal fibres was

performed by following the same muscle spindle on serial sec-

tions through the entire length of the muscle. Intrafusal fibre

cross-sectional area measurements were performed manually

using Zeiss AXIOVISION software version 4.7. All FDS and FDB fibres

negative for MHC IIa and IIb on double-stained sections were

considered to be of a type IIX phenotype by subtraction of the

number of type I-stained fibres from serial muscle sections.

Statistical analysis

Quantification of the total axon number and cross-sectional

area of both genotype (wild type vs. myostatin null) and age

(6 vs. 12 months old) was analysed using two-way analysis of

variance (ANOVA). Assessment of the number of proprioceptors,

the number of intrafusal fibres and the CSA of intrafusal fibres

was carried out using a two-tailed Student t-test, where

P £ 0.05 was considered to be significant.

Results

Myostatin deletion increases the number of the

axons in fore- and hindlimb nerves

It is well established that myostatin (GDF-8) inhibition

increases the muscle mass through hyperplasia and ⁄ or

hypertrophy (McPherron et al., 1997; Elashry et al., 2009).

Development of muscle has been shown to influence motor

neurons but this paradigm has not been examined in

mstn) ⁄ ) animals. To this end we chose two major nerves

that innervate the muscles of the fore- (radial) and hindlimb

(ischiatic). Our analysis showed that myostatin deletion

resulted in 21.4 ± 2.8% increases in the number of axons in

the radial nerve and 14.4 ± 0.8% increases in the ischiatic

nerve at 6 months of age. A similar increase was observed

at 12 months of age (Fig. 1A–C).

Myostatin deletion delays the age-related reduction

of axon number

Previous analysis of axon number in the median nerve of

humans demonstrated a reduction with age (Brown, 1972).

Here we examined the effect of deleting myostatin on the

age-related influence on axon number. Our results revealed

that the radial nerve of 12-month-old wild-type mice

showed a significant 18.5 ± 3.1% reduction in axon number

compared to 6-month-old animals. In contrast, the mstn) ⁄ )

mice showed a non-significant decrease of 3.4 ± 2.3% dur-

ing this period (Fig. 1B). Similarly, the ischiatic nerve of

mstn+ ⁄ + 12-month-old mice showed a significant reduction

in axon number of 8.2 ± 2.7% compared to a non-signifi-

cant reduction of 3.7 ± 0.8% in mstn) ⁄ ) mice (Fig. 1C).

Interestingly, analysis of 24-month-old mstn) ⁄ ) showed a

highly significant reduction in the total number of axons

(22.2 ± 2.9% and 11.1 ± 1.4% for radial and ischiatic nerves,

respectively) compared to 6-month-old mstn) ⁄ ) mice

(Fig. 1D). Our results demonstrate that myostatin deletion

does not cause a significant reduction in the number of the

axons at 12 months of age compared to the wild type.

However, this was not permanent, as nerve fibre depletion

became evident by 24 months.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al. 175

Hypertrophy followed by age-related hypotrophy in

myostatin null axons

Previous studies have demonstrated that myostatin deletion

induces a shift from small to large fast myofibres (Girgen-

rath et al., 2005; Elashry et al., 2009). Therefore, we exam-

ined whether the muscle fibre size shift induced by

myostatin loss is accompanied by changes in the CSA of the

nerve fibres. Our results for the radial and ischiatic nerves

from 6-month-old mstn) ⁄ ) animals showed increases in the

CSA of the individual nerve fibres compared to tissue from

age-matched mstn+ ⁄ + controls (Fig. 2A,D, also Fig. 2G and I;

quantification in Table 1).

We then addressed the question of whether the CSA

increase resulted from expansion of the axon or its sur-

rounding myelin sheath. Our analysis of the 6-month-old

radial and ischiatic nerves of mstn) ⁄ ) animals revealed sig-

nificant increases in the CSA of both the axon and myelin

sheath (Fig. 2B,C,E,F, compare G and I). At 6 months of age,

the axonal expansion was greater in the ischiatic nerve than

in the radial nerve (Fig. 2B,E).

Radial and ischiatic nerves from both mstn) ⁄ ) and mstn+ ⁄ +

genotypes exhibited age-related hypotrophy in the CSA of

individual fibres by 12 months (Fig. 2A,D). The degree of

hypotrophy depended on the identity of the nerve. The

radial nerve from the mstn) ⁄ ) mice showed less hypertrophy

compared to age-matched mstn+ ⁄ + mice (Fig. 2A). The

opposite was found for the ischiatic nerve (Fig. 2D). Detailed

analysis of the axon and myelin sheath characteristics

revealed some interesting findings. The radial nerve of

mstn+ ⁄ + mice showed greater age-related hypotrophy in

both the axon and myelin sheath compared to mstn) ⁄ ) mice

(Fig 2B,C). In contrast, the ischiatic nerve of the mstn) ⁄ ) ani-

mals displayed a greater reduction in the diameter of both

axon and myelin sheath compared to mstn+ ⁄ +animals

(Fig. 2E,F, also compare G with H and I with J).

The CSA of motor neurons is directly related to the size of

the motor unit. During aging, loss of (large-diameter) fast

motor neurons leads to muscle fibres being re-innervated by

(small-diameter) slow motor neurons. We therefore assessed

whether the size of nerve fibres changed over time. We

A B

C D

Fig. 1 Myostatin deletion increases the

number of nerve fibres. (A) Representative

images showing transverse sections through

the ischiatic nerve of 6-month-old mstn+ ⁄ +

and mstn) ⁄ ) mice. Myostatin deletion-

induced hyperplasia results in an increased

cross-sectional area. Scale bar: 20 lm. (B,C)

Quantification of the average nerve fibre

number in the radial (B) and ischiatic (C)

nerves of 6- and 12-month-old mstn+ ⁄ + (red)

and mstn) ⁄ ) (green) mice (n = 3 mice per

group). Higher values were observed in

mstn) ⁄ ) nerves at both ages compared to

age-matched mstn+ ⁄ + counterparts. Two-way

ANOVA shows a significant genotype-

dependent increase in nerve fibre number

(P < 0.001). mstn+ ⁄ + animals 12 months old

showed decreases in the number of nerve

fibres for both radial (B) and ischiatic (C)

nerves when compared to 6-month-old

animals of equivalent genotype (n = 3 mice

per group), but not mstn) ⁄ ) animals. Two-

way ANOVA shows significant age-dependent

decreases in nerve fibre number (P = 0.001

for both nerves). (D) Average nerve fibre

number in the radial and ischiatic nerves of

6-, 12- and 24-month-old mstn) ⁄ ) mice.

One-way ANOVA was performed to measure

the effect of age on nerve fibre number of

the mstn) ⁄ ) mice (6- vs. 12- vs. 24-month-

old mice). Significant reductions in the

number of axons from 6 to 24 m were

observed (n = 3 animals at each time point;

*P < 0.001). Error bars are mean ± SEM.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al.176

grouped nerve fibres into three categories – small (0–

20 lm2), medium (20–60 lm2) and large (more than 60 lm2)

– to evaluate the percentage of each population present at a

given time. (All data are shown as percentage ± SEM in

Table 2). Our analysis for the radial nerve showed that the

mstn) ⁄ ) mice contained fewer small-sized nerve fibres but

more large-sized nerve fibres compared to the mstn+ ⁄ + ani-

mals (Fig. 3A). A similar situation was found at 12 months,

with the major difference being that the proportion of small

nerves had increased with age for both genotypes. Following

this trend, similar significant changes in the percentage of

large and small nerve fibres with both genotype and age

were revealed following examination of the ischiatic nerve

(Fig. 3B). These results suggest that myostatin deletion

induced a nerve fibre shift towards large-sized units as com-

pared to age-matched wild types. On the other hand, both

genotypes gained more small-sized units at the expense of

large-sized units during aging. To confirm and extend these

results, we examined the radial nerve from 24-month-old

mstn) ⁄ ) mice and compared it with younger mutant animals.

One-way ANOVA revealed that the radial nerve from 24-

month-old mstn) ⁄ ) mice showed a significant increase in the

percentage of small-sized axons compared to 6- and 12-

month-old mice and a significant decrease in the percentage

of large-sized axons, with no significant difference in the per-

centage of medium-sized axons (Fig. 3C, compare D–F, and

Tables 1 and 2). Our results suggest that myostatin deletion

reduces the rate of age-related nerve fibre hypotrophy but

does not in any way halt this process. The mstn) ⁄ ) nerve does

undergo significant hypotrophy by 24 months (Fig. 3F).

A B C

D E

G H I J K

F

Fig. 2 Myostatin deletion increases nerve fibre CSA. Average nerve fibre CSA (axons and myelin sheath) of the radial (A) and ischiatic (D) nerves

of 6- and 12-month-old mstn+ ⁄ + and mstn) ⁄ ) mice. (A) mstn) ⁄ ) mice show a significant increase in nerve fibre CSA compared to mstn+ ⁄ +

counterparts (P < 0.001). Significant age-related changes in CSA were observed in both genotypes analysed (P < 0.001). Average axon CSA of the

radial (B) and ischiatic (E) nerves of 6- and 12-month-old mstn+ ⁄ + and mstn) ⁄ ) mice. Two-way ANOVA for both nerves showed significant changes

by genotype, age and interaction of the two parameters. Average myelin sheath CSA of the radial (C) and ischiatic (F) nerves of 6- and 12-month-

old mstn+ ⁄ + and mstn) ⁄ ) mice. Two-way ANOVA for both nerves showed significant changes by genotype, age and interaction of the two

parameters. (G–J) TEM images showing sections of radial nerve from 6- and 12-month-old mstn+ ⁄ + and mstn) ⁄ ) mice. Images from 6-month-old

mstn) ⁄ ) mice show an increase in the CSA of axons compared to age-matched mstn+ ⁄ + animals. Fibres from 12-month-old mstn) ⁄ ) radial nerves

show a reduction in the CSA compared to those from 6-month-old animals. AX, axon; MS, myelin sheath. (K) Diagrammatic illustration showing

the technique used for nerve fibre (NF), axon (AX) and myelin sheath (MS) cross-sectional area measurements. Scale bar: 5 lm.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al. 177

Unfortunately, comparisons with wild types were not possi-

ble due to the premature death of age-matched litter mates.

Hyperplasia of muscle spindles in the myostatin

null mice

It is well established that myostatin deletion increases skele-

tal muscle mass through varying degrees of hyperplasia and

hypertrophy (McPherron et al., 1997; Elashry et al., 2009).

However, it is not clear whether the hyperplasia of extrafus-

al fibres is accompanied by an increase in the number of

muscle spindles. Previous studies have shown that postural

muscles contain a high proportion of muscle spindles.

Therefore, to analyse the effect of myostatin loss on muscle

spindle number, we carried out a study on the flexor digito-

rum superficialis and the flexor digitorum brevis as repre-

sentatives of postural muscles of the fore- and hindlimbs,

respectively. We counted every muscle spindle in these two

muscles using histological (Fig. 4A) and immunohistochemi-

cal techniques (Fig. 4A’–A’’), based either on the presence

of nuclear bag ⁄ chain structures (Fig 4A’) or the expression

of MHC within intrafusal fibres (contractile apparatus) at

both polar ends of each muscle spindle (Fig. 4A’’). Our

results show that most of the spindles were distributed in

the mid-belly region of the muscle, with only a few located

at the periphery. Mstn) ⁄ ) muscles contained significantly

increased numbers of the muscle spindles in both muscles

(increases of 26 and 41% for the FDS and FDB muscles,

respectively, P < 0.05) (Fig. 4B). Analysis of the number of

extrafusal fibres revealed that mstn) ⁄ ) mice had 16 ± 1%

and 69 ± 3% more muscle fibres in their FDS and FDB mus-

cles, respectively.

Next, we investigated whether myostatin deletion

increases the number of intrafusal fibres within each spin-

dle. The average number of intrafusal fibres varied from

4.1 ± 0 and 4.26 ± 0 for the FDB and 3.8 ± 0 and 4 ± 0 for

the FDS in the mstn+ ⁄ + and mstn) ⁄ ) mice, respectively. Sur-

prisingly, we failed to find any significant differences in the

Table 1 Average cross-sectional area (CSA) of whole nerve fibre, axon and myelin sheath from 6- and 12-month-old mstn+ ⁄ + and 6-, 12- and 24-

month-old mstn) ⁄ ) mice. All data are shown as mean ± SEM.

Motor unit CSA (lm)2

Radial Ischiatic

Mstn+ ⁄ + Mstn) ⁄ ) Mstn+ ⁄ + Mstn) ⁄ )

6 months 12 months 6 months 12 months 24 months 6 months 12 months 6 months 12 months

Nerve fibre 40.4 ± 1.4 27.1 ± 0.7 48.9 ± 1.2 40.6 ± 1.1* 22.3 ± 0.5§# 29.8 ± 0.7 26.8 ± 0.6 48.2 ± 0.9 31 ± 0.6*

Axon 18.8 ± 0.8 10.9 ± 0.3 20.1 ± 0.6 17.1 ± 0.5* 9.5 ± 0.3§# 10.2 ± 0.3 9.7 ± 0.3 18.5 ± 0.5 11.8 ± 0.3*

Myelin sheath 21.7 ± 0.7 16.2 ± 0.3 28.9 ± 0.7 23.5 ± 0.6* 12.8 ± 0.3§# 19.6 ± 0.4 17.1 ± 0.4 29.7 ± 0.5 19.2 ± 0.3*

Radial nerve – nerve fibre: significant difference (P < 0.001) from *6-month-old mstn) ⁄ ), §6-month-old mstn) ⁄ ) and # 12-month-old

mstn) ⁄ ) mice; axon: significant difference (P < 0.001) from *6-month-old mstn) ⁄ ), §6-month-old mstn) ⁄ ) and #12-month-old mstn) ⁄ )

mice; myelin sheath: significant difference (P < 0.001) from *6-month-old mstn) ⁄ ), §6-month-old mstn) ⁄ ) and # 12-month-old mstn) ⁄ )

mice.

Ischiatic nerve – nerve fibre: *significant difference (P < 0.001) from 6-month-old mstn) ⁄ )mice; axon: *significant difference

(P < 0.001) from 6-month-old mstn) ⁄ ); myelin sheath: *significant difference (P < 0.001) from 6-month-old mstn) ⁄ ).

Table 2 Average relative percentage of small, medium and large nerve fibres (lm2) from 6- and 12-month-old mstn+ ⁄ + and 6-, 12- and 24-

month-old mstn) ⁄ ) mice. All data are shown as mean ± SEM.

Motor unit percentage (%)

Radial Ischiatic

mstn+ ⁄ + mstn) ⁄ ) mstn+ ⁄ + mstn) ⁄ )

6 m 12 m 6 m 12 m 24 m 6 m 12 m 6 m 12 m

Small (0-20 lm2) 38.2 ± 8.9 46.5 ± 3.1 25.2 ± 2.1 37.1 ± 5.5 59.6 ± 5.9*§ 38.1 ± 2.3 49.8 ± 3.5 6.2 ± 1.1 34.9 ± 3.0*

Medium (20–60 lm2) 40.5 ± 2.6 44.4 ± 2.6 41.6 ± 1.1 36.3 ± 1.1 35.5 ± 4.5 53.0 ± 2.7 40.3 ± 1.1 67.8 ± 4.7 58.8 ± 2.4

Large (> 60 lm2) 21.3 ± 9 9.2 ± 3.5 33.2 ± 2.7 26.6 ± 6.3 4.9 ± 1.8*§ 8.8 ± 2.3 9.9 ± 3.7 26.0 ± 5.3 8.8 ± 1.6*

Radial nerve – small: *significant difference from 6-month-old mstn) ⁄ ) mice (P = 0.007); §significant difference (P = 0.04) from 12-

month-old mstn) ⁄ ) mice; medium: no significant changes; large: *significant difference from 6-month-old mstn) ⁄ ) mice (P = 0.008),§12-month-old mstn) ⁄ ) mice (P = 0.02).

Ischiatic nerve – small: *significant difference from 6-month-old mstn) ⁄ ) mice (P = 0.001); medium: no significant changes; large:

*significant difference from 6-month-old mstn) ⁄ ) mice (P = 0.02).

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Myostatin null nerve fibre phenotype, M. I. Elashry et al.178

number of intrafusal fibres of both muscles in the absence

of myostatin when compared to the age-matched mstn+ ⁄ +

control (Fig. 4C). These studies show that myostatin dele-

tion results in an increase in the number of muscle spindles

but not in an increase in the number of intrafusal myofibres

present within each of the muscle spindles.

Myostatin deletion has no effect on the fibre-type

properties of intrafusal fibres

We determined the molecular properties of the intrafusal

fibres to establish whether the deletion of myostatin

induces a fibre-type conversion similar to that observed in

the extrafusal fibres (Elashry et al., 2009). Interestingly, mus-

cle spindles of mstn) ⁄ ) animals showed no significant differ-

ence in the fibre-type composition in either the FDS or the

FDB muscles, compared to age-matched mstn+ ⁄ + controls

(Fig. 4D, see also Fig. 5A–D).

Intrafusal fibre hypotrophy in myostatin null mice

Our results demonstrated that myostatin inhibition had no

effect on the total number and MHC profile of the intra-

fusal fibres. Lastly, we examined whether myostatin dele-

AD

E

F

B

C

Fig. 3 Myostatin deletion causes a shift from small to large nerve fibres. Average radial and ischiatic nerve fibres were categorized into small

(0–20 lm2), medium (20–60 lm2) and large (>60 lm2). (A) Mstn) ⁄ ) (light and dark green) mice showed a tendency towards a reduction in the

percentage of small nerve fibres and a significant increase in the percentage of large nerve fibres in the radial nerve compared to mstn+ ⁄ + (pink

and red) (P = 0.07 and P < 0.03, respectively). A significant interaction between genotype and age was apparent for changes in medium-sized

nerve fibres (P = 0.05). (B) mstn) ⁄ ) ischiatic nerves (light and dark green) showed significant increases in the percentage of medium and large

nerve fibres, respectively, compared with mstn+ ⁄ + nerves (P < 0.001 and P = 0.05, respectively), with a tendency towards a reduction in small

nerve fibres (P = 0.06). In 12-month-old animals, significant genotype-independent changes were seen in the percentage of small nerve fibres

during aging (P < 0.02). A significant interaction between genotype and age was apparent for changes in small and large nerve fibres (P < 0.001

and P < 0.05, respectively). (C) Average percentage of small, medium and large radial nerve axons from 6-, 12- and 24-month-old mstn) ⁄ ) mice.

The 24-month-old radial nerves (black) harbour significant increases in small nerve fibres and decreases in large nerve fibres. Error bars displayed

as mean ± SEM. (D–F) Representative images of the radial nerve from mstn) ⁄ ) animals at 6- (D), 12- (E) and 24-months of age (F). Scale

bar: 20 lm.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al. 179

tion had any effect on the regulation of the CSA of the

intrafusal fibres. Surprisingly, the mstn) ⁄ ) FDS muscle intra-

fusal fibres showed a significant reduction in the CSA of

type IIb and type I intrafusal fibres (Fig. 5E) compared to

age-matched mstn+ ⁄ + mice. Similarly, there was a signifi-

cant reduction in the CSA of type IIb myofibres in the FDB

compared to age-matched mstn+ ⁄ + mice (Fig. 5F) but not in

type I fibres. These data suggest that the mechanisms regu-

lating intrafusal fibre-type development and hypertrophy

differ from those that control these processes in extrafusal

fibres.

Discussion

In this study we report the effect of a genetic manipulation

that causes an increase in skeletal muscle fibre number on

axonal numbers and sizes in two major limb nerves. We

show that following the deletion of myostatin, a potent

inhibitor of skeletal muscle development, the number and

size of axons in both the radial and ischiatic nerves of the

mouse increase compared to wild-type litter mates. Further-

more, we show that the age-related decrease in the num-

ber of axons is attenuated in the mstn) ⁄ ) compared to

A A’

B

C

D

A”

Fig. 4 Myostatin deletion causes an increase

in the number of muscle spindles but no

changes in number of the intrafusal fibres or

MHC-expressing fibres. (A) Haematoxylin and

eosin staining. (A’–A’’) Double

immunofluorescent labelling (right) against

MHC IIa (green) and MHC IIb (red) stained

with 4’,6-diamidino-2-phenylindole (DAPI,

blue) showing the equatorial nuclear region

(A’, white arrowhead) and the myosin heavy

chain region at the polar end showing type

IIa (A’’, green arrowhead) and type IIb (A’’,

red arrowhead). Scale bar: 50 lm. (B)

Average number of muscle spindles in the

FDS and FDB muscles. Significant increases in

muscle spindle number were observed in

muscles from both mstn) ⁄ ) (green bars) and

age-matched mstn+ ⁄ + mice (red bars)

(*P < 0.05). (C) Average total number of

intrafusal fibres in muscle spindles from the

FDS and FDB of mstn+ ⁄ + (red bars) and

mstn) ⁄ ) (green bars) mice. No significant

differences were present. (D) The average

number of MHC type I (blue), IIa (green) and

type IIb (red) intrafusal fibres revealed no

significant change in distribution in the

mstn) ⁄ ) compared to mstn+ ⁄ + mice.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al.180

A

B

C

D

E

F

A’

B’

C’

D’

A”

B”

C”

D”

Fig. 5 Myostatin deletion caused a decrease in the CSA of the intrafusal fibres but no changes in the histochemical properties of the spindle.

(A-D) Double immunofluorescent staining for MHC IIa and IIb (A–A’’ and C–C’’) or IIa and I (B–B’’ and D–D’’) on transverse section of FDB muscle

from mstn+ ⁄ + (A–B’’) and mstn) ⁄ ) (C–D’’) highlighting intrafusal fibres (arrows). Scale bar: 50 lm. Quantification of the average CSA of MHC

type I, IIa and IIb positive intrafusal fibres from the FDS (E) and FDB (F). (E) The average CSA of type I and type IIb intrafusal fibres was significantly

smaller in the FDS muscle in the mstn) ⁄ ) than in age-matched mstn+ ⁄ + mice (*P = 0.001). (F) The FDB muscle only displays a significant reduction

in the average CSA of type IIb intrafusal fibres compared to mstn+ ⁄ + counterpart (*P = 0.005). All data are shown as mean ± SEM.

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Myostatin null nerve fibre phenotype, M. I. Elashry et al. 181

mstn+ ⁄ + animals. Finally, we show that the increase in extra-

fusal fibres that results in the absence of myostatin is

matched by an increase in the number of muscle spindles.

Investigations going back over 100 years have docu-

mented the link between motor neuron number and the

number of muscle fibres. The general consensus is that the

developing vertebrate embryo generates many more motor

neuron cells than are required, a finding that has been

interpreted as a fail-safe mechanism that insures that all

muscle fibres are innervated.

Three mechanisms are used during prenatal and post-

natal life to control the number of motor neurons. During

early embryogenesis, the motor neuron population is

restricted to the ventral portion of the neural tube follow-

ing its induction by signals originating from the floor plate.

Recent work suggests that any motor neurons that have

been incorrectly induced along the dorsal ventral axis of

the neural tube are subsequently eliminated (Oppenheim

et al., 1999). The second phase of motor neuron number

control occurs later in life but still at the prenatal stages

when motor neurons compete for trophic factors on

recently differentiated myofibres. Motor neurons unable to

secure a source of trophic factors are thereafter eliminated.

Finally, motor neuron number is also regulated after birth.

It is worth noting that in invertebrates, neural activity is

required at an earlier stage of muscle development and is

responsible for promoting myoblast proliferation and sub-

sequent patterning (Fernandes & Keshishian, 2005).

Classical experiments carried out by Hollyday & Ham-

burger (1976) and Pittman & Oppenheim (1979) demon-

strated that increasing the number of potential motor

neuron targets or applying neuromuscular blockading

chemicals greatly increases the number of surviving motor

neurons by preventing normal cell death, a process termed

hypothanasia (Hollyday & Hamburger, 1976). These and

other studies led to the development of the trophic factor

access hypothesis that large numbers of motor neurons are

initially generated, and that these could at early stages of

development access trophic factors produced over extended

regions of the muscle fibres (reviewed by Banks & Chamber-

lain, 2005 and Landmesser, 1992). Trophic factors, possibly

including IGF-1, glial derived neurotrophic factor and CNTF,

are retrogradely transported from the muscle to the motor

neuron cell body (Bartlett et al., 1998; Reynolds et al.,

2000). However, muscle contraction activity thereafter leads

to the remodelling (constriction) of the neuromuscular junc-

tion(s) and concentrates the production of trophic factors in

this region.

Classical studies examined the increase in axon number

only up to later stages of prenatal development and there-

fore did not address whether sufficient trophic factors would

be present to maintain them into adult life. These stud-

ies also did not address whether mechanisms that control

the number of motor neurons in adult life would correct the

extra cells that survived the prenatal cull. Here we show that

deletion of myostatin results in a sustained increase in axon

numbers that extends into adult life. We suggest that the

additional fibres that are generated in the absence of myost-

atin are able to produce sufficient trophic factors to sustain

neurons not only at early stages but also into adulthood.

According to the trophic factor access hypothesis, muscle

fibres are initially poly-innervated and then the remodelling

of the neuro-muscular junction results in all but one axon

terminal maintaining contact with the muscle fibre. We

suggest that the large increase in muscle fibres during both

primary and secondary myogenesis (Matsakas et al., in press)

may lead to fewer poly-innervated myofibres, as there are

more target cells available to the developing motor neurons.

Work is ongoing to investigate this line of thinking.

An important finding from our analysis of the two nerves

examined was that the deletion of myostatin delays the

decrease in axon number and size seen in the wild-type mice

at 12 months of age. Aging has long been known to be

associated with the progressive loss of motor neurons. Fast

motor neurons are preferentially lost, resulting in the loss of

type II muscle fibres. These subsequently are re-innervated

by surviving slow motor neurons, which ultimately leads to a

change in the muscle profile towards an oxidative pheno-

type (Lexell et al., 1988; Lexell, 1997; Degens, 2007). Our

finding that the loss of axons is delayed in the myostatin null

could be explained by the fact that the muscle fibres in the

mutant are considerably larger than in the wild type and

therefore may produce more trophic factors. Indeed, addi-

tional productions of trophic factors have been demon-

strated to promote motor neuron survival (Dobrowolny

et al., 2005). These could counter the detrimental effects of

aging taking place in the fast motor neuron, thereby delaying

axon degeneration. However, it should be noted that the

number and size of axons do eventually decrease, and the

extra production of trophic factors are unable to prevent the

detrimental effects of aging in the long term. Interestingly,

even though the large-diameter axons were lost in the

mstn) ⁄ )animals, themuscle seemstoretainfastfibres (Elashry

et al., 2009). These results would suggest that the genetic

deletion of myostatin prevents the fibres from responding

tothechangeininnervationthattakesplaceduringaging.

It is of interest that although both the radial and ischiatic

nerves of the mstn) ⁄ ) mice were resistant to age-related

axonal loss, the ischiatic axons from the null mice displayed

a greater degree of hypotrophy compared with the radial

axons. At present we do not understand the mechanism

responsible for this difference. However, we propose a pos-

sible explanation based on the muscles that each nerve

innervates. The radial nerve mainly innervates the triceps

and extensor muscles in the forearm. In contrast, the ischiat-

ic nerve innervates large muscles in the back of the leg and

shank. We suggest that these large muscles contain a

greater proportion of fibres that have undergone extensive

hypertrophy following deletion of myostatin and that,

during ageing, they undergo a greater reduction in size

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Myostatin null nerve fibre phenotype, M. I. Elashry et al.182

compared with those in the forelimb. A possible conse-

quence of this hypotrophy is the decreased production of

factors maintaining axon anabolic processes.

Our study also addresses the development of the muscle

spindles in a model that induces fibre hyperplasia and hyper-

trophy. We found that there was an increase in the number

of muscle spindles that develop in both the FDS and FDB

muscles examined. Our detailed analysis of muscle spindles

of the myostatin null mice led to three unexpected findings.

First, we show that the intrafusal fibres within the spindles

from mstn) ⁄ ) mice had not undergone hyperplasia, even

though the extrafusal fibres had increased in number.

Secondly, we found that the intrafusal fibres from mstn) ⁄ )

mice had undergone hypotrophy compared to the intrafusal

fibres of mstn+ ⁄ + mice. Thirdly, we show that the intrafusal

fibres from mstn) ⁄ ) animals did not show the fibre-type

switch towards type IIb that is seen in extrafusal fibres.

Much progress has been made regarding our understand-

ing of fibre-type development. Key molecules regulating

this process (MEF2C and MEF2D) were recently identified by

Olson’s group (Potthoff et al., 2007). Overexpression of

either molecule leads to the expression of slow MHC and

the deletion of these genes leads to loss of slow fibres and

development of fast fibres (Potthoff et al., 2007). Interest-

ingly, myostatin has been shown positively to regulate the

expression of these genes (Hennebry et al., 2009). Therefore

the loss of myostatin would lead to the lack of MEF2 expres-

sion, allowing a fast fibre default of the muscle fibres. How-

ever, this does not seem to be the case for muscle spindle

fibres and we suggest either that other mechanisms are

used in these cells or that the cells are able to over-ride the

loss of myostatin. Investigations are currently underway to

identify the mechanisms regulating fibre development in

muscle spindles.

In summary, our work shows that increasing the number

of muscle fibres leads to an increase in axon number. Fur-

thermore, we demonstrate that the axons also increase in

diameter and this possibly reflects the fast fibre composi-

tion of the muscle. Interestingly, we show that, in the

absence of myostatin, there is a delay in the loss of axons.

We suggest that this could be due to the enlarged muscle

producing elevated levels of neurotrophic factors. This

could be of clinical importance, as a number of diseases

involve motor neuron loss, e.g. amyotrophic lateral sclerosis

(ALS), a fatal disease in humans. Motor neuron loss in ani-

mal models of ALS can be delayed through the overexpres-

sion of IGF-1. Recent studies have shown that inhibition of

myostatin results in beneficial outcomes in rodent models

of ALS, promoting studies of this molecule for therapeutic

uses (Holzbaur et al., 2006; Morrison et al., 2009).

Acknowledgements

We would like to thank the Egyptian Ministry of Higher Educa-

tion, the Wellcome Trust (078649), and University of Reading

for generous funding, which allowed this work to be carried

out.

References

Banks GB, Chamberlain JS (2005) Relevance of motoneuron

specification and programmed cell death in embryos to

therapy of ALS. Birth Defects Res C Embryo Today 75, 294–

304.

Bartlett SE, Reynolds AJ, Hendry IA (1998) Retrograde axonal

transport of neurotrophins: differences between neuronal

populations and implications for motor neuron disease.

Immunol Cell Biol 76, 419–423.

Brown WF (1972) A method for estimating the number of motor

units in thenar muscles and the changes in motor unit count

with ageing. J Neurol Neurosurg Psychiatry 35, 845–852.

Carmeli E, Reznick AZ (1994) The physiology and biochemistry

of skeletal muscle atrophy as a function of age. Proc Soc Exp

Biol Med 206, 103–113.

Degens H (2007) Age related skeletal muscle dysfunction: causes

and mechanisms. J Musculoskelet Neuronal Interact 7, 246–

252.

Dobrowolny G, Giacinti C, Pelosi L, et al. (2005) Muscle

expression of a local Igf-1 isoform protects motor neurons in

an ALS mouse model. J Cell Biol 168, 193–199.

Elashry MI, Otto A, Matsakas A, et al. (2009) Morphology and

myofiber composition of skeletal musculature of the forelimb

in young and aged wild type and myostatin null mice.

Rejuvenation Res 12, 269–281.

Fernandes JJ, Keshishian H (2005) Motoneurons regulate

myoblast proliferation and patterning in Drosophila. Dev Biol

15, 493–505.

Forger NG, Prevette D, deLapeyriere O, et al. (2003)

Cardiotrophin-like cytokine ⁄ cytokine-like factor 1 is an

essential trophic factor for lumbar and facial motoneurons in

vivo. J Neurosci 23, 8854–8858.

Girgenrath S, Song K, Whittemore LA (2005) Loss of myostatin

expression alters fiber-type distribution and expression of

myosin heavy chain isoforms in slow- and fast-type skeletal

muscle. Muscle Nerve 31, 34–40.

Gray H, Williams PL, Bannister LH (1995) Gray’s Anatomy: The

Anatomical Basis of Medicine and Surgery, 38th edn. New

York: Churchill Livingstone.

Grieshammer U, Lewandoski M, Prevette D, et al. (1998) Muscle-

specific cell ablation conditional upon Cre-mediated DNA

recombination in transgenic mice leads to massive spinal and

cranial motoneuron loss. Dev Biol 197, 234–247.

Habgood MD, Hopkins WG, Slack JR (1984) Muscle size and

motor unit survival in mice. J Physiol 356, 303–314.

Hamburger V (1934) The effects of wing bud extirpation on the

development of the central nervous systerm in chick embryos.

J Exp Zool 68, 449–494.

Hennebry A, Berry C, Siriett V, et al. (2009) Myostatin regulates

fiber-type composition of skeletal muscle by regulating MEF2

and MyoD gene expression. Am J Physiol Cell Physiol 296,

C525–C534.

Hollyday M, Hamburger V (1976) Reduction of the naturally

occurring motor neuron loss by enlargement of the periphery.

J Comp Neurol 170, 311–320.

Holzbaur EL, Howland DS, Weber N, et al. (2006) Myostatin

inhibition slows muscle atrophy in rodent models of

amyotrophic lateral sclerosis. Neurobiol Dis 23, 697–707.

ªª 2011 The AuthorsJournal of Anatomy ªª 2011 Anatomical Society of Great Britain and Ireland

Myostatin null nerve fibre phenotype, M. I. Elashry et al. 183

Illanes O, Henry J, Skerritt G (1990) Light and electron

microscopy studies of the ulnar, saphenous, and caudal

cutaneous sural nerves of the dog. Am J Anat 187, 158–164.

Lance-Jones C (1982) Motoneuron cell death in the developing

lumbar spinal cord of the mouse. Brain Res 256, 473–479.

Landmesser L (1992) The relationship of intramuscular nerve

branching and synaptogenesis to motoneuron survival.

J Neurobiol 23, 1131–1139.

Lexell J (1997) Evidence for nervous system degeneration with

advancing age. J Nutr 127, 1011S–1013S.

Lexell J, Taylor CC, Sjostrom M (1988) What is the cause of the

ageing atrophy? Total number, size and proportion of

different fiber types studied in whole vastus lateralis muscle

from 15- to 83-year-old men. J Neurol Sci 84, 275–294.

Matsakas A, Otto A, Elashry MI, et al. (in press) Altered primary

and secondary myogenesis in the myostatin-null mouse.

Rejuvenation Res, in press.

Maier A (1997) Development and regeneration of muscle

spindles in mammals and birds. Int J Dev Biol 41, 1–17.

McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal

muscle mass in mice by a new TGF-beta superfamily member.

Nature 387, 83–90.

Morrison BM, Lachey JL, Warsing LC, et al. (2009) A soluble

activin type IIB receptor improves function in a mouse model

of amyotrophic lateral sclerosis. Exp Neurol 217, 258–268.

Oppenheim RW, Homma S, Marti E, et al. (1999) Modulation of

early but not later stages of programmed cell death in

embryonic avian spinal cord by sonic hedgehog. Mol Cell

Neurosci 13, 348–361.

Phelan KA, Hollyday M (1991) Embryonic development and

survival of brachial motoneurons projecting to muscleless

chick wings. J Comp Neurol 311, 313–320.

Pittman R, Oppenheim RW (1979) Cell death of motoneurons in

the chick embryo spinal cord. IV. Evidence that a functional

neuromuscular interaction is involved in the regulation of

naturally occurring cell death and the stabilization of

synapses. J Comp Neurol 187, 425–446.

Potthoff MJ, Wu H, Arnold MA, et al. (2007) Histone

deacetylase degradation and MEF2 activation promote the

formation of slow-twitch myofibers. J Clin Invest 117, 2459–

2467.

Pu SF, Zhuang HX, Marsh DJ, et al. (1999) Time-dependent

alteration of insulin-like growth factor gene expression during

nerve regeneration in regions of muscle enriched with

neuromuscular junctions. Brain Res Mol Brain Res 63, 207–216.

Reynolds AJ, Bartlett SE, Hendry IA (2000) Molecular

mechanisms regulating the retrograde axonal transport of

neurotrophins. Brain Res Brain Res Rev 33, 169–178.

Sendtner M, Pei G, Beck M, et al. (2000) Developmental

motoneuron cell death and neurotrophic factors. Cell Tissue

Res 301, 71–84.

Shorey M (1909) The effect of the destruction of peripheral

areas on the differentiation of the neuroblasts. J Exp Zool 7,

25–64.

Steinberg DA (2002) Scientific neurology and the history of the

clinical examination of selected motor cranial nerves. Semin

Neurol 22, 349–356.

Strelau J, Strzelczyk A, Rusu P, et al. (2009) Progressive

postnatal motoneuron loss in mice lacking GDF-15. J Neurosci

29, 13640–13648.

Tanaka H, Landmesser LT (1986) Cell death of lumbosacral

motoneurons in chick, quail, and chick-quail chimera embryos:

a test of the quantitative matching hypothesis of neuronal

cell death. J Neurosci 6, 2889–2899.

ªª 2011 The AuthorsJournal of Anatomy ªª 2011 Anatomical Society of Great Britain and Ireland

Myostatin null nerve fibre phenotype, M. I. Elashry et al.184