The effect of variation in expression of the candidate dyslexia susceptibility gene homolog Kiaa0319 on neuronal migration and dendritic morphology in the rat.

Abstract

We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct ("rescue"), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non-transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non-cell autonomous mechanisms. Of the Kiaa0319 shRNA-transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites.

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Cerebral Cortex
doi:10.1093/cercor/bhp154
The Effect of Variation in Expression of the
Candidate Dyslexia Susceptibility Gene
Homolog Kiaa0319 on Neuronal Migration
and Dendritic Morphology in the Rat
Veronica J. Peschansky1, Timothy J. Burbridge1, Amy J. Volz1,
Christopher Fiondella2, Zach Wissner-Gross3, Albert
M. Galaburda1, Joseph J. Lo Turco2and Glenn D. Rosen1
1The Dyslexia Research Laboratory, Division of Behavioral
Neurology, Department of Neurology, Beth Israel Deaconess
Medical Center, Boston, MA 02215, USA,2Department of
Physiology and Neurobiology, University of Connecticut, Storrs,
CT 06268, USA and3Department of Physics, Harvard University,
Cambridge, MA 02138, USA
Veronica J. Peschansky, Timothy J. Burbridge, and Amy J. Volz
contributed equally to this manuscript.
We investigated the postnatal effects of embryonic knockdown and
overexpression of the candidate dyslexia gene homolog Kiaa0319.
We used in utero electroporation to transfect cells in E15/16 rat
neocortical ventricular zone with either 1) small hairpin RNA
(shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression
construct, 3) Kiaa0319 shRNA along with KIAA0319 expression
construct (‘‘rescue’’), or 4) a scrambled version of Kiaa0319 shRNA.
Knockdown, but not overexpression, of Kiaa0319 resulted in
periventricular heterotopias that contained large numbers of both
transfected and non--transfected neurons. This suggested that
Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as
well as non--cell autonomous mechanisms. Of the Kiaa0319 shRNA--
transfected neurons that migrated into the cortical plate, most
migrated to their appropriate lamina. In contrast, neurons trans-
fected with the KIAA0319 expression vector attained laminar
positions subjacent to their expected positions. Neurons trans-
fected with Kiaa0319 shRNA exhibited apical, but not basal,
dendrite hypertrophy, which was rescued by overexpression of
KIAA0319. The results provide additional supportive evidence
linking candidate dyslexia susceptibility genes to migrational
disturbances during brain development, and extends the role of
Kiaa0319 to include growth and differentiation of dendrites.
Keywords: cerebral cortex, dendritic hypertrophy, heterotopias,
malformation, RNAi
Introduction
Developmental dyslexia is a language--based learning disability
that affects between 4% and 10% of the population, and has
a strong genetic component (Fisher and Francks 2006). Post
mortem examination of the brains of developmental dyslexics
demonstrated the presence of neuronal migration anomalies,
including molecular layer ectopias, laminar dysplasia, and
occasional focal microgyria (Galaburda and Kemper 1979;
Galaburda et al. 1985; Humphreys et al. 1990). More recently,
an association between periventricular nodular heterotopias
and developmental dyslexia has been reported (Chang et al.
2005; Sokol et al. 2006).
Recent reports proposing candidate dyslexia susceptibility
genes have opened up new lines of investigation into the genetic
modulation of this disorder. A number of these genes, in
specific—DCDC2 and KIAA0319 on Chr 6 (Francks et al. 2004;
Cope et al. 2005; Meng et al. 2005; Harold et al. 2006; Paracchini
et al. 2006; Schumacher et al. 2006; Luciano et al. 2007;
Paracchini et al. 2008), and DYX1C1 on Chr 15 (Taipale et al.
2003; Brkanac et al. 2007; Marino et al. 2007)—have been shown
to have roles in neuronal migration. Thus, we have previously
demonstrated that in utero electroporation of shRNA targeted
against rat homologs of DCDC2, KIAA0319, or DYX1C1 in the
rat disrupts the process of neuronal migration to the cerebral
cortex as assessed during the prenatal period (Meng et al. 2005;
Paracchini et al. 2006; Wang et al. 2006). Further evaluation
of the postnatal consequences of embryonic knockdown of
Dyx1c1 and Dcdc2 function in rats revealed the presence of
a variety of neocortical malformations, including molecular layer
ectopias and periventricular heterotopias (PVHs) (Rosen et al.
2007; Burbridge et al. 2008). In addition to these frank
neuronal migration anomalies, we found evidence of more
subtle disruptions, with some transfected neurons migrating to
the cortical plate (CP), albeit past their expected laminar
locations (Rosen et al. 2007; Burbridge et al. 2008).
As mentioned above, embryonic knockdown of Kiaa0319
disrupted neuronal migration when assessed 4--7 days after
transfection (Paracchini et al. 2006). Unknown were the long-
term effects, both in terms of neuronal migration and
subsequent neuronal morphology, of embryonic knockdown
or overexpression of this candidate dyslexia susceptibility
gene. In the current report, therefore, we examined the brains
of postnatal rats that were embryonically transfected by in
utero electroporation with plasmids containing 1) shRNA
targeted against Kiaa0319 (knockdown), 2) a construct
expressing KIAA0319 protein (overexpression), or 3) a combi-
nation of the shRNA and expression constructs (rescue). We
qualitatively assessed these brains for the presence or absence
of neuronal migration anomalies and the laminar disposition of
transfected and non--transfected neurons. In addition, we
quantified migration distances and neuronal morphology of
the transfected cells.
Experimental Procedures
In Situ Hybridization
In order to better interpret the knockdown and overexpres-
sion findings, we first determined the expression of Kiaa0319
in the prenatal brain by in situ hybridization. We obtained time-
mated pregnant females (Charles River Laboratory, Wilmington,
MA) and sacrificed the litters on E14/15, E16/17, or E19/20.
Three embryos from each litter were immediately frozen and
they were cut in either the horizontal, sagittal, or coronal plane
on a cryostat at 18 lm, and the slides were processed for in situ
hybridization of Kiaa0319 as described below.
The cDNAs prepared from frontal, parietal, and occipital lobes
of human embryonic brain (20 weeks, Biochain Institute,
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Cerebral Cortex Advance Access published August 13, 2009

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Hayward, CA) were used as template to amplify the full-length
coding sequence of Kiaa0319 using forward (5#ATGGCGCCC-
CCCACAGGTGTG3#) and reverse (5# TTATCTGTCCTTTGAG-
CAATAACTG 3#) primers. All fragments were then cloned into
pGEMT-Easy vector (Promega, Madison, WI). Rat embryonic
(E14) brain cDNA was synthesized from total RNA using
SuperScript III Reverse Transcriptase enzyme (Invitrogen, CA).
Full-length coding sequence of Kiaa0319 was amplified from
E14 cDNA using forward primer (5# ATGGTGTCCCCACCAG-
GAGTAC 3#) and reverse primer (5# TTATCTGTCCTTTGAG-
TAATAACCA 3#). The amplified product was cloned into
pGEMT-Easy vector. All the plasmids generated by PCR were
sequenced at WM Keck sequencing facility. Nonradioactive in
situ hybridizations were done by UB-In Situ (Natick, MA), as
previously described (Berger and Hediger 2001) using a digox-
igenin-labeled cRNA probe. The antisense and sense probes
were generated by using T7 and SP6 promoters flanking pGEMT-
Easy-Kiaa0319 of human and rat plasmids.
In Utero Electroporation
In utero electroporations of litters designated for postnatal
analysis were performed at the Beth Israel Deaconess Medical
Center. One litter that was analyzed in the prenatal period was
transfected at the University of Connecticut. The Institutional
Animal Care and Use Committees of these institutions
approved all procedures.
A total of 11 pregnant Wistar rats were obtained (Charles
River Laboratory) and each litter was assigned to 1 of 3 groups:
Kiaa0319 shRNA, KIAA0319 Overexpression, and Rescue.
Within each litter, pups were randomly assigned to receive 1
of 2 treatments (see Table 1). This balanced design was
essential for the analysis of migrational distance as it controlled
for between--litter variation in gestational age. In utero
electroporation of plasmid DNA was performed at E15/16 as
described previously (Bai et al. 2003; Rosen et al. 2007;
Burbridge et al. 2008). The concentration of enhanced green
fluorescent protein (eGFP) and monomeric red fluorescent
protein (mRFP) plasmids was 0.75 lg/lL, the shRNA was 1.5
lg/lL, and expression plasmids were 1.5 lg/lL.
Plasmids
For the Kiaa0319 shRNA condition, plasmids encoding shRNA
(pU6shRNA-Kiaa0319) and plasmids encoding eGFP (pCAGGS-
eGFP) were cotransfected into the ventricular zone (VZ). We
have previously demonstrated that cotransfection is highly
efficient, as virtually all neurons cotransfected with eGFP and
mRFP were colabeled when sacrificed 4 days post-transfection
(Rosen et al. 2007). Littermates were cotransfected with
plasmidsencodingascrambled
(pU6shRNA-Kiaa0319 scram) along with plasmids encoding
mRFP (pCAGGS-mRFP) and plasmid encoding eGFP. Pups in
the KIAA0319 overexpression group were cotransfected with
an IRES construct coding both for the human KIAA0319
versionofthe shRNA
protein and eGFP (pCAG-KIAA0319-IRES-eGFP) and pCAGGS-
mRFP, whereas their littermates were transfected with
pU6shRNA-Kiaa0319 + pCAGGS-eGFP. In the Rescue condition,
subjectswerecotransfected
pCAGGS-KIAA0319-eGFP,and
were transfected with pU6shRNA-Kiaa0319 + pCAGGS-eGFP.
The effectiveness of these plasmids in knocking down
exogenous Kiaa0319 function was validated by Western blot
(Supplemental Fig. 1).
with
pCAGGS-mRFP.
pU6shRNA-Kiaa0319,
Littermates
BrdU Injection
Pregnant dams at E18/19 were anesthetized with isoflurane (5%)
and intraperitoneally injected with 50 mg/kg of 5-bromo-2#-
deoxyuridine (Sigma Aldrich, St. Louis, MO, 10 mg/mL solution).
Histology
One litter transfected with Kiaa0319 shRNA was sacrificed 4
hours after BrdU, whereas the remaining litters were sacrificed
at P21. Animals were deeply anesthetized (Ketamine/Xylazine
10:1, 100 mg/mL) and sacrificed by transcardial perfusion with
0.9% saline followed by 4% paraformaldehyde. The brains were
removed from the skull and postfixed for 24 h before being
cryoprotected in 10% and then 30% sucrose phosphate buffer.
The brains were sectioned coronally at 40 lm on a freezing
microtome. Sections were then mounted and coverslipped
with VECTASHIELD Mounting Medium (Vector Labs, Burlin-
game, CA) and visualized under fluorescence for the presence
of eGFP and/or mRFP. One series of every tenth section was
stained for Nissl substance using Thionin. One adjacent series
of free-floating sections was processed for immunohistochem-
ical detection of eGFP (Chemicon, 1:200) using ABC protocols.
Immunohistochemistry
Adjacent series of sections were processed for immunofluo-
rescence detection of laminar markers. These included Cux1
(CDP (M-222), Santa Cruz Biotechnology, Santa Cruz, CA,
1:1000) and FoxP2 (FoxP2 (N--16), Santa Cruz Biotechnology,
1:50). Cux1 is a transcription factor that predominantly labels
layer 2--4 neurons, especially in the in parietal cortex (Nieto
et al. 2004), whereas FoxP2 labels neurons in layer 6
throughout the cortex (e.g., Keays et al. 2007). An antibody
for the connective tissue growth factor Ctgf (Heuer et al.
2003), (L-20 Santa Cruz Biotechnology, 1:50) was used to label
neurons in layer 6b (Molyneaux et al. 2007), and cells that
contained BrdU were labeled with Anti-BrdU (BD Bioscences,
San Jose, CA, 1:100). Primary antibodies were detected with
one of the following secondary antibodies: Alexa Fluor 555,
Alexa Fluor 594 (Invitrogen, Carlsbad, CA, 1:200), or Cy5
(Jackson ImmunoResearch, West Grove, PA, 1:50).
Additional sections were stained for gamma-aminobutyric
acid-ergic (GABAergic) antibodies Calretinin (MAB1468, Mil-
ipore Corp., Billerica, MA, 1:1000) and Parvalbumin (MAB353,
Millipore, 1:200). The presence of progenitor cells was assessed
by staining for Nestin (MAB1572, Millipore, 1:1000).
Table 1
Summary of treatments (N)
GroupTreatment 1Treatment 2
Kiaa0319 shRNA
KIAA0319, overexpression
Rescue
pU6shRNA-Kiaa0319 þ pCAGGS-eGFP (12)
pU6shRNA-Kiaa0319 þ pCAGGS-eGFP (8)
pU6shRNA-Kiaa0319 þ pCAGGS-eGFP (9)
pU6shRNA-Kiaa0319 scram þ pCAGGS-mRFP þ pCAGGS-eGFP (11)
pCAG-KIAA0319-IRES-eGFP þ pCAGGS-mRFP (7)
pU6shRNA-Kiaa0319 þ pCAG-KIAA0319-IRES-eGFP þ pCAGGS-mRFP (10)
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Analysis
In Situ Quantification
Individual sense and antisense sections from horizontally
prepared brains (1 each from E14/15, E16/17, and E19/20
rats) were imaged with monochrome digital camera (Insight,
Diagnostic Instruments, Sterling Heights, MI) on a light box
(Aristo Grid Lamp Products, Waterbury, CT) and interfaced via
firewire to Macintosh G4 computer (Apple Computer, Cuper-
tino, CA). Each antisense section image and its corresponding
sense section were captured using SPOT software (Diagnostic
Instruments)withcommon
ImageJ
<http://rsb.info.nih.gov/ij/>;, optical density values
were measured for the combined CP and VZ. A total of 9--13
sections were measured for each brain. The average difference
in optical density between sense and antisense images were
computed and expressed as a percent of sense density.
exposuresettings. Using
Postnatal Assessment of Pathology
All analyses of postnatal brains were performed blind with
respect to condition. Nissl-stained sections were surveyed for
the presence of neocortical and/or hippocampal malforma-
tions, and their location noted.
Migration Analysis
Quantitative analysis of migrational distance was conducted
using a custom Matlab (Mathworks, Natick, MA) program. The
location of eGFP+ cells was charted in 4 randomly chosen
immunohistochemically stained series using Neurolucida (MBF
Biosciences, Williston, VT). The program then determined the
location of each cell in a user--defined region of interest as the
percentage of cortical depth, with 0% being the white matter/
subplate border and 100% being the pial surface. Frequency
distributions were determined for each animal, and the mean
value across all animals within each condition was determined
(Supplemental Fig. 2). Differences in the distribution of
migrated neurons were assessed using ANOVA. Initial analysis
determined that there were no differences among the
Kiaa0319 shRNA-transfected groups (F2,24= 1.4, NS), and so
their data were pooled for all analyses.
Neuronal Morphology
Five neurons from each brain were randomly selected from all
laminar locations for morphological analysis. The cell body and
the extent of each apical and basal dendrite were traced with
Neurolucida (MBF Biosciences). Using Neurolucida Explorer
(MBF Biosciences), Sholl analysis and branch analysis (dendritic
length and numbers of nodes, ends, and dendrites) were then
performed, and a single measure derived for each animal (mean
value from 5 neurons for each of the dependent measures).
Statistical differences were determined by single factor and
repeated measures ANOVA. Initial analysis determined that
there were no differences among the Kiaa0319 shRNA--
transfected groups (F2,27< 1, NS) and so their data were
pooled for all analyses.
Image Processing
Fluorescent images were obtained on a confocal microscope
(Zeiss LSM 510 Meta, Carl Zeiss, Inc., Thornwood, NY).
Photomicrographs were adjusted for exposure and sharpened
(unsharp mask filter) using Adobe Photoshop (Adobe Inc., San
Jose, CA). Some brightfield images were acquired using the
Virtual Slice Module of Neurolucida. Image montages were
created in Canvas X (ACD Systems, Miami, FL).
Results
Kiaa0319 is Highly Expressed in a Regionally Distinct
Manner
In situ hybridization revealed that Kiaa0319 was expressed in
a regionally distinct manner (Fig. 1). At all ages examined, there
was increased expression in the VZ, intermediate zone (IZ),
CP, striatum, hippocampus, and brain stem. Expression in the
CP increased over time, and there was evidence of Kiaa0319
expression in migrating neurons (Fig. 1). Quantitatively, differ-
ences in optical density between the antisense and sense
probes ranged from 0.5-fold (brain stem) to 1.2-fold (striatum).
In addition, there was increased expression in the mitral cell
layer of the olfactory bulb at E18/19. In situ hybridizations
performed on E14.5 mouse embryos by GenePaint (www.
genepaint.org, GeneID = D130043K22Rik) and in adult mice by
theAllen BrainAtlas (www.brain-map.org,
D130043K22Rik) confirmed this, although expression in the
adult striatum was diminished compared that of the embryo.
GeneID
=
There are PVHs in Kiaa0319 shRNA--Transfected Subjects
Nissl- and eGFP-stained sections from each of the 57 animals
were examined for the presence of neuronal migration
anomalies, including molecular layer ectopias, hippocampal
dysplasias, and PVHs. There were molecular layer ectopias
associated with the embryonic injection site in half of the
animals in all conditions, but there was no evidence of separate
molecular layer malformations that suggested a migrational
disorder. There were hippocampal dysplasias in 3 of the 29
animals transfected with Kiaa0319 shRNA (Treatment 1 in
Table 1), which were identical to those previously reported
(Rosen et al. 2007; Burbridge et al. 2008).
All of the Kiaa0319 shRNA--transfected animals had
evidence of disruption of neuronal migration to the neocortex
(Fig. 2A). Transfected neurons were seen throughout the
neocortex, with the highest concentrations at the border with
the white matter and in layer 2/3. In approximately 75% of the
cases, there were PVHs (Fig. 2E--H) that were visible for much
of the rostral--caudal extent of the brain. In contrast, there were
few unmigrated neurons in the Kiaa0319 scram and KIAA0319
overexpression conditions (Fig. 2B, C). One animal in the scram
condition did have a small collection of neurons at the white
matter border that resembled a PVH, but the frequency and
severity of these malformations were significantly greater in the
Kiaa0319 shRNA condition (v2= 91.2, df =1, P <0.001). There
were no malformations in the brains of any animals in the
KIAA0319 overexpression condition.
In order to determine the specificity of the shRNA for
Kiaa0319 we cotransfected animals with a plasmid encoding
human KIAA0319 along with the Kiaa0319 shRNA plasmid.
Human KIAA0319 nucleotide sequence does not match rat
Kiaa0319 sequence in the region targeted by the Kiaa0319
shRNA, and therefore it is not susceptible to RNAi. Of the rats
simultaneously transfected with Kiaa0319 shRNA and the
human KIAA0319 expression construct, 4 out of 10 had small,
focal regions of PVHs, which were not as extensive as those
seen in the Kiaa0319 shRNA treatment condition (Fig. 2D).
The remainder had no obvious malformations (Fig. 2C, D).
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There was a significant difference in the number of PVHs (v2=
4.3, df = 1, P < 0.05), which suggests that overexpressing the
human KIAA0319 protein in Kiaa0319 shRNA-treated rats at
least partially rescued this phenotype, and indicates that the
effects of the RNAi are not due to off-target effects.
We assessed the PVH for the presence of Nestin-positive
progenitor cells. There was no evidence of progenitor cells
within the PVH, but there was a marked increase of Nestin-
positive fibers (Fig. 3). Thus, although there were sparse
Nestin-positive fibers contained within homologous regions of
the nontransfected hemisphere (Fig. 3E), there were dense
collections of these fibers within the PVH (Fig. 3D) that
resembled radial glial fibers. Some of fibers could be seen to
invade the upper layers of the cerebral cortex. These results
suggest that there is an undue preservation of radial glial-like
morphology within the PVH.
There are Non--Cell Autonomous Effects of Embryonic
Kiaa0319 Knockdown
In order to assess whether embryonic transfection with
Kiaa0319 shRNA affects the normal laminar position of
neurons, we stained Kiaa0319 shRNA-transfected brains with
laminar markers. Staining with Cux1, a marker of layer 2--4
neurons in parietal and other medial cortices, revealed
a number of Kiaa0319 shRNA--transfected neurons in both
the PVHs and layer 2/3 that were colabeled with Cux1. In
contrast, staining for Ctgf, a marker of layer 6b neurons, did not
disclose any colabeled neurons (Fig. 4). Similarly, staining with
the layer 6 marker Foxp2 did not reveal any transfected cells
that were colabeled (Fig. 5). These results suggest that PVHs
and some of the deeper cortical areas contain neurons that are
normally destined for supragranular layers.
Intriguingly, there were large numbers of Cux1+ neurons in
the PVHs that were not colabeled with eGFP (indicating that
they were not transfected with Kiaa0319 shRNA) that failed to
migrate (Fig. 4). This suggested that there were non--cell
autonomous effects of transfection with Kiaa0319 shRNA.
Alternatively, it could be that these Cux1+ neurons were
originally transfected with Kiaa0319 shRNA + eGFP, and that
the fluorescent protein had been subsequently lost from the
cells. In order to test this alternative, we examined the brains of
animals transfected with Kiaa0319 shRNA + eGFP on E15/16
and subsequently injected with BrdU at E18/19. We first
assessed the distribution of BrdU+ and eGFP+ cells following
a 4-h post-BrdU injection interval (Fig. 6A--F). We found that
there were very few colabeled cells, indicating that cells
Figure 1. In situ hybridization of Kiaa0319 in embryonic rat brains. Photomontages of in situ hybridization of Kiaa0319 antisense probes in E14/15 (A,D), E16/17 (B,E), and E19/
20 (C,F) rat embryos. (G) High power photomicrographs of developing cerebral cortex indicating CP, subventricular zone (SVZ) and VZ/IZ. Kiaa0319 is expressed highly in the CP,
striatum, and hippocampus at all ages. There is increased expression in the mitral cell layer of the olfactory bulb at E19/20, and generalized moderate expression in the brain stem
at all ages. Bar in panels A--F 5 1 mm. Bar in panel G 5 100 lm.
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labeled by BrdU at E18/19 were largely nontransfected cells
(Fig. 6C,F; Supplemental Movies 1 and 2), and that the BrdU+
and eGFP+ populations belong to distinct populations. We then
examined similarly treated animals in the postnatal period, and
examined for the colocalization of BrdU+, Cux1+, and eGFP+
neurons in layer 2/3 and the PVH (Fig. 6G--N).
As expected, there were large numbers of neurons colabeled
for BrdU and Cux1 as well as Cux1 and eGFP colabeled cells in
layer 2. However, there were no neurons colabeled with eGFP
and BrdU. In the PVH, the results were identical to those
described in Figure 5. Specifically, there were large numbers of
Cux1+ neurons that were not colabeled with eGFP. Moreover,
there were large numbers of E18/19 BrdU+ neurons in the PVH
and none of these were colabeled with eGFP (although some
coexpressed Cux1). Taken together, these results indicate that
the nontransfected neurons in the PVH did not lose the eGFP
label, but rather represent non--cell autonomous effects of
Kiaa0319 knockdown in the developing rat brain.
We sought further confirmation of the non--cell autonomous
effects of embryonic knockdown of Kiaa0319 by staining for
GABAergic interneurons in the PVH using antibodies against
Calretinin and Parvalbumin. GABAergic interneurons are
generated in the medial ganglionic eminence, and are therefore
not likely to have been transfected following in utero electro-
poration. Thus, the presence of positive immunoreactive cells
in the PVH would argue strongly for non--cell autonomous
effects. We found both Calretinin- and Parvalbumin-positive
interneurons within the PVH (Fig. 7), which supports the
notion that embryonic transfection with shRNA targeted
against Kiaa0319 has non--cell autonomous effects on both
radially migrating and tangentially migrating neurons.
Altered Expression of KIAA0319 Disrupts the Laminar
Position of Pyramidal Neurons
Previously, we demonstrated a distinctive pattern of migrational
disturbance following transfections with shRNA targeted against
Dyx1c1 or Dcdc2. Specifically, we found a bimodal pattern of
migration, with approximately 7--20% of the transfected neurons
not migrating past the cortical--white matter border, and the
remaining neurons ‘‘overmigrating’’ past their expected laminar
locations (Rosen et al. 2007; Burbridge et al. 2008). In the
current experiment, we sought to determine whether this
overmigration phenotype occurred following either knockdown
of Kiaa0319 or overexpression of its protein.
Of the 57 animals, 4 were excluded from this analysis
because the transfections were outside the main region of
Figure 2. Neuronal migration following embryonic knockdown or overexpression of Kiaa0319. (A--D) Position of transfected neurons in 2 representative sections from brains
embryonically transfected with plasmids containing Kiaa0319 shRNA (A), scrambled Kiaa0319 shRNA (B), KIAA0319 protein (C), or Kiaa0319 shRNA along with KIAA0319
protein (D). (E) Photomicrograph of cerebral cortex of Nissl-stained section illustrating region of PVH (arrows). This animal was embryonically transfected with Kiaa0319 shRNA þ
eGFP. Bar 5 250 lm. (F) Photomicrograph of section adjacent to Panel E immunohistochemically stained for eGFP. Transfected neurons are located within the PVH. Bar 5 250
lm. (G and H) High-power photomicrograph of PVH (arrows) illustrated in panels (E) and (F). Bar 5 125 lm.
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analysis (somatosensory cortex). The results of the migration
distance analysis for all groups are summarized in Figure 8.
Virtually, all (98.4%) of the neurons in the Kiaa0319 scram
(control) transfected group migrated into the neocortex, with
the majority peaking in layer 2/3, at approximately 75% of the
cortical depth, with an average of 69.8 ± 1.0%. In comparison,
10% of the Kiaa0319 shRNA—transfected cells remained at
the cortical/white matter border, which is significantly
different from the Kiaa0319 scram group (F1,36= 9.1, P <
0.01). Interestingly, the distribution of those neurons that did
migrate is virtually identical to that of the Kiaa0319 scram
group, with an average migration distance of 68.8 ± 1.0%
(F1,36< 1, NS). Thus, although the bimodal migration distance
phenotype is consistent among all 3 candidate dyslexia
susceptibility genes examined, unlike Dcdc2 and Dyx1c1
(Rosen et al. 2007; Burbridge et al. 2008) there is no
overmigration phenotype following knockdown of Kiaa0319.
Virtually all (98.7%) of the neurons transfected with the
KIAA0319 expression construct migrated into the neocortex,
with the major peak at lower layer 2/3 at a cortical depth of
65%, (average = 63.6 ± 2.5%). This average migration distance
differs from the Kiaa0319 scram group (F1,16= 5.1, P < 0.05),
suggesting that KIAA0319 overexpression disrupts the normal
migration of neurons. Of the neurons that were co--transfected
with Kiaa0319 shRNA and KIAA0319 expression construct
(the rescue condition), 5.5% did not migrate in the CP. This
percentage of unmigrated cells is significantly smaller than that
in the Kiaa0319 shRNA group (F1,33= 5.6, P < 0.05), which
indicates that the presence of the KIAA0319 protein rescues
the ‘‘nonmigration’’ phenotype. Interestingly, the migration
distance for the rescue condition peaks at the same point as the
KIAA0319 expression vector alone group, at around 65% of the
cortical depth, with an average of 63.3% ± 1.0. This differs
significantly from the Kiaa0319 scram group (F1,17= 12.6, P <
0.01), and does not significantly differ from the KIAA0319
overexpression group (F1,13< 1, NS). Taken together, the
evidence suggests that neurons that are transfected with the
KIAA0319 expression construct (both the overexpression and
rescue groups) tend to migrate to lower laminar positions than
would be expected for normal cohort of migrating cells. As the
KIAA0319 expression construct used to rescue is insensitive to
complete knockdown by the Kiaa0319 shRNA this would
suggest that overexpression of KIAA0319 alters the position of
cells within cortical lamina. It is not yet clear whether this shift
Figure 3. Nestin-positive fibers located in PVH in the brain of a rat embryonically transfected with Kiaa0319 shRNA. (A) Brightfield photomontage of Nissl-stained section
showing PVH (small arrows). This section also has an ectopic collection of neurons in the molecular layer, which is an artifact of the injection (large arrow). Bar 5 500 lm. (B)
Brightfield photomontage of section adjacent to (A) immunohistochemically stained for GFP illustrating PVH (arrows). Bar 5 250 lm. (C) Brightfield photomontage of section
adjacent to (B) (arrows for orientation with B) immunohistochemically stained for Nestin. Nestin stains fibers in the PVH as well as blood vessels throughout the brain. Box
indicates region illustrated in (D). Bar 5 250 lm. (D) High-power brightfield photomicrograph of region denoted in (C) illustrating dense plexus of Nestin-positive fibers (arrows).
Bar 5 25 lm. (E) High-power brightfield photomicrograph of region homologous to that in (D). Compared with (D), there are far fewer Nestin-positive fibers (arrows). (F) (Mall
arrows), blood vessels (large arrows), and eGFP-positive neurons (arrowheads) present. There are no cells that are colabeled. Bar 5 100 lm.
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in position by overexpression of KIAA0319 results from
impairment of migration or from impairment of laminar sorting,
which could result from changes in cell adhesion.
Knockdown of Kiaa0319 Causes Increased Arborization
of Apical Dendrites
The results from the quantitative analysis of dendritic
arborization are summarized in Figure 9. We analyzed the data
from the Sholl analysis (Fig. 9A,B) using a repeated measures
ANOVA, with dendrite length within each 10-lm concentric
circle as a dependent measure, treatment groups as the
independent measure, and the concentric circles (‘‘bins’’) as
the repeated measure. For apical dendrites, there were
significant main effects for both treatment group (F3,52= 5.7,
P < 0.01) and bins (F332,4316= 171.2, P < 0.001), as well as
a significant bins 3 treatment group interaction (F249,4316 =
6.33, P < 0.001). We further analyzed the treatment group
main effect, and found that the Kiaa0319 shRNA group
significantly differed from each of the other 3 groups (shRNA
vs. Overexpression: F1,35= 9.3, P < 0.01; shRNA vs. Rescue:
F1,37= 8.9, P <0.01; shRNA vs. Scram: F1,38= 4.3, P <0.05). This
indicated that there was significant dendritic hypertrophy in
neurons embryonically transfected with Kiaa0319 shRNA. In
contrast, there was no significant difference among the 4
treatment conditions for basal dendrite length (F3,52= 2.0, NS),
although there were significant effects for bins (F48,2496 =
351.2, P < 0.001) and bin 3 treatment group interaction
(F144,2496= 2.5, P < 0.001). These results suggest a trophic
effect on apical, but not basal, dendritic arborization caused by
Kiaa0319 interference.
In order to dissect the components of the dendritic tree that
differed among the treatment groups, we analyzed the number
of dendrites (for basal only), nodes, ends, and total length (Fig.
9C). For apical dendrites, we found that the Kiaa0319 shRNA--
transfected neurons had more ends, nodes, and longer
dendritic length than neurons in the Rescue group (F1,35=
7.5, P < 0.01; F1,35 = 8.0, P < 0.01; F1,35 = 7.1, P < 0.05,
respectively). In addition, we found that there were more ends
(F1,37= 7.6, P < 0.01) and nodes (F1,37= 7.8, P < 0.01) in the
Kiaa0319 shRNA group when compared with the Kiaa0319
scram group. There were no significant differences in any of
these apical dendrite measures between neurons embryoni-
cally transfected with Kiaa0319 shRNA neurons and those
transfected with the KIAA0319 expression construct. More-
over, there were no significant differences among any of the
basal dendrite measures. Taken together, these results support
the Sholl analysis, and indicate that knockdown of Kiaa0319
results in apical, but not basal, dendritic hypertrophy.
Figure 4. Confocal microscopy of lamina specific markers Cux1 and Ctgf in the brain of rats embryonically transfected with shRNA targeted against Kiaa0319. The white line
delineates the border between a PVH and the white matter. The 4 panels illustrate cells transfected with eGFP (A), cells immunopositive for the upper lamina specific marker Cux1
(B), cells immunopositive for the subplate specific marker Ctgf (C) and a merged panel (D). There are eGFP (shRNA--transfected) cells that are Cux1 positive (straight sided
arrowheads), and some that are not (concave arrowheads). There are, in addition, a large number of Cux1-positive cells that are not colabeled with eGFP (arrows). This suggests
that these nontransfected cells arrive in the PVH by non--cell autonomous mechanisms. There are no cells that colabel with Ctgfþ cells. Bar 5 100 lm.
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Discussion
Previous reports had demonstrated that embryonic transfection
with shRNA targeted against the rat homolog of the candidate
dyslexia susceptibly gene Kiaa0319 resulted in significant
arrest of neuronal migration when assessed 4 days following
transfection, with the majority of transfected cells remaining in
the VZ (Paracchini et al. 2006). The current experiment
indicates that this disruption of neuron migration causes
specific types of neuronal migration anomalies in the postnatal
brain—specifically PVHs and abnormal laminar locations, and
embryonically transfecting neurons with both the Kiaa0319
shRNA and KIAA0319 expression construct rescues this
phenotype. In contrast, overexpressing the KIAA0319 protein
does not cause gross neuronal migration disorders.
In humans, PVHs and other anomalies of neuronal migration
are relatively easy to visualize with computed tomography and
magnetic resonance imaging and are confirmed post mortem
using standard histological techniques. It has been hypothe-
sized, however, that these malformations may simply act as
flags for other, more widespread, disturbances in neocortical
organization. For example, disturbances in white matter con-
nectivity associated with neuronal migration disorders have
been revealed using diffusion tensor imaging in humans (Lee
et al. 2005; Huppi and Dubois 2006), and severe disruptions in
connectivity have been demonstrated in various animal models
of neuronal migration disorders (Giannetti et al. 1999, 2000;
Jenneretal.2000;Rosenetal.2000).Moreover,theabundanceof
radial glia-like morphology in Nestin-positive fibers within the
PVH, supports the existence of a more widespread disturbance
in neocortical organization. Interestingly, this maintenance of
radial glia--like morphology is remarkably similar to that seen
following induction a malformation resembling microgyria via
a P1 freezing lesion, where Nestin-positive fibers were seen in
the microgyria well into adulthood (Rosen et al. 1994).
Inducing neuronal migration anomalies by embryonic trans-
fection with shRNA has enabled us to identify disruptions of
laminar positioning that would otherwise not be visualized. In
the current experiment, we have demonstrated that following
embryonic transfection with Kiaa0319 shRNA, Cux1+ and
E18/19 BrdU+ neurons that are normally destined for layer 2/3
Figure 5. Distribution of the lower lamina specific marker Foxp2 in the brain of a rat embryonically transfected with Kiaa0319 shRNA. (A) Photomicrograph of cerebral cortex of
Nissl-stained section illustrating region of PVH (arrows). This animal was embryonically transfected with Kiaa0319 shRNA þ eGFP. Bar 5 500 lm. (B) Photomicrograph of
section adjacent to panel (A) immunohistochemically stained for eGFP. Transfected neurons are located within the PVH. Bar 5 500 lm. (C, D, and E) Confocal microscopic
images of PVH (white lines) seen in (A) and (B). The 3 panels illustrate cells transfected with eGFP and Kiaa0319 shRNA (C), cells immunopositive for the lower lamina specific
marker Foxp2 (D), and a merged panel (E). There are no cells double labeled for eGFP and Foxp2, nor are there any Foxp2 labeled cells in the PVH. This suggests that lower lamina
cells are not disrupted by embryonic transfection with Kiaa0319 shRNA. Arrow in (C) is for orientation. Box in each panel indicates region magnified in panels (F, G, and H). Bar 5
200 lm. (F, G, and H) Magnified regions of panels (C, D, and E), respectively. Bar 5 100 lm.
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collect in PVHs. Although some of these neurons were
transfected with Kiaa0319 shRNA, there were large numbers
that were not. This replicates and extends previous reports,
where we found that embryonic transfection with shRNA
targeted against the rat homologue of the candidate dyslexia
susceptibility gene Dcdc2 resulted in nontransfected Cux1+
neurons in the PVH (Burbridge et al. 2008).
It could be argued, however, that the Cux1+ and BrdU+
neurons that are eGFP negative, were in fact transfected with
Kiaa0319 shRNA but that the cell simply stopped expressing
eGFP in the postnatal period. To test this hypothesis, we
examined animals that received E15/16 transfections of
Kiaa0319 shRNA, an E18/19 injection of BrdU, and were then
sacrificed 4 hours later. In these animals, the eGFP+ and BrdU+
cells made up separate and unique populations—there were
few cells that were double labeled (Fig. 6A--F). When we
examined the brains of identically treated animals in the
postnatal period, then, any BrdU+ neuron was not transfected
with Kiaa0319 shRNA + eGFP. Because we found large
numbers of BrdU+ neurons in the PVH, this strongly supports
the hypothesis that these cells failed to migrate due to non--cell
autonomous mechanisms. This argument is further bolstered
by the presence of GABAergic interneurons in the PVH.
Because these neurons are not generated in the VZ (Anderson
et al. 2001), it is unlikely that they were transfected with
Kiaa0319 shRNA + eGFP. The presence, therefore, of
Parvalbumin- and Calretinin-positive interneurons, which are
not colabeled with eGFP in the PVH, strongly supports the
notion that there are non--cell autonomous effects of embry-
onic transfection with Kiaa0319 shRNA.
The results reported here are similar to previous reports that
examined the pre- and postnatal phenotypes associated with
embryonic transfection of candidate dyslexia susceptibility
genes. Thus, embryonic knockdown of Dcdc2 (Meng et al.
Figure 6. Non--cell autonomous effects of Kiaa0319 shRNA transfection. Distribution of cells in the border between the subventricular zone (SVZ) and the IZ (A--C) and the VZ/
SVZ border (D--F) 4 h after an injection of BrdU at E18/19 in rats transfected at E15/16 with Kiaa0319 shRNA þ eGFP. There are few cells double labeled for eGFP and BrdU
(arrow), indicating that these are 2 separate populations. Z-axis movie of panels C and F are available as supplements. Bar 5 20 lm. (G--N) Distribution of Cux1- and E18/19
BrdUþ cells in the layer 2/3 (G--J) and the PVH (delineated by dashed white lines; K--N) of a rat embryonically transfected with Kiaa0319 shRNA þ eGFP at E15/16. There are
large numbers of eGFPþ (G and K), Cux1þ (H and L), and BrdUþ (I and M) cells in both layer 2/3 and the PVH. A significant subset of cells in both in layer 2 and the PVH are
both Cux1þ and BrdUþ. These are seen as purple cells in the merged panels (J and N; arrows). As in Figure 3, there is another subset of cells that colabel for both eGFP and
Cux1 (yellow cells in the merged panel, arrowheads). There are, however, no cells colabeled for eGFP and BrdU in either layer 2/3 or the PVH. The lack of double-labeled cells in
the PVH support the notion that there are non--cell autonomous effects of embryonic transfections with Kiaa0319 shRNA. Bar 5 200 lm.
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2005; Paracchini et al. 2006; Burbridge et al. 2008) and Dyx1c1
(Wang et al. 2006; Rosen et al. 2007) disrupted neuronal
migration. As with Kiaa0319 embryonic knockdown, the
majority of brains transfected with Dcdc2 or Dyx1c1 shRNA
develop PVHs, although Dyx1c1 and Kiaa0319 knockdown
groups have more extensive malformations. We did find
hippocampal malformations following embryonic transfections
with shRNA targeted against Kiaa0319, but the incidence was
smaller than that associated with knockdown of either Dyx1c1
or Dcdc2. As with the case of Dcdc2 transfected brains, we did
not find any evidence of molecular layer ectopias in the current
study, whereas they were present in about 25% of the brains
transfected with Dyx1c1 shRNA.
Although embryonic knockdown of the 3 candidate dyslexia
susceptibility genes always resulted in a population of
unmigrated neurons, the disposition of those neurons that
did migrate into the cerebral cortex differed. In the case of the
previous reports, the peak locations of transfected neurons in
the cerebral cortex were superficial to their expected lamina
(Rosen et al. 2007; Burbridge et al. 2008). This was not the case
in the present report, as neurons embryonically transfected
with Kiaa0319 shRNA were distributed in the cerebral cortex
identically to control animals transfected with the scrambled
version of the Kiaa0319 shRNA. In contrast, we did find
disruptions of laminar position following overexpression of
KIAA0319, with transfected neurons migrating to positions
below their expected location. This was true both for the
overexpression and ‘‘rescue’’ group, which were simulta-
neously transfected with the overexpression construct and
Kiaa0319 shRNA. This ‘‘undermigration’’ of neurons trans-
fected in both conditions could either represent the effects of
overexpressing the protein, or could be due to the effect of the
human protein on the migrating neurons. Because we lack an
effective antibody against the protein, we cannot directly
distinguish between these 2 possibilities.
Figure 7. GABAergic interneurons are present in PVHs. Distribution of Calretinin-positive (A--C) and Parvalbumin-positive (E--F) interneurons in a PVH (dashed white lines) from
a brain embryonically transfected with Kiaa0319 shRNA. There are no neurons that colabel for eGFP (arrowheads, neurons that were transfected with Kiaa0319 shRNA) and
either of the 2 GABAergic antibodies (arrows). Bar 5 100 lm.
Figure 8. Migration distance analysis following embryonic knockdown and/or
overexpression of Kiaa0319. Quantitative migration analysis of the percent of neurons
migrating (X-axis) against the normalized depth of the cerebral cortex. Photomicro-
graph of cerebral cortex is included as an aid for laminar delineation. There are
significantly more unmigrated neurons in the shRNA group (blue) when compared
with the other 3 groups. There is no difference in the upper layer migration between
the Kiaa0319 shRNA and Kiaa0319 scrambled (green) groups, nor is there
a difference between the Overexpression (red) and Rescue (light blue) groups.
Neurons in brains embryonically transfected with KIAA0319 protein migrate below
their expected laminar position based on comparison with the scrambled group.
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Figure 9. Neuronal morphology and quantitative analysis following embryonic knockdown and/or overexpression of Kiaa0319. (A--D) Representative tracing of neurons
embryonically transfected with plasmids expressing Kiaa0319 shRNA (A), KIAA0319 protein (B), scrambled Kiaa0319 shRNA (C), and both Kiaa0319 shRNA and KIAA0319
protein (D). (E) Sholl analysis of apical dendrites for each condition. The dendrites of Kiaa0319 shRNA--transfected neurons are longer within 200 lm of the cell body when
compared with the other 3 groups. (F) Sholl analysis of basal dendrites for each condition reveals no significant differences among the 4 groups. (G) Quantitative analysis of
specific dendritic features confirms the Sholl analysis. Apical dendrites of neurons embryonically transfected with Kiaa0319 shRNA have more nodes, ends, and overall length
when compared with the scrambled and rescue groups. In contrast, there are no significant differences in any of these measures in the basal dendrites. There is no difference in
the number of basal dendrites. *Differs from Kiaa0319 shRNA, P \ 0.05.
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It is possible that changes in the methods of quantification
may explain the disparity between the previous reports and the
current experiment. In the original 2 reports, migration
distance of transfected neurons was assessed by determining
the number of neurons in successive deciles in a 250-lm-wide
sampling grid. In the current experiment, the actual distance
that each neuron migrated (as a percent of the total depth of
the cortex) was recorded (Supplemental Fig. 2). It could be,
then, that the results from the previous reports resulted from
undersampling. We have reanalyzed the data from one of these
experiments using the present methodology (Burbridge et al.
2008), and found that the results were identical to the previous
report (unpublished observations). It is therefore likely that the
results reported here reflect a real difference in the postnatal
phenotypes between Kiaa0319 and the other candidate
dyslexia susceptibility genes.
There is little currently known concerning the functions of
these genes, but what is known suggests that they may well
disrupt neuronal migration by distinct mechanisms. DCDC2 is
one of a group of proteins distinguished by the presence of
tandem or single dcx domains, which are critical for binding and
stabilizing microtubules (Allen et al. 1998; des Portes et al. 1998;
Gleeson et al. 1999; Graham et al. 2004; LoTurco 2004; Reiner
et al. 2004; Schaar et al. 2004). The function of Dyx1c1 is not
well known, but previous reports suggest that Dyx1c1 is
localized in the cytoplasm along microtubules as well (Wang
et al. 2006). KIAA0319, on the other hand, is a gene that codes
for an integral membrane protein with a large glycosylated
(Velayos-Baeza et al. 2008) extracellular region containing
multiple PKD domains, a single transmembrane domain, and
a small cytoplasmic C-terminus. Recently 3 KIAA0319 splice
variants have been identified and one of these codes for
a secreted protein (Velayos-Baeza et al. 2008). We do not yet
know which splice variant may be responsible for function in
migration or dendritic differentiation, however future experi-
ments using different splice variants to rescue the RNAi
phenotypes described here can now be used to distinguish
among their functions. The full-length KIAA0319 may be
involved in cellular adhesion, as PKD domains in polycistin--1
are necessary for adhesion of renal epithelial cells (Wilson 2001).
Consistent with a function in cell adhesion in developing cortex,
Parrachini et al. (2006) showed a change in the relationship
between radial glia and migrating neurons following interfer-
ence of Kiaa0319 in embryonic neocortex. Similarly, the
significant non--cell autonomous component of migration
disruption we show here is consistent with a role for the
secreted splice variant of Kiaa0319 (Velayos-Baeza et al. 2008).
Prior to the current experiment, the effects of embryonic
knockdown and overexpression of candidate dyslexia suscep-
tibility genes on neuronal morphology had not been addressed.
Here we find that transfection with Kiaa0319 shRNA results in
hypertrophy of apical, but not basal, dendrites when compared
with the other 3 conditions. It could be that our results could
be affected by biases in the orientation of the sections, the
laminar positions of the neurons being measured, or in the
relatively narrow section thickness. There was no difference in
the laminar distribution of neurons that were measured
between the groups, and the analysis of the subsection of
those neurons in the upper laminae were identical to those
from the entire population. Our results could be explained by
biased sectioning only if the orientations of Kiaa0319 shRNA--
transfected neurons were systematically different than those of
neurons in the other groups. We have no evidence of
differential orientation between the experimental groups.
The relatively narrow section thickness does result in a less
elaborate dendritic branching when compared with, for
example, biocytin injection. Although there is no evidence of
systematic bias between the groups, replication of these results
in thicker sections of biocytin-injected neurons would be
warranted. Further confirmation of the effect of Kiaa0319
expression on dendritic outgrowth awaits experiments in
cultured neurons.
The mechanisms underlying the dendritic differences are
not known, but could reflect direct effects of Kiaa0319 on
both neuronal migration and dendritic outgrowth. Along these
lines, the relationship between neuronal migration disorders
and dendritic hypertrophy has been previously established.
Takashima et al. (1991) reported the coexistence of hemi-
megalencephaly, a disorder of neuronal migration, and den-
dritic hypertrophy in 6 post mortem patients. More recently,
PTEN deficient mice—a model for macrocephaly and Lher-
mitte--Dudos disease—exhibit both ectopic neurons and
hypertrophic dendrites in the cerebral cortex (Kwon et al.
2006). The p21-activated kinase (Pak1), a cytoskeletal regula-
tor, has significant effects on neuronal migration, elaboration of
axons and dendrites, and the formation of dendritic spines
(Banerjee et al. 2002; Causeret et al. 2009), and Lis-1 deficient
mice have hippocampal migration disorders as well as altered
dendritic arborization (Fleck et al. 2000). ROBO1, a candidate
dyslexia susceptibility gene, plays important roles in both
neuronal migration and process outgrowth (Long et al. 2004;
Hannula-Jouppi et al. 2005; Andrews et al. 2008). Alternatively,
it could be that the changes in the elaborations of the dendrites
are secondary to the neuronal migration disorder that is
induced by Kiaa0319 knockdown. For example, long-term
potentiation has been shown to increase various measures of
dendritic morphology in the cerebral cortex (Monfils et al.
2004), and it is well known that damage to the developing brain
can affect dendritic arborization in connected regions (Marin-
Padilla 1997). One way to distinguish between these possibil-
ities would be to assess dendritic morphology of both trans-
fected and nontransfected neurons in the same brain. Future
experiments involving sequentially transfecting pups will
enable this question to be directly addressed. Alternatively,
one could conditionally re-express Kiaa0319 in animals
embryonically transfected with Kiaa0319 shRNA after the
completion of neuronal migration and during the dendritic
differentiation (P7--P21).
In summary, we have demonstrated that embryonic knock-
down of the candidate dyslexia susceptibility gene homolog
Kiaa0319 results in a neuronal migration disorder and
hypertrophy of apical dendrites. Both cell autonomous and
non--cell autonomous mechanisms appear to play a role in the
formation of the neuronal migration disturbances, but their
relative contribution to apical dendritic hypertrophy is not
known. Overexpression of KIAA0319 does not produce PVH or
other gross disturbances in neuronal migration, but does act to
limit the distance that neurons migrate into the CP. At present,
all of the candidate dyslexia susceptibility genes whose
functions have been investigated have been found to play a role
in neuronal migration. This is the first demonstration that one
of these genes has an effect on subsequent neuronal
morphology, which may provide another biological substrate
for the functional differences seen in this disorder. Whether
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other dyslexia susceptibility genes have similar effects on
neuronal morphology is not known, but will provide a fruitful
avenue for future investigations.
Supplementary Material
Supplementary material can be found at: http://www.cercor.
oxfordjournals.org/.
Funding
National Institutes of Health (HD20806) to A.M.G., J.J.L., and
G.D.R; and Department of Defense (DoD) through the National
Defense Science & Engineering Graduate Fellowship (NDSEG)
Program and Fannie and John Hertz Foundation/Myhrvold
Family fellowship supported Z.W.G.
Notes
The authors thank Ankur Thomas for the validation of the plasmids. The
authors thank the reviewers for their helpful comments on a previous
version of the manuscript. Conflict of Interest: None declared.
Address correspondence to Glenn D. Rosen, PhD, Department of
Neurology, E/CLS-643, Beth Israel Deaconess Medical Center, 330
Brookline Ave., Boston, MA 02215, USA. Email: grosen@bidmc.harvard.
edu.
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