Disruption of Neuronal Migration by RNAi of Dyx1c1 Results in Neocortical and Hippocampal...

Disruption of Neuronal Migration by RNAiof Dyx1c1 Results in Neocortical andHippocampal Malformations

Glenn D. Rosen1,2, Jilin Bai3, Yu Wang3, Christopher G.

Fiondella3, Steven W. Threlkeld4, Joseph J. LoTurco3 and Albert

M. Galaburda1,2

1Dyslexia Research Laboratory and Charles A. Dana Research

Institute, Department of Neurology, Division of Behavioral

Neurology, Beth Israel Deaconess Medical Center, Boston, MA

02215, USA, 2Harvard Medical School, Boston, MA 02115, USA,3Deparmentof PhysiologyandNeurobiology and 4Departmentof

Psychology, University of Connecticut, Storrs, CT 06268, USA

The brains of individuals with developmental dyslexia have neo-cortical neuronal migration abnormalities including molecular layerheterotopias, laminar dysplasias, and periventricular nodular het-erotopias (PNH). RNA interference (RNAi) of Dyx1c1, a candidatedyslexia susceptibility gene, disrupts neuronal migration in de-veloping embryonic neocortex. Using in utero electroporation, wecotransfected cells in the rat neocortical ventricular zone (VZ) atE14/15 with short hairpin RNA vectors targeting Dyx1c1 along witheither plasmids encoding enhanced green fluorescent protein orplasmids encoding monomeric red fluorescent protein only. RNAi ofDyx1c1 resulted in pockets of unmigrated neurons resembling PNH.The pattern of migration of transfected neurons was bimodal, withapproximately 20% of the neurons migrating a short distance fromthe VZ and another 40% that migrated past their expected lamina.Approximately 25% of the transfected brains had hippocampalpyramidal cell migration anomalies. Molecular layer ectopias,which were not related to injection site artifacts, were also seenin 25% of the animals. These results support the hypothesis thattargeted disruption of the candidate dyslexia susceptibility gene,Dyx1c1, results in neuronal migration disorders similar to thoseseen in the brains of dyslexics.

Keywords: dyslexia, Dyx1c1, molecular layer ectopias, neuronal migration,periventricular nodular heterotopias, RNAi

Introduction

Developmental dyslexia is a language-based learning disability

affecting 4--10% of the population, which is characterized by

a difficulty with learning to read despite adequate motivation,

intelligence, and educational opportunity. The complex nature

of this disorder is reflected in the wide-ranging differences

reported between the brains of dyslexics and nondyslexics,

including symmetry of language-related regions (Galaburda

et al. 1985; Shapleske et al. 1999), reduced gray matter volume

(Silani et al. 2005; Vinckenbosch et al. 2005), and altered

patterns of white matter organization (Klingberg et al. 2000;

Schwartzman et al. 2005). We have reported small neocortical

malformations in the brains of dyslexics (Galaburda and Kemper

1979; Galaburda et al. 1985; Humphreys et al. 1990). These

neuronal migration anomalies, consisting of nests of neurons

and glia in the molecular layer (ectopias), intracortical laminar

dysplasias, and occasional instances of focal microgyria, are

located primarily in the lateral surface of the left hemisphere,

including the perisylvian, temporo-occipital, temporoparietal,

and frontal regions. Similarly, recent reports have demonstrated

an increased incidence of developmental dyslexia in patients

with periventricular nodular heterotopias (PNH) (Chang et al.

2005; Sokol et al. 2006).

The known genetic etiology of dyslexia similarly reflects the

complexity of this disorder. Thus, linkage analysis points to

dyslexic susceptibility loci on chromosomes 1, 2, 3, 6, 7, 11, 15,

18, and X (see Fisher and Francks 2006). Recently, candidate

dyslexia susceptibility genes have been proposed at some of

these intervals. ROBO1, an axon guidance and neuronal

migration gene located on Chr 3, has been reported to be

a candidate dyslexia susceptibility gene (Hannula-Jouppi et al.

2005), and 2 genes,DCDC2 and KIAA0319, have been identified

on Chr 6 (Francks et al. 2004; Cope et al. 2005; Meng, Smith,

et al. 2005; Paracchini et al. 2006; Schumacher et al. 2006).

DYX1C1 (also known as EKN1), a candidate dyslexia suscepti-

bility gene located on Chr 15, has been proposed, although

there is controversy regarding its generalizability outside of

Finnish populations (Taipale et al. 2003; Chapman et al. 2004;

Scerri et al. 2004; Wigg et al. 2004; Bellini et al. 2005; Marino

et al. 2005).

These genetic results have informed research on the ana-

tomic substrates of developmental dyslexia. We have demon-

strated thatDCDC2, KIAA0319, andDYX1C1 are genes involved

in neuronal migration (Meng, Smith, et al. 2005; Paracchini et al.

2006; Wang et al. 2006). Specifically, we used in utero electro-

poration to transfect premigratory neurons in the ventricular

zone (VZ) with short hairpin RNA (shRNA) targeted against

each of these candidate dyslexia susceptibility genes. Examina-

tion of the brains 4 days post transfection revealed in each case

that transfected neurons were arrested in migration. What is not

yet known is the adult neocortical phenotype of animals trans-

fected with these shRNAs, which would be of interest for com-

parison with the adult dyslexic brain. Here we report the results

of a study in which we examined the brains of adult rats that had

in utero electroporation of shRNA targeted against Dyx1c1. We

hypothesized that the neuronal migration abnormalities in-

duced by this treatment would result in neocortical malforma-

tions similar to those seen in postmortem dyslexic brains.

Materials and Methods

In Utero ElectroporationIn utero electroporation was performed at the University of Connect-

icut, and all procedures were approved by the Institutional Animal Care

and Use Committee of that institution. In all Dyx1c1 shRNA treatments,

plasmids encoding shRNA (pU6DyxHPB) and plasmids encoding en-

hanced green fluorescent protein (eGFP) (pCAGGS-eGFP) were co-

transfected into the VZ. The remaining animals received transfection

only with plasmids encoding monomeric red fluorescent protein

(mRFP) (pCAGGS-RFP). Ten Wistar dams (Charles River Laboratory,

Wilmington, MA) were anesthetized at E14/15, the uterine horns were

exposed, and approximately half of the pups were randomly assigned to

receive Dyx1c1 shRNA plasmids (1.5 lg/lL) plus eGFP (0.5 lg/lL),whereas the remaining pups were injected with mRFP (0.4 lg/lL).

Cerebral Cortex November 2007;17:2562--2572

doi:10.1093/cercor/bhl162

Advance Access publication January 11, 2007

� The Author 2007. Published by Oxford University Press. All rights reserved.

For permissions, please e-mail: [email protected]

by guest on January 5, 2016http://cercor.oxfordjournals.org/

Dow

nloaded from

http://cercor.oxfordjournals.org/

The plasmids were microinjected by pressure (General Valve picos-

pritzer) through the uterine wall into one randomly chosen lateral

ventricle of each embryo, using a pulled glass capillary (Drummond

Scientific, Broomall, PA). Equal numbers of mRFP and Dyx1c1 shRNA +eGFP injections were made. Electroporation was achieved by discharge

of a 500 lF 250 V capacitor charged to 50--100 V (Bai et al. 2003). A pair

of copper alloy plates (1 3 0.5 cm) pinching the head of each embryo

through the uterus were the conduit for the voltage pulse. For each

embryo, plasmids were injected in one hemisphere, and diffusion of the

plasmids into the opposite hemisphere was encouraged by gently

tapping the embryo skull until dye was seen to migrate into the

opposite hemisphere. A voltage pulse was discharged across the embryo

skull with the positive electrode adjacent to the transfected hemi-

sphere, the electrodes were repositioned to the opposite hemisphere,

and a second pulse was delivered. This resulted in the transfection of

cortex in both hemispheres. Five embryos were harvested 2 days post

transfection for comparison of the size of the initial transfection in both

the mRFP and Dyx1c1 shRNA + eGFP conditions. Thirty-six males were

weaned on P21 and were housed into pairs. Animals were reared under

a 12:12 light/dark cycle with food and water available ad lib and were

behaviorally tested (Threlkeld et al. forthcoming). In order to assess the

efficiency of cotransfection (i.e., what proportion of cells are cotrans-

fected when unequal molar ratios similar to those used in the Dyx1c1

shRNA + eGFP condition), 4 animals were cotransfected with eGFP (3.2

lg/lL) and mRFP (0.5 lg/lL). As shown in the results (and similar to

previous reports, e.g., Bai et al. 2003), essentially all cells are cotrans-

fected with both plasmids even when one plasmid is used at a much

lower concentration.

Histology

Adults

At P90--100, animals were deeply anesthetized (Xylanzine/Ketamine

100 mg/mL) and sacrificed by transcardial perfusion with 0.9% saline

followed by 4% paraformaldehyde. The brains were removed from the

skull and visualized whole under fluorescence for the presence of eGFP

(N = 21) or mRFP (N = 15). Twelve brains (6 Dyx1c1 shRNA + eGFP and

6 mRFP) were coronally sectioned at 40 lm on a freezing microtome

after cryoprotection in 30% sucrose buffer. The remaining 24 brains (15

Dyx1c1 shRNA + eGFP and 9 mRFP) were dehydrated in graded ethanol

and embedded in 12% celloidin before being sectioned in the coronal

plane at 30 lm. One series of every 10th section was stained for Nissl

substance (Thionin for the frozen sections, cresyl violet for the

celloidin). An adjacent series of free-floating sections was processed

for immunohistochemical detection of either eGFP (Chemicon, 1:200)

or mRFP (Chemicon, Temecula, CA, 1:1000) using ABC protocols. The

celloidin was first removed from the celloidin-embedded tissue by

incubation in a 1:1 solution of ethyl ether and 100% ethanol for 30 min.

Embryonic

For assessment of the number of cells initially transfected in the Dyx1c1

shRNA + eGFP and mRFP conditions, embryos were harvested 2 days

after transfection, a time when previous experiments have indicated

that proliferation in the transfected population has ceased (Wang et al.

2006). For the investigation of cotransfection efficiency at molar ratios

similar to those used in the current experiment, embryos were

harvested 4 days post transfection. Pregnant dams were anesthetized

as above and embryos harvested. Brains were quickly removed, fixed by

4% paraformaldyhyde, and stored in sodium phosphate buffer. They

were sectioned on a vibratome (Leica VT1000S, Leica Microsystems Inc.,

Bannockburn, IL) at 80 lm, and the sections mounted on glass slides and

coverslipped.

In Situ HybridizationIn order to better interpret the knockdown findings, we determined the

expression of Dyx1c1 in the prenatal brain by in situ hybridization

during various stages of embryonic development. We obtained time-

mated pregnant females (Charles River) and sacrificed the litters on E13,

E15, E17, or E19. Embryos from these litters were immediately frozen,

their brains were cut in the sagittal or coronal plane on a cryostat at 18

lm thickness, and the slides were processed for in situ hybridization of

Dyx1c1 as described below.

The cDNA prepared from frontal, parietal, and occipital lobes of

human embryonic brain (20 weeks, Biochain Institute, Hayward, CA)

was amplified with respective forward and reverse primers (GGG AGA

AAT TCA GAA AAT ATA TTT AC and TTA AGA TTT TAG TTC TGT TCC

TTG AATT) for 35 cycles. All fragments were then cloned into t vector

(Invitrogen, Carlsbad, CA) and sequenced to verify Dyx1c1 amplifica-

tion. Rat embryonic and postnatal brain cDNAs were synthesized from

total RNA and amplified with the primers ATG CCG GTG CGA GTG AGC

GAG and CAT CAT CTC GCC TAG GGC GTA TC to rat Dyx1c1. The

amplified DNA was gel-purified, cloned, and sequence verified to be

Dyx1c1. Nonradioactive in situ hybridizations were done by UB-In Situ

(Natick, MA), as previously described (Berger and Hediger 2001), using

a digoxigenin-labeled cRNA probe. The antisense and sense probes were

obtained from the polymerase chain reaction (PCR) products, amplified

from rat E14 brain cDNA, and cloned in pGEMT-Easy flanking T7 and SP6

promoters. Two probes, one from the first 400 bp generated from PCR

primer pairs and the second full-length cDNA yielded similar results.

Analysis

Adult

All analyses of adult brains were performed blind with respect to con-

dition. Nissl-stained sections were surveyed for the presence of malfor-

mations and the location noted. The location of immunohistochemically

labeled cells was charted in an adjacent series from 5 Dyx1c1 shRNA +eGFP and 4 mRFP brains using Neurolucida (MBF Biosciences, Williston,

VT), and the average number of labeled cells per section was computed.

The distance of labeled cell migration from the white matter to the pial

surface was assessed on 4 Dyx1c1 + eGFP and 4 mRFP brains. Section

drawings by Neurolucida were imported into Canvas X (ACD Systems,

Miami, FL), and a counting rectangle subdivided into 10 equal-size bins

was sized to extend from the white matter to the pial surface. The width

of the counting box was held constant at approximately 500 lm. The

number of labeled cells within each decile was manually counted and

recorded. For eachbrain,wemeasuredone rectangle in eachhemisphere

from 8 sections. A total of 2929 cells were counted in animals co-

transfected with Dyx1c1 shRNA + eGFP and 5678 in those transfected

with mRFP alone. The percentage of labeled cells in each decile was

determined for each animal, and themean value across all animals within

each condition was determined. Differences in frequency distribution

between the 2 conditions were assessed using chi-square analysis.

Embryonic

In order to determine the size of the initial transfection in transfected

(mRFP) and cotransfected (Dyxt1c1 shRNA + eGFP) conditions, images

of embryonic brains harvested 2 days post transfection were obtained by

confocal microscopy (Leica TCS SP2, Leica Microsystems) with a 403

objective. Image J (http://rsb.info.nih.gov/ij/) was used to count the

number of labeled neurons in each of 3 sections in each brain

automatically. Three brains were assessed in the mRFP condition and

2 brains in the Dyx1c1 shRNA + eGFP condition.

To determine the efficiency of cotransfection at molar ratios similar to

those used in shRNA transfections, we examined 4 brains that were

harvested 4 days following transfection of eGFP and mRFP plasmids at

approximately 6:1 molar ratio. Confocal microscopy and sequential

scanning for the 2 flours was used to assess single labeling and double

labeling (indicating cotransfection with both plasmids).

Image ProcessingPhotomicrographs were adjusted for exposure and sharpened (unsharp

mask filter) using Adobe Photoshop (Adobe Inc., San Jose, CA). Some

images were acquired using the Virtual Slice Module of Neurolucida.

Image montages were created in Canvas X (ACD Systems).

Results

Dyx1c1 Is Ubiquitously Expressed at a Modest Level inthe Forebrain

In situ hybridization of Dyx1c1 in embryonic rat embryos

revealed that this gene is expressed relatively ubiquitously in

Cerebral Cortex November 2007, V 17 N 11 2563


Dow

nloaded from

http://rsb.info.nih.gov/ij/


the forebrain during forebrain development (Fig. 1A). The level

of expression is relatively modest, with higher concentrations in

the neocortex, hippocampus, and choroid plexus. There is no

literature regarding Dyx1c1 expression in rat brain, but

examination of in situ hybridizations of adult mouse brains

at the Allen Brain Atlas (http://www.brainatlas.org/aba/) also

demonstrated a relatively modest level of ubiquitous expression

of Dyx1c1 in the forebrain, with higher levels in the neocortex,

hippocampus, and choroid plexus. We also queried The

GeneNetwork (http://genenetwork.org/search.html), an on-

line, publicly accessible database of transcript expression

(from Affymetrix 430 v2 microarrays) from dissected hippo-

campus, cerebellum, eye, striatum, and forebrain for a variety of

mouse strains, to assess Dyx1c1 transcript expression in adult-

hood. These data confirmed relatively modest expression of the

transcript in all 5 regions assayed (Fig. 1B).

Cotransfection Is Highly Efficient

Examination of embryos cotransfected with eGFP and mRFP at

an approximately 6:1 molar ratio 4 days post transfection

revealed that nearly all transfected neurons were transfected

with both plasmids (Fig. 2). Examination of thousands of

neurons in all 4 animals revealed only an occasional neuron

that was transfected with only one of the plasmids. This

indicates a near perfect efficiency of cotransfection using molar

ratios even greater than those employed in the Dyx1c1 shRNA +eGFP condition (approximately 5:1). This supports the conten-

tion that nearly every eGFP-labeled neuron in this condition was

also transfected with Dyx1c1 shRNA.

There Are Fewer Dyx1c1 RNA interference--TransfectedNeurons in the Neocortex

We counted the number of transfected neurons 2 days post

transfection in 3 mRFP and 2Dyx1c1 shRNA + eGFP animals and

found similar numbers of labeled neurons in these conditions

( �X±SEM=492±13:5 vs: 478±1:0; respectively; F1,4 < 1, not sig-

nificant; Fig. 3A). These results support the contention that the

initial number of transfected neurons did not differ between the

2 conditions. Examination of the adult brains, however, revealed

fewer labeled cells in Dyx1c1 shRNA + eGFP--transfected

animals as compared with those transfected with mRFP alone

(see Fig. 3B,C,D). This was confirmed quantitatively: the average

number of labeled cells in each section was significantly higher

in the mRFP than in the Dyx1c1 shRNA + eGFP condition

( �X±SEM=954:6±118:0 vs: 347:0±90:7; respectively; t = 4.2, de-

grees of freedom [df] = 7, P < 0.01). Taken together, these

results suggest a loss of neurons in Dyx1c1 shRNA + eGFP--

transfected animals in adulthood.

There Are Pockets of Unmigrated Cells in the WhiteMatter

There were periventricular clusters of unmigrated cells in all

Dyx1c1 shRNA + eGFP (see Fig. 4) but in none of the mRFP

animals. These PNH were located directly either at the

ependymal layer deep to the white matter, within the white

matter itself, or at the cortical white matter border and were

easily visible in Nissl-stained sections. Examination of these cells

in immunohistochemically stained sections revealed that only

a subset was immunopositive for eGFP. This suggested that

these neurons did not migrate because of a secondary rather

than a direct effect of Dyx1c1 RNA interference (RNAi) trans-

fection. The morphology of the unmigrated immunopositive

cells was clearly neuronal, although their normal radial orien-

tation was disturbed (Fig. 4D,D9).

Laminar Displacement of shRNA-Transfected Neurons

In all the cases examined, mRFP+ neurons migrated to supra-

granular locations, predominantly to neocortical layer 3. Rela-

tively few mRFP-labeled neurons were located in infragranular

layers. In contrast, eGFP+ neurons in the Dyx1c1 shRNA

condition were commonly found in infragranular layers, and

those that migrated supragranularly were found superficial to

the layer 3 location seen in mRFP-transfected animals (see Fig.

5A,B). This was confirmed quantitatively by migration distance

analysis (Fig. 5C). The distribution of labeled cells through

the thickness of the neocortex in Dyx1c1 shRNA + eGFP--

transfected animals differed significantly from that of neurons

transfected with mRFP (v2 = 3920.0, df = 9, P < 0.0001). Thus,

Figure 1. Expression of Dyx1c1 in the developing and adult rodent brain. (A) In situhybridization of Dyx1c1 of E15 and E17 rat embryos. Dyx1c1 gene expression ismodest and relatively ubiquitous in the forebrain. Bar 5 1 mm. (B) Dyx1c1 transcriptexpression levels in the 5 brain regions in the adult mouse as assessed by Affymetrix430 v2 microarray. Expression data are transformed to a log 2 scale and normalizedsuch that the mean ± standard error of mean (SEM) for all transcripts is 8 ± 2. Dataare expressed as the mean ± SEM from at least 30 mouse strains contained in TheGeneNetwork (http://genenetwork.org/).

2564 Dyx1c1 Knockdown and Neocortical Malformations d Rosen et al.


Dow

nloaded from

http://www.brainatlas.org/aba/

http://genenetwork.org/search.html

http://genenetwork.org/


whereas the distribution in mRFP animals was Gaussian, with

a peak at 70--80% of the distance to the pial surface, in the

Dyx1c1 shRNA + eGFP cases, it was essentially bimodal, with

peaks at the white matter border and at a location closer to the

pial surface. A chi-square analysis of just the neurons in the

upper 50% of the cortex was also significant (v2 = 2554.9, df = 4,

P < 0.0001), indicating that the neurons in the Dyx1c1 shRNA +eGFP condition migrated to more superficial locations than

neurons transfected with mRFP only.

Malformations of the Hippocampus

Of the 21 animals cotransfected with eGFP and Dyx1c1 shRNA,

5 had malformations of the hippocampus, one of which was

bilateral. There were no hippocampal malformations seen in any

of the brains of animals transfected with mRFP only. The

hippocampal malformations consisted predominantly of dis-

placed cells from the pyramidal layer into the stratum radia-

tum and stratum oriens (see Fig. 6A,B,D,E). There were also

ectopic collections of cells in the stratum radiatum that

appeared to be pyramidal in morphology. Interestingly, the

morphology of the eGFP+ cells was more typical of neocorti-

cal pyramidal cells than of hippocampal pyramidal cells (Fig.

6C,F,G). Specifically, these neurons had extended apical den-

drites and less elaborate dendritic arborization than typically

seen in hippocampal pyramidal cells. Examination of immuno-

histochemically stained sections revealed that only a small

percentage of the displaced neurons were eGFP+. Thus, some

of the displaced neurons were not transfected with shRNA

targeted against Dyx1c1, thereby suggesting a secondary effect

of the shRNA transfection in the hippocampus.

Molecular Layer Ectopias in the Cerebral Cortex

Collections of ectopic neurons in the molecular layer were seen

in both mRFP (13/15) and Dyx1c1 shRNA + eGFP (13/21)

animals. These ectopic neurons were often accompanied by

laminar dysplasias in the subjacent cortex, which appear to be

related to the disturbances associated with the injection of

plasmids into the ventricle at E14 (Rosen et al. 1992, 1995). This

is confirmed by examination of immunohistochemically stained

sections where the physical displacement of labeled cells can be

seen (see Fig. 7).

Five of the 21 brains cotransfected with eGFP and Dyx1c1

shRNA, and none of the mRFP-transfected cases, had 2 separate

collections of ectopic neurons in layer I of the cortex: one

Figure 2. Cotransfection at high molar ratios is highly efficient. Embryos were cotransfected with eGFP and mRFP at an approximately 6:1 molar ratio and harvested 4 days later.(A--C) Photomicrographs illustrating eGFP (A) and mRFP (B) transfected neurons from the VZ to the pial surface. Nearly every transfected neuron is colabeled with eGFP and mRFP,as can be seen in the overlaid image (C). Bar for (A--C)5 100 lm. (D--F) High-power photomicrographs illustrating transfected neurons labeled with eGFP (D), mRFP (E), and bothfluorescent proteins (F). Here every transfected neuron is double labeled. Bar 5 10 lm.



Dow

nloaded from


Figure 3. Disposition of labeled neurons in the brains of animals transfected in utero with Dyx1c1 shRNA þ eGFP or mRFP alone. (A) When examined 2 days post transfection,there is no apparent difference in the number of transfected neurons between the 2 groups. Bar 5 20 lm. (B) The number of Dyx1c1 shRNA þ eGFP neurons expressed asa percentage of mRFP neurons in short survival (2 days following transfection) and adult (P90--100) rats. Whereas there is no difference 2 days post transfection, there isa significant decrease in labeled neurons in the Dyx1c1 shRNAþ eGFP condition in adulthood. (C, D) In adulthood, there are fewer labeled neurons in the Dyx1c1 shRNA þ eGFPcondition, and they are scattered throughout the neocortical laminae when compared with the laminarly delimited location of labeled neurons in the mRFP condition.



Dow

nloaded from


collection resembled the disturbance at the injection site

described above, whereas the other showed a different mor-

phology. In each case where a second collection of ectopic

neurons was found, it was in an area distant from the site of

injection. In addition, these malformations were distinguishable

from the injection-related malformations by the general lack in

the latter of disturbed cortex in the subjacent layers (see Fig.

8A,B,C). The second type of molecular layer ectopias was

remarkably similar in appearance to ectopias seen in human

dyslexic brains (Fig. 8D). Examination of the immunohisto-

chemically stained sections in this second set of ectopias

revealed that, in contrast to injection site ectopias, there was

no displacement of labeled neurons into more superficial lamina

in the area of the malformation. Moreover, transfected neurons

surrounded the ectopia, but there were few, if any, labeled

neurons within the cluster of cells (see Fig. 8E). This suggested

once again that at least some component of the malformations

caused by Dyx1c1 shRNA might arise from changes in cell

interactions rather than from direct RNAi effects on cells.

Discussion

The results of the current experiment support previous work

suggesting that RNAi of the rodent homolog of the candidate

dyslexia susceptibility gene DYX1C1 disrupts neuronal migra-

tion in developing cerebral cortex (Wang et al. 2006). Four days

following transfection, there was a nearly complete block of

migration into the cortical plate. Moreover, this block was

rescued by concurrent overexpression of Dyx1c1 indicating

that the RNAi effect is specific to knockdown of Dyx1c1

expression. When examined in adulthood, the pattern of the

disturbance in the cerebral cortex has evolved. There is an

approximately 60% reduction of transfected cells in Dyx1c1

shRNA--transfected brains as compared with those transfected

with mRFP alone. Neurons transfected with shRNA targeted

against Dyx1c1 exhibit an essentially bimodal pattern of

neuronal migration, with approximately 20% of the surviving

neurons remaining in the white matter and in layer VI and 60%

migrating to supragranular layers. Interestingly, two-thirds of

these supragranular neurons appear to have migrated to laminae

beyond those seen in animals transfected with fluorescent

protein alone at the same age. In addition to these migrational

disturbances, molecular layer ectopias not related to the

injection site are seen in approximately 25% of the Dyx1c1

shRNA--transfected animals, which appear to be similar to those

seen in the brains of postmortem dyslexics. We unexpectedly

found that targeted knockdown of Dyx1c1 also disrupts the

anatomic organization of the hippocampus.

Neuronal Migration and Developmental Dyslexia

Of the 4 candidate dyslexia susceptibility genes currently

identified, all 4 have been shown to play a role in neuronal

migration. Using in utero electroporation of shRNA targeted

against the genes DCDC2, KIAA0319, and DYX1C1, we have

demonstrated that each of these genes disrupts neuronal

migration to the neocortex (Meng, Smith, et al. 2005; Paracchini

et al. 2006; Wang et al. 2006). The fourth dyslexia candidate

gene, ROBO1 (Hannula-Jouppi et al. 2005), has also been well

characterized as being important for axon guidance and

Figure 4. Packets of unmigrated neurons in the white matter and VZ of Dyx1c1 shRNA þ eGFP subjects. (A) Photomicrograph of Nissl-stained section illustrating packets ofunmigrated cells in the white matter (white arrow and black arrowheads) and along the ventricle (black arrow). Bar 5 250 lm. (B) Section adjacent to Panel (A)immunohistochemically stained for eGFP. Arrows align with Panel (A). Bar 5 250 lm. (C) Higher power magnification of large packet of unmigrated cells. Bar 5 100 lm. (D)Section adjacent to Panel (C) immunohistochemically stained for eGFP. There are labeled neurons in the malformation, but comparison with Panel (C) indicates that there are someneurons that are unlabeled in this malformation. Bar5 100 lm. (D’) High-power magnification of white rectangle in Panel (D) illustrating the neuronal morphology of labeled cellscontained in the malformation. Bar 5 10 lm.



Dow

nloaded from


neuronal migration (Nguyen Ba-Charvet et al. 1999; Zhu et al.

1999; Hivert et al. 2002). Combined roles in neuronal migration

and the establishment of neural circuits make these genes

nicely placed to be behind the abnormally functioning cortex

seen in developmental dyslexia.

In previous experiments involving in utero electroporation of

shRNA targeted against dyslexia susceptibility genes, brains

were examined within 4 days of transfection. As a result, the

effect on the eventual organization of the forebrain was not

assessed. In this experiment, we found forebrain malformations

in the brains of animals sacrificed in adulthood. Intriguingly, we

found molecular layer ectopias that are remarkably similar to

those seen in postmortem dyslexic brains (Galaburda and

Kemper 1979; Galaburda et al. 1985; Humphreys et al. 1990).

In addition, the clusters of unmigrated neurons seen in the

Dyx1c1 shRNA + eGFP condition closely resemble PNH, the

presence of which has been associated with an increased

incidence of developmental dyslexia (Chang et al. 2005; Sokol

et al. 2006). This lends support the purported link between

neuronal migration disorders and developmental dyslexia.

As a cautionary note, it must be mentioned that the Dyx1c1

locus described in the initial report in a Finnish population

(Taipale et al. 2003) was also found in a Canadian sample, but

with different alleles and haplotypes (Wigg et al. 2004). Also,

Chapman et al. (2004) found linkage to the same region of Chr

15 in a US population, but again did not directly replicate the

role of Dyx1c1. Other studies have failed to provide support for

the Dyx1c1 locus in Italian (Bellini et al. 2005; Marino et al.

2005), English (Scerri et al. 2004), and US (Meng, Hager, et al.

2005) populations. Thus, although our data support a role of

Dyx1c1 in neuronal migration, definitive support for its role as

a candidate dyslexia susceptibility gene outside the Finnish

population awaits further studies.

Dyx1c1 Disrupts Neuronal Migration

There was a significant diminution of labeled neurons in the

cortex of the Dyx1c1 shRNA + eGFP condition when compared

with the mRFP condition, with the latter having nearly 3-fold as

many labeled neurons. This could be the result of changes in the

proliferation of neurons or in subsequent cell death. Wang et al.

(2006) demonstrated that there was no change in the pro-

liferation of cells after treatment with RNAi against Dyx1c1, and

our analysis did not demonstrate any difference in size of the

initial transfection between animals cotransfected with shRNA

and eGFP and those transfected with fluorescent protein alone

(Fig. 2A,B). Because relatively equal numbers of neurons are

transfected in both conditions, the diminution of labeled cells in

the Dyx1c1 shRNA + eGFP condition in this study most likely

represents an increase in cell death in this population of

transfected cells.

The periventricular collections of unmigrated neurons were

not surprising given the results from the previous experiments,

where few neurons from Dyx1c1 shRNA + eGFP cases had

migrated away from the VZ when examined 4 days after trans-

fection. It was, however, unexpected that the unmigrated neu-

rons in this study comprised only 20% of the labeled neurons,

with the overwhelming majority of labeled neurons continuing

on to migrate into the cortical plate. But it is also clear that the

migration of the labeled cells in Dyx1c1 shRNA + eGFP animals

differs from those transfected only with mRFP. In mRFP animals,

the distribution of labeled neurons throughout the depth of

the neocortex was Gaussian, with approximately 75% of the

cells located in layer 3. In contrast, labeled cells in the Dyx1c1

shRNA+eGFPconditionweredistributed essentially in abimodal

manner, with one collection of cells remaining in the white mat-

ter and another group migrating up to neocortical layer 2.

Figure 5. Laminar disposition of labeled neurons in Dyx1c1 shRNA þ eGFP (A) andmRFP (B) subjects. The majority of mRFP transfected are situated in layer 3 of theneocortex. In contrast, labeled neurons in the Dyx1c1 shRNA þ eGFP conditions tendto either not migrate from the VZ or arrive in layer 2 of the neocortex. Bars5 250 lm.(C) Histogram of mean (±standard error of mean) average percentage of neurons ineach decile of neocortex ranging from the white matter to the pial surface. Thequantitative data confirm the observations suggesting that in mRFP cases (red bars),the majority of the labeled cells are concentrated in the mid-upper deciles. In contrast,the labeled cells in the Dyx1c1 shRNAþ eGFP condition are distributed bimodally, withsome not migrating much beyond the VZ and other neurons migrating past theexpected lamina.



Dow

nloaded from


The reason for the migration of labeled cells to layer 2 of

the cerebral cortex in the Dyx1c1 shRNA + eGFP condition as

compared with layer 3 in the mRFP condition is not known. It

could be that knockdown of Dyx1c1 function serves mostly to

delay neuronal migration. Thus, transfected neurons remain in

the VZ in the immediate aftermath of the transfection. They

then begin their migration at the same time that later generated

neurons begin theirs. The position in the upper layers of the

cerebral cortex would be consistent with the possibility that

these cells have been respecified to upper layer fates similar to

the results from transplantation experiments of early generated

neurons into latter VZ environments (e.g., McConnell 1985,

1990). Alternatively, it could be that knockdown of Dyx1c1

function in these neurons fundamentally changes their ability to

migrate. In this case, neurons migrate throughout the cortical

layers and collect in layer 2 because they cannot migrate past

the external glial limiting membrane. At this point, we cannot

distinguish between these possibilities.

Malformations in the Forebrain

We have previously shown that puncture wounds to the cortical

plate lead to ectopic collections of neurons in the molecular

layer (Rosen et al. 1992, 1995). In the current experiment,

ectopic collections of neurons were seen in the majority of

cases and were related to the puncture of the cortical plate by

the injection of plasmids. In addition to these obvious traumatic

malformations, 25% of the Dyx1c1 shRNA + eGFP animals had

other ectopic collections of neurons in the molecular layer,

which are similar to those seen in humans with developmental

dyslexia (see above).

The malformations of the hippocampus were an unexpected

finding. These malformations are remarkably similar to those

seen in the progeny of mothers injected with methylazoxyme-

thanol (MAM) at E15 (Chevassus-Au-Louis et al. 1998). In the

case of these induced malformations, the neurons in the hippo-

campus exhibit neocortical-like neuronal activity and are hy-

pothesized to have migrated from the neocortex (Castro et al.

Figure 6. Hippocampal malformations associated with RNAi-targeted knockdown of Dyx1c1. (A) Displacement of cells from the pyramidal layer of CA1 into the stratum radiatum(arrow). Arrowheads denote clusters of ectopic pyramidal neurons. Bar 5 250 lm. (A’) Higher power magnification of ectopic cells in panel (A). Bar 5 100 lm. (B) Sectionadjacent to that in panel (A) immunohistochemically stained for eGFP with arrows matching for orientation. Note that only a small percentage of the ectopic neurons are eGFPþ.Bar5 250 lm. (B’) Higher power magnification of ectopic neurons in panel (B). White box is magnified area in Panel (C). (C) High-power magnification of immunopositive ectopicneuron in the hippocampus that morphologically resembles a neocortical pyramidal cell. Bar 5 100 lm. (D) Another example of hippocampal migration disturbances of thepyramidal layer. Here, pyramidal cells are displaced into both the stratum radiatum as well as the stratum oriens (arrowhead). Box is magnified area in Panel (D’). Bar 5 250 lm.(D’). Higher power magnification of malformation in panel (A) (arrow). Bar5 100 lm. (E) Section adjacent to that in panel (D) immunohistochemically stained for eGFP with arrowsmatching for orientation. Note that only a small percentage of the ectopic neurons are eGFPþ. Black box is magnified in Panel (E’), white box is magnified in Panel (G). Bar5 250lm. (E’) Higher power magnification of ectopic neurons in panel (E). Gray box is magnified in Panel (F). Bar 5 100 lm. (F) High-power magnification of immunopositive ectopichippocampal neurons exhibiting morphology similar to neocortical pyramidal cell. Bar 5 10 lm. (G) High-power magnification of neuron in the immunopositive neuron in thepyramidal layer of the hippocampus with neocortical pyramidal morphology. Bar 5 50 lm.



Dow

nloaded from


2002). In our case, it could be that the in utero electroporation-

labeled neurons from the VZ destined to be hippocampal.

The paucity of labeled neurons in the hippocampus of mRFP-

transfected animals, however, argues against this interpretation.

In addition, the long apical dendrites and modest dendritic

arborization of these ectopic immunopositive neurons more

closely resemble neocortical pyramidal cells. It is more likely,

therefore, that the labeled neurons in the hippocampal malfor-

mations are neocortical neurons that have mismigrated from

the VZ.

Secondary Effects of Dyx1c1 Knockdown

There are a number of indications that there were secondary

effects of Dyx1c1 knockdown in the developing forebrain. Few

of the neurons that comprise the nontraumatic molecular layer

ectopias, the periventricular collections of unmigrated neurons,

and the hippocampal malformations were eGFP+. This result

could theoretically be due to the possibility that some eGFP-

negative cells are transfected only with Dyx1c1 shRNA but not

eGFP plasmids. This is unlikely given the high degree of

cotransfection efficiency at the molar ratios used in this

experiment. It is more likely that there are secondary effects

of the knockdown of Dyx1c1 function that act to disrupt

neuronal migration in the forebrain beyond the direct effects on

transfected cells.

It is tempting to speculate that disturbances to radial glial

cells may underlie these secondary effects. Radial glial cells

function not only as the scaffold by which neocortical neu-

rons migrate from the VZ to the cortical plate (Rakic 1988) but

also serve as neuronal precursors in the neocortex (Noctor

et al. 2002; Fishell and Kriegstein 2003). In addition, radial

glial cells are involved in the maintenance of the early external

glial limiting membrane (Marin-Padilla 1995). We have pre-

viously shown that disruptions of the external glial limiting

membrane can cause molecular layer ectopias to occur (Rosen

et al. 1992; Sherman et al. 1992). Prenatal exposure to MAM

not only induces hippocampal malformations but also results

in periventricular nodules and other neocortical heterotopias

(Zhang et al. 1995). MAM exposure directly affects the radial

glia by disrupting their attachment to the cortical surface

(Zhang et al. 1995) and hastening their transition to astro-

cytes (Noctor et al. 1999; Gierdalski and Juliano 2003), and it

has been hypothesized that this disruption of the radial glial

cells is the main cause of the malformations in MAM-exposed

animals (Paredes et al. 2006). Thus, disturbances of radial glial

cells during the period of neuronal migration have been

associated with all 3 types of malformations exhibited by our

model. It is therefore tempting to hypothesize that Dyx1c1

acts not only to disrupt the machinery important for neu-

ronal migration in neurons (Wang et al. 2006) but also may,

perhaps indirectly through the injury to the migrating neurons,

disrupt the radial glia. Future research will directly test this

hypothesis.

Notes

This work was supported, in part, by National Institutes of Health grant

HD20806. The authors wish to acknowledge the expert technical

assistance of Shira Anconina, Cullen Owens, and Zachary Snow.

Conflict of Interest: None declared.

Address Correspondence to Glenn D. Rosen, Department of Neurol-

ogy, Beth Israel Deaconess Medical Center, 330 Brookline Avenue,

Boston, MA 02215, USA, Email: [email protected].

Figure 7. Ectopic collections of neurons associated with injection site. (A) Low-power photomicrograph of Nissl-stained section illustrating ectopic collections of neurons in themolecular layer of the neocortex (arrowheads). As is typical of malformations associated with injection sites, there is also a cell-free region below the ectopic collection (arrow).Compare with undisturbed cortex in panel (C). (B) Section adjacent to panel (A) immunohistochemically stained for eGFP. The deviation of the band of labeled neurons is typical of aninjection site malformation. Compare with control section in panel (D). Bar 5 250 lm.



Dow

nloaded from


References

Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ. 2003. RNAi

reveals doublecortin is required for radial migration in rat neocortex.

Nat Neurosci. 6:1277--1283.

Bellini G, Bravaccio C, Calamoneri F, Donatella Cocuzza M, Fiorillo P,

Gagliano A, Mazzone D, del Giudice EM, Scuccimarra G, Militerni R,

et al. 2005. No evidence for association between dyslexia and

DYX1C1 functional variants in a group of children and adolescents

from Southern Italy. J Mol Neurosci. 27:311--314.

Berger UV, Hediger MA. 2001. Differential distribution of the glutamate

transporters GLT-1 and GLAST in tanycytes of the third ventricle.

J Comp Neurol. 433:101--114.

Castro P, Pleasure S, Baraban S. 2002. Hippocampal heterotopia with

molecular and electrophysiological properties of neocortical neu-

rons. Neuroscience. 114:961.

Chang BS, Ly J, Appignani B, Bodell A, Apse KA, Ravenscroft RS, Sheen VL,

Doherty MJ, Hackney DB, O’Connor M, et al. 2005. Reading

impairment in the neuronal migration disorder of periventricular

nodular heterotopia. Neurology. 64:799--803.

Figure 8. Molecular layer ectopia in Dyx1c1 shRNA þ eGFP subject. (A) Line drawings of 2 sections from the same subject illustrating location of injection site ectopia (panel B)and noninjection site ectopia (panel C). Bar 5 1 mm. (B) Low-power photomicrograph demonstrating injection site ectopia (arrows). (C) Low-power photomicrograph of Nissl-stained section of ectopic collections of neurons in the molecular layer (black arrows) in a more caudal section. In this same section, there is also a collection of unmigrated neurons(white arrows) and hippocampal pyramidal cell dysplasia. (D) Photomicrograph of molecular layer ectopia (arrows) in a human dyslexic brain. Compare with panel (C). This brain wasembedded in celloidin, which shrinks the brain by approximately 60%. (E) Section adjacent to panel (C) immunohistochemically stained for eGFP. Arrows and arrowheads are fororientation with panel (C). Note the absence of labeled neurons in the ectopic collection of neurons in layer I of the neocortex (arrows). Bar for (B--E) 5 250 lm.



Dow

nloaded from


Chapman NH, Igo RP, Thomson JB, Matsushita M, Brkanac Z, Holzman T,

Berninger VW, Wijsman EM, Raskind WH. 2004. Linkage analyses of

four regions previously implicated in dyslexia: confirmation of a locus

on chromosome 15q. Am J Med Genet B Neuropsychiatr Genet.

131:67--75.

Chevassus-Au-Louis N, Congar P, Represa A, Ben-Ari Y, Gaiarsa JL. 1998.

Neuronal migration disorders: heterotopic neocortical neurons in

CA1 provide a bridge between the hippocampus and the neocortex.

Proc Natl Acad Sci USA. 95:10263--10268.

Cope N, Harold D, Hill G, Moskvina V, Stevenson J, Holmans P, Owen MJ,

O’Donovan MC, Williams J. 2005. Strong evidence that KIAA0319 on

chromosome 6p is a susceptibility gene for developmental dyslexia.

Am J Hum Genet. 76:581--591.

Fishell G, Kriegstein AR. 2003. Neurons from radial glia: the consequen-

ces of asymmetric inheritance. Curr Opin Neurobiol. 13:34--41.

Fisher SE, Francks C. 2006. Genes, cognition and dyslexia: learning to

read the genome. Trends Cognit Sci. 10:250--257.

Francks C, Paracchini S, Smith SD, Richardson AJ, Scerri TS, Cardon LR,

Marlow AJ, MacPhie IL, Walter J, Pennington BF, et al. 2004. A 77-

kilobase region of chromosome 6p22.2 is associated with dyslexia in

families from the United Kingdom and from the United States. Am J

Hum Genet. 75:1046--1058.

Galaburda AM, Kemper TL. 1979. Cytoarchitectonic abnormalities in

developmental dyslexia; a case study. Ann Neurol. 6:94--100.

Galaburda AM, Sherman GF, Rosen GD, Aboitiz F, Geschwind N. 1985.

Developmental dyslexia: four consecutive cases with cortical anom-

alies. Ann Neurol. 18:222--233.

Gierdalski M, Juliano SL. 2003. Factors affecting the morphology of radial

glia. Cereb Cortex. 13:572--579.

Hannula-Jouppi K, Kaminen-Ahola N, Taipale M, Eklund R, Nopola-

Hemmi J, Kaariainen H, Kere J. 2005. The axon guidance receptor

gene ROBO1 is a candidate gene for developmental dyslexia. PLoS

Genet. 1:e50.

Hivert B, Liu Z, Chuang CY, Doherty P, Sundaresan V. 2002. Robo1 and

Robo2 are homophilic binding molecules that promote axonal

growth. Mol Cell Neurosci. 21:534--545.

Humphreys P, Kaufmann WE, Galaburda AM. 1990. Developmental

dyslexia in women: neuropathological findings in three cases. Ann

Neurol. 28:727--738.

Klingberg T, Hedehus M, Temple E, Salz T, Gabrieli JDE, Moseley ME,

Poldrack RA. 2000. Microstructure of temporo-parietal white matter

as a basis for reading ability: evidence from diffusion tensor magnetic

resonance imaging. Neuron. 25:493--500.

Marin-Padilla M. 1995. Prenatal development of fibrous (white matter),

protoplasmic (gray matter), and layer I astrocytes in the human

cerebral cortex: a Golgi study. J Comp Neurol. 357:554--572.

Marino C, Giorda R, Luisa Lorusso M, Vanzin L, Salandi N, Nobile M,

Citterio A, Beri S, Crespi V, Battaglia M, et al. 2005. A family-based

association study does not support DYX1C1 on 15q21.3 as a candi-

date gene in developmental dyslexia. Eur J Hum Genet. 13:491--499.

McConnell SK. 1985. Migration and differentiation of cerebral cortical

neurons after transplantation into the brain of ferrets. Science.

229:1268--1271.

McConnell SK. 1990. The specification of neuronal identity in the

mammalian cerebral cortex. Experientia. 46:922--929.

Meng H, Hager K, Held M, Page GP, Olson RK, Pennington BF, DeFries JC,

Smith SD, Gruen JR. 2005. TDT-association analysis of EKN1 and

dyslexia in a Colorado twin cohort. Hum Genet. 118:87--90.

Meng H, Smith SD, Hager K, Held M, Liu J, Olson RK, Pennington BF,

DeFries JC, Gelernter J, O’Reilly-Pol T, et al. 2005. DCDC2 is

associated with reading disability and modulates neuronal develop-

ment in the brain. Proc Natl Acad Sci USA. 102:17053--17058.

Nguyen Ba-Charvet KT, Brose K, Marillat V, Kidd T, Goodman CS,

Tessier-Lavigne M, Sotelo C, Chedotal A. 1999. Slit2-mediated

chemorepulsion and collapse of developing forebrain axons. Neu-

ron. 22:463--473.

Noctor SC, Flint AC, Weissman TA, Wong WS, Clinton BK, Kriegstein AR.

2002. Dividing precursor cells of the embryonic cortical ventricular

zone have morphological and molecular characteristics of radial glia.

J Neurosci. 22:3161--3173.

Noctor SC, Palmer SL, Hasling T, Juliano SL. 1999. Interference with the

development of early generated neocortex results in disruption of

radial glia and abnormal formation of neocortical layers. Cereb

Cortex. 9:121--136.

Paracchini S, Thomas A, Castro S, Lai C, Paramasivam M, Wang Y, Keating

BJ, Taylor JM, Hacking DF, Scerri T, et al. 2006. The chromosome

6p22 haplotype associated with dyslexia reduces the expression of

KIAA0319, a novel gene involved in neuronal migration. Hum Mol

Genet. 15:1659--1666.

Paredes M, Pleasure SJ, Baraban SC. 2006. Embryonic and early postnatal

abnormalities contributing to the development of hippocampal

malformations in a rodent model of dysplasia. J Comp Neurol.

495:133--148.

Rakic P. 1988. Specification of cerebral cortical areas. Science.

241:170--176.

Rosen GD, Sherman GF, Richman JM, Stone LV, Galaburda AM. 1992.

Induction of molecular layer ectopias by puncture wounds in

newborn rats and mice. Dev Brain Res. 67:285--291.

Rosen GD, Waters NS, Galaburda AM, Denenberg VH. 1995. Behavioral

consequences of neonatal injury of the neocortex. Brain Res.

681:177--189.

Scerri TS, Fisher SE, Francks C, MacPhie IL, Paracchini S, Richardson AJ,

Stein JF, Monaco AP. 2004. Putative functional alleles of DYX1C1 are

not associated with dyslexia susceptibility in a large sample of sibling

pairs from the UK. J Med Genet. 41:853--857.

Schumacher J, Anthoni H, Dahdouh F, Konig IR, Hillmer AM, Kluck N,

Manthey M, Plume E, Warnke A, Remschmidt H, et al. 2006. Strong

genetic evidence ofDCDC2 as a susceptibility gene for dyslexia. Am J

Hum Genet. 78:52--62.

Schwartzman A, Dougherty RF, Taylor JE. 2005. Cross-subject compar-

ison of principal diffusion direction maps. Magn Reson Med.

53:1423--1431.

Shapleske J, Rossell SL, Woodruff PW, David AS. 1999. The planum

temporale: a systematic, quantitative review of its structural,

functional and clinical significance. Brain Res Brain Res Rev.

29:26--49.

Sherman GF, Rosen GD, Stone LV, Press DM, Galaburda AM. 1992. The

organization of radial glial fibers in spontaneous neocortical ectopias

of newborn New-Zealand black mice. Dev Brain Res. 67:279--283.

Silani G, Frith U, Demonet JF, Fazio F, Perani D, Price C, Frith CD, Paulesu

E. 2005. Brain abnormalities underlying altered activation in dyslexia:

a voxel based morphometry study. Brain. 128:2453--2461.

Sokol DK, Golomb MR, Carvahlo KS, Edwards-Brown M. 2006. Reading

impairment in the neuronal migration disorder of periventricular

nodular heterotopia. Neurology. 66:294.

Taipale M, Kaminen N, Nopola-Hemmi J, Haltia T, Myllyluoma B,

Lyytinen H, Muller K, Kaaranen M, Lindsberg PJ, Hannula-Jouppi K,

et al. 2003. A candidate gene for developmental dyslexia encodes

a nuclear tetratricopeptide repeat domain protein dynamically

regulated in brain. Proc Natl Acad Sci USA. 100:11553--11558.

Threlkeld SW, McClure MM, Bai J, Wang Y, LoTurco JJ, Rosen GD, Fitch

RH. Forthcoming. Developmental disruptions and behavioral impair-

ments in rats following in utero RNAi of Dyx1c1. Brain Res Bull.

Vinckenbosch E, Robichon F, Eliez S. 2005. Gray matter alteration in

dyslexia: converging evidence from volumetric and voxel-by-voxel

MRI analyses. Neuropsychologia. 43:324--331.

Wang Y, Paramasivam M, Thomas A, Bai J, Kaminen N, Kere J, Voskul J,

Rosen G, Galaburda A, LoTurco J. 2006. Dyx1c1 functions in

neuronal migration in developing neocortex. Neuroscience.

143:515--522.

Wigg KG, Couto JM, Feng Y, Anderson B, Cate-Carter TD, Macciardi F,

Tannock R, Lovett MW, Humphries TW, Barr CL. 2004. Support for

EKN1 as the susceptibility locus for dyslexia on 15q21. Mol Psy-

chiatry. 9:1111--1121.

Zhang LL, Collier PA, Ashwell KW. 1995. Mechanisms in the induction of

neuronal heterotopiae following prenatal cytotoxic brain damage.

Neurotoxicol Teratol. 17:297--311.

Zhu Y, Li H, Zhou L, Wu JY, Rao Y. 1999. Cellular and molecular

guidance of GABAergic neuronal migration from an extracortical

origin to the neocortex. Neuron. 23:473--485.



Dow

nloaded from


Disruption of Neuronal Migration by RNAi of Dyx1c1 Results in Neocortical and Hippocampal...

Documents

Transcript of Disruption of Neuronal Migration by RNAi of Dyx1c1 Results in Neocortical and Hippocampal...