Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia
Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in ‘Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315–1325, 1998; Sheen et al. in ‘Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69–76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in ‘MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789–801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventicular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity.
Disruption of neural progenitors along the
ventricular and subventricular zones in
Russell J. Ferland
, Luis Federico Batiz
, Jason Neal
, Gewei Lian
, Elizabeth Bundock
, Yi-Chun Hsiao
, Rachel Diamond
, Davide Mei
, Alison H. Banham
, Philip J. Brown
Charles R. Vanderburg
, Jeffrey Joseph
, Jonathan L. Hecht
, Rebecca Folkerth
, Christopher A. Walsh
, Esteban M. Rodriguez
and Volney L. Sheen
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,
Troy, NY 12180, USA,
The Wadsworth Center, Albany, NY 12201, USA,
Instituto de Anatomı
a, Universidad Austral de Chile, Valdivia, Chile,
Department of Neurology and,
Neuropathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA,
Department of Neuropathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA,
Advanced Tissue Resource Center, Center for Molecular Pathology, Harvard Center for Neurodegeneration and
Repair, Massachusetts General Hospital, Boston, MA 02114, USA,
Department of Child Neurology and Psychiatry,
University of Pisa and IRCCS Fondazione Stella Maris 56018, Calambrone, Pisa, Italy,
Nufﬁeld Department of
Clinical Laboratory Sciences, University of Oxford, Level 4 Academic Block, John Radcliffe Hospital, Oxford,
Oxfordshire OX3 9DU, UK and
Howard Hughes Medical Institute and Division of Genetics, Children’s Hospital,
Harvard Medical School, Boston, MA 02115, USA
Received October 13, 2008; Revised and Accepted November 5, 2008
Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along
the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-
ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in ‘Mutations in ﬁlamin 1 pre-
vent migration of cerebral cortical neurons in human periventricular heterotopia’. Neuron, 21, 1315–1325,
1998; Sheen et al. in ‘Mutations in ARFGEF2 implicate vesicle trafﬁcking in neural progenitor proliferation
and migration in the human cerebral cortex’. Nat. Genet., 36, 69–76, 2004). Recent studies have shown
that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with
FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in ‘MEKK4 signaling regulates ﬁla-
min expression and neuronal migration’. Neuron, 52, 789–801, 2006). Here we show that neurons in post-
mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily
composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in
the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal
zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with
hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attach-
ment protein alpha (a-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to
periventicular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafﬁcking
and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafﬁcking
of transport vesicles. Our current ﬁndings suggest that PH formation arises from a ﬁnal common pathway
involving disruption of vesicle trafﬁcking, leading to impaired cell adhesion and loss of neuroependymal
The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors.
To whom correspondence should be addressed. Tel: þ1 6176674078; Fax: þ1 6176677919; E-mail: email@example.com
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Human Molecular Genetics, 2009, Vol. 18, No. 3 497–516
Advance Access published on November 7, 2008
Impairments in neuronal migration, particularly in cell motility,
are thought to give rise to several genetic malformations of the
developing cortex: lissencephaly (smooth brain), subcortical
band heterotopia (heterotopic neurons arrested under the
normal cerebral cortex) and periventricular heterotopia (PH;
nodules of neurons lining the lateral ventricles). In each of
these disorders, early post-mitotic neuroblasts fail to migrate
(or have impairments in migration) from the ventricular zone
(VZ) into the cortical plate. These impairments in neuronal moti-
lity are thought to give rise to a thickened cortex with loss of
normal cortical lamination, in lissencephaly (1,2); an abnormal
layer of neurons below a normal cortex, in subcortical band
heterotopia (3–5); and nodules of neurons along the lateral
ventricles and beneath a normal-appearing cortex, PH (6,7).
Understanding the genetic mechanisms underlying impaired
neuronal motility have aided in elucidating the causes of many
human cortical malformations. Neurons harboring a hetero-
zygous LIS1 mutation (the genetic cause of classical lissence-
phaly) show abnormal dynein localization and reduced cell
motility (8–10). Lis1 interactions with dynein and doublecortin
support a role of some type for this protein in cell motility
(11– 15). In subcortical band heterotopia, neurons harboring
a mutation in the X-linked gene doublecortin (DCX) also
undergo delayed neuronal migration (16,17). One role for
DCX in neuronal migration could be in polymerization of
microtubules (18). After X-inactivation, hemizygous males
with no normal DCX developed lissencephaly, whereas females
with a DCX mutation developed subcortical band heterotopia
(16,17). In mice, mutations in Dcx and in the related Dclk gene
disrupt neuronal migration to the cortex (19,20). In addition,
cell-autonomous inhibition of Dcx by RNAi was found to
induce abnormal neuronal morphology and was sufﬁcient to
impair neuronal migration (19,21). Therefore, the band hetero-
topia with DCX mutations appeared to reﬂect cell-autonomous
roles for DCX in neuronal migration. Lastly, a recent case of
human mosaicism in LIS1 also resulted in subcortical band
heterotopia (22), consistent with the view that both DCX and
LIS1 are involved in some aspect of cell motility.
As is the case for DCX mutations, PH due to mutations in the
X-linked ﬁlamin-A (FLNA) gene also is characterized as having
a cell motility defect (23– 26), but the evidence for this is not
deﬁnitive. Mutations in FLNA resulted in PH in females and
lethality in hemizygous males (27,28). FLNA encodes a 280
kDa actin-binding phosphoprotein that is widely expressed
throughout (and outside of) the nervous system (29). FLNA
homodimers regulate the actin cytoskeleton through inter-
actions via the protein’s multiple receptor-binding regions,
with effects on regulating cell stability, protrusion and moti-
lity (30). Moreover, FLNA-deﬁcient melanoma cells show
impaired cell motility (31). FLNA also promotes actin
branching, tethers large actin ﬁlaments and holds them in a
perpendicular arrangement (30,32 –34). The resulting three-
dimensional orthogonal network of actin ﬁlaments represents
a characteristic cortical actin structure at the leading edge of
the migrating cell. Thus, FLNA is believed to be essential for
mammalian cell locomotion through its stabilization of loose
microﬁlament nets. However, neither null mutations in FlnA,
nor cell-autonomous removal of FlnA in neural crest cells,
has resulted in cell-autonomous defects in neural migration
(35,36), suggesting that some as yet unknown function of
FlnA is the essential function in neural development.
Other mechanisms for the formation of periventricular
nodules have been proposed in light of the identiﬁcation of a
second cause of PH: mutations in the ADP-ribosylation factor
guanine exchange factor 2 (ARFGEF2) gene (37). ARFGEF2
encodes the brefeldin A-inhibited guanine nucleotide-exchange
factor 2 (BIG2), a protein that regulates exchange of GDP for
GTP, as required by the ADP ribosylation factors (ARFs).
Since the ARF proteins are primarily involved in intracellular
membrane and vesicular trafﬁcking, it is unknown how FLNA
and ARFGEF2, two genes with notably divergent functions,
both give rise to the CNS disorder PH. The congenital
microcephaly observed in affected individuals with ARFGEF2
mutations would suggest a failure in development within the
neural precursor population, rather than a failure in post-mitotic
neurons with impaired migration (37,38). Finally, while the
clusters of neurons along the lateral ventricles in PH could still
be indicative of a failure of neurons to migrate from the VZ,
PH with microcephaly is an autosomal recessive disorder,
meaning that all progenitors and neurons harbor the mutation.
The relative preservation of the overlying cortex implies that
the actual motility of neurons is preserved (37). Thus, if FLNA
and ARFGEF2 give rise to PH by a common pathway (as radio-
graphic studies of subjects with the two types of the disorder
strongly suggest), the cellular and molecular bases of that
pathway remain unexplained.
To pursue mechanisms underlying PH formation, we exam-
ined post-mortem human brains in four cases of PH, and corre-
lated these observations with ﬁndings from both in vitro and
in vivo mouse studies in which FlnA and Big2 function was dis-
rupted. We found that many neurons, including those that har-
bored a mutation in FLNA, appeared to be positioned properly
in the human cerebral cortex. Moreover, closer examination of
the lateral ventricular lining suggested that the integrity of the
neuroependyma was invariably compromised along the ventri-
cular lining, in the four cases. After inhibition of FlnA function
in mice, many affected cells were found to be restricted to the
ventricular lining or were found to be situated adjacent to a dis-
rupted neuroependymal lining, with loss of cell-to-cell contacts.
A similar loss of cell adhesion within the affected cells was seen
in vitro. Disruption of Big2 through brefeldin-A (BFA) treat-
ment led to a comparable disruption of the neuroependymal
lining, and to periventricular nodule formation in mice.
Finally, loss of alpha-SNAP function in the hydrocephalus
with hop gait (hyh) mouse, which harbors a mutation in Napa,
leads to denudation of the neuroepithelium, and to periventricu-
lar nodule formation. Overall, these observations raise the possi-
bility that PH does not merely reﬂect a problem in neuronal
motility; rather, it also entails a disruption of cell morphology
and cell–cell integrity along the neuroependymal lining,
mediated through vesicle trafﬁcking-dependent pathways.
Heterotopic neurons within human PH
In the current studies, we examined the cerebral cortex and the
ventricular lining from four human cases of PH by standard
498 Human Molecular Genetics, 2009, Vol. 18, No. 3
histology and immunostaining. Case 1 was an 83-year-old male
with PH (no FLNA mutation analysis could be performed, given
the poor DNA quality). Case 2 was a 27-year-old female with PH
(no FLNA mutation analysis could be performed, given the poor
DNA quality; however, she had glomeruloid vasculopathy
consistent with a FLNA mutation). Case 3 was a 2-month-old
female with PH (with a heterozygous point mutation (G!A)
at bp 5290, resulting in an Ala!Thr substitution at amino
acid position 1764, but it remains unclear whether this mutation
was pathological). Case 4 was a 37 week gestational age (GA)
male with PH [from a previously reported family with FLNA
Neuronal nodules bilaterally lining the lateral ventricles (the
hallmark of PH) were observed in Case 3, the 2-month-old
female, by MRI (Fig. 1A, white arrows) and by examination
of post-mortem brain tissue (Fig. 1B, black arrow). The
cerebral cortex and the basal ganglia otherwise appeared
normal. Hemotoxylin–eosin (H–E) staining showed a normal
six-layered cerebral cortex with nodules lining the underlying
lateral ventricle (Fig. 1C). Occasional regions of dysplasia
were observed in the cortex (Fig. 1D), with large pyramidal
neurons seen inappropriately in the more superﬁcial layers of
the cortex, although the lamination across the vast majority of
the cortex appeared normal. Higher magniﬁcation of the hetero-
topia revealed the presence of disorganized cells residing in
these nodules (Fig. 1E). Closer examination of the heterotopia,
using various neuron-speciﬁc markers (neuroﬁlament, NeuN,
Tbr1, c-Neu, calbindin and Tle-1), demonstrated the presence
of heterogeneous types of post-mitotic neurons in these
nodules (Fig. 1E), consistent with prior ﬁndings (40).
Preserved cerebral cortical lamination and interneuron
migration in human PH brain
The lamination of the cerebral cortex in individuals with PH
has been shown to be grossly normal (41,42). However,
those studies did not use layer-speciﬁc markers. Neurons
that are born early in cerebral cortical development take up
ﬁnal positions in the lower layers of the mature cerebral
cortex and are detected in the human cerebral cortex by the
neuronal marker FOXP1 (43 –45). In the embryonic brain,
however, FOXP1 is seen in the more superﬁcial layers II/III
of the brain (45). Neurons that are born later during cerebral
cortical development migrate past these deep-layer cells and
take their ﬁnal positions in the upper layers of the mature cer-
ebral cortex. These cells are detected with the neuronal marker
CUX1 (46). CUX1 staining was only successfully performed
on frozen sections from the 2-month-old female (Case 3),
given the formalin ﬁxation of the other brain samples.
In the PH cerebral cortex of the 2-month-old female (Case 3),
FOXP1 immunoreactivity was noted in the deep cortical layers,
whereas CUX1 was observed in the upper superﬁcial layers of
the cortex, with no blurring of the cortical layer boundaries
(Fig. 2A). Similar ﬁndings of largely preserved lamination by
Nissl staining were seen in the other documented cases of PH
(Cases 1 and 2) in which the clinical and MRI features were con-
sistent with FLNA mutations (Fig. 2C and D). FOXP1 staining in
the samples from the 27-year-old woman (Case 2) with PH also
demonstrated normal expression of FOXP1 in the deeper cortical
layers, consistent with the interpretation that cortical lamination
is preserved in PH (Fig. 2D). Preserved lamination was observed
even in a male (Case 4; 37-week GA male) with a presumed
X-linked FLNA mutation [the family of this child had documen-
ted FLNA mutations and PH (39)] in whom all neurons harbored
the mutation (Fig. 2E). Examination of the brain from the male
case of PH (Case 4) revealed FOXP1 expression in a narrow
band across the superﬁcial cortical layers II/III, consistent with
other reports of FOXP1 staining in the upper layers of the cer-
ebral cortex in embryonic human brain (45), and once again
suggesting appropriate migration during this early stage in devel-
opment (Fig. 2E). Overall, these ﬁndings indicate that cortical
lamination and therefore migration were largely preserved in
the cerebral cortex of individuals with PH.
Several other observations suggest that abnormal neuronal
motility is not entirely responsible for periventricular nodule
formation. In both the cortex and the nodules lining the
lateral ventricles of PH brains, cells expressed the inhibitory
neuronal markers GABA and GAD, in addition to the excit-
atory neuronal marker glutamate (Supplementary Material,
Fig. S1), demonstrating that both inhibitory and excitatory
neurons are found within the periventricular heterotopic
nodules. Since many inhibitory interneurons are derived
from the ganglionic eminence, the GABAergic neurons
likely migrated long distances to arrive within the nodules.
Secondly, CUX1 expression was observed in 60% of the
neurons forming the nodules lining the lateral ventricles, but
there was an almost complete absence of FOXP1 expression
(only 2%) (Fig. 2B). FOXP1 is largely expressed in the
earlier-born, deep-layer neurons of human cortex, while
CUX1 is seen in the more superﬁcial, later-born neurons of
layers II/III of the cortex. Absence of FOXP1-positive
neurons in the nodules suggests that earlier-born neurons
were more capable of migrating into the cortex than were
later-born neurons. Conversely, the predominance of CUX1-
positive neurons in the nodules suggests impairments in neur-
onal migration later during cortical development. Taken
together, these data indicate that nodule formation does not
solely reﬂect a cell-autonomous neuronal motility deﬁciency,
but also additionally a non-cell autonomous defect, such as a
disruption of some developmental process along the VZ, that
becomes more apparent later in development and that affects
later-born neurons more severely.
Disruption of the neuroependyma in human PH brain
Since a mutation in either FLNA or ARFGEF2 results in
human PH, we examined whether expression and/or localiz-
ation of these proteins was disrupted in human PH brain
tissue. Our prior studies had shown that FLNA and BIG2
(coded for by ARFGEF2) were co-expressed along the ventri-
cular lining, the region where both of these proteins had
their highest expression (47). In the brain of Case 3, the
2-month-old female, neurospheres isolated from the lining of
the lateral ventricles demonstrated higher expression levels
of FLNA than did the differentiated neurons/glia in the
cortex and in the nodules (Supplementary Material, Fig. S2),
suggesting that this protein serves a developmental function
within the neural stem cell (NSC) population.
Because NSCs typically reside along the germinal matrix of
the lateral ventricles, we closely examined the VZ and subven-
Human Molecular Genetics, 2009, Vol. 18, No. 3 499
Figure 1. Histopathology in human periventricular heterotopia (PH). (A) T2-weighted MR image of the brain from a 2-month-old female with PH (Case 3).
Small gray matter nodules of neurons (white arrowheads) can be seen lining the lateral ventricles. (B) Gross specimen of the same case demonstrates a periven-
tricular nodule (black arrow) with otherwise normal appearing cortex and basal ganglia. (C) Hemotoxylin –eosin staining shows a normal six-layered cortex and
contiguous nodules (asterisks) along the lateral ventricle (higher magniﬁcation to the right). (D) Occasional regions of cortical dysplasia are seen with larger
pyramidal neurons ectopically located in the more superﬁcial layers of the cortex. However, the vast majority of the cortical layers appear normal. (E) The
nodule is comprised of neurons, expressing a variety of neuronal-speciﬁc markers.
500 Human Molecular Genetics, 2009, Vol. 18, No. 3
tricular zone (SVZ). In our gross histological examination of
the PH brain, we noted a marked disruption of the neuroepen-
dymal lining next to the periventricular nodules. In control,
age-matched brain, FLNA was tightly localized to the apical
side of the neuroependyma of the lateral ventricles [Fig. 3A
and B (left column)]. However, in the PH brain, FLNA
Figure 2. Neurons expressing the mutant FLNA protein migrate appropriately into the cerebral cortex. (A) Photomicrographs of the cerebral cortex and the nodular
heterotopia from the 2-month-old female, immunostained with the superﬁcial layer marker (CUX1) and the deep-layer marker (FOXP1), demonstrate a normal cortical
lamination with no blurring of the cortical layer boundaries. (B) Photomicrograph of the heterotopic nodules in the 2-month-old female demonstrate increased numbers
of CUX1-positive neurons, as compared to FOXP1-positive neurons, suggesting that these nodules are composed of later-born neurons. (C) Bright ﬁeld photomicro-
graph of the cortex of an 83-year-old male with PH. The cortical layers appear discrete with no blurring of laminae on Nissl stain. (D) Nissl stained cortical section of a
27-year-old female similarly shows preservation of the layers. Immunostaining for FOXP1, which labels the deep-layer neurons, shows positive and appropriate stain-
ing in laminae V/VI. Glomeroid vascular anomalies were also seen in the cortex and have previously been reported in PH cases due to FLNA mutations. (E)Nissl
stained cortical section of a 37-week GA male derived from a familial PH pedigree with a previously reported FLNA mutation shows appropriate cortical layering
by Nissl stain. Positive immunostaining for FOXP1 which in embryonic human brain labels the neurons of the superﬁcial layers (45).
Human Molecular Genetics, 2009, Vol. 18, No. 3 501
immunoreactivity appeared diffusely localized and often
discontinuous, particularly along the ependymal lining of the
lateral ventricles and adjacent to the nodules [Fig. 3A and B
(right column)]. Similar results were obtained with the neural
adhesion markers a-catenin and b-catenin (Fig. 3C and D),
the PH-related BIG2 protein (Fig. 3E) and the neuroepithelial
markers vimentin and S100B (Fig. 3F and G): disruption of
the normal contiguous expression of these markers was seen
along the neuroepithelium in PH brains. Disruption of the neu-
roependymal lining also was observed, as a discontinuity in the
staining of FLNA and b-catenin along the neuroependyma
in the brain of Case 4 (37-week-old GA male PH brain;
Fig. 3H), and in regions immediately below the nodules and
in areas with a normal-appearing overlying white matter and
cortex without nodules, in both Cases 1 and 2 of PH (Sup-
plementary Material, Fig. S3). The distinct co-localization of
FLNA and BIG2 (the proteins responsible for PH) (27,37) to
the neuroepithelium, and the apparent disruption of both these
and other neuroepithelial markers along the neuroependyma,
raised the possibility that PH results from a breakdown of the
integrity of the neuroepithelial lining of the lateral ventricle,
thereby giving rise to the ectopic neuronal nodules via defects
in neuronal migration. Areas of ependymal breakdown
without associated nodules could have arisen after the period
of neuronal migration. Similarly, areas where the ependyma
remained intact suggested that normal radial glial differentiation
into the ependyma could occur (Fig. 3G).
Loss of FlnA function impairs onset of neural precursor
migration and disrupts the neuroependyma
To begin to explore experimentally the molecular mechanisms
behind these observations in the human cases of PH, we con-
ducted experiments using the mouse as a model system. Since
FLNA has been implicated in neuronal motility, we directly
examined the migration of early neuroblasts following
in utero electroporation of a FLNA dominant-negative con-
struct (EGFP-DABD-FilaminA), to determine whether a
neuronal migration defect is the result of a cell-autonomous,
or a non-cell-autonomous, defect. Prior studies had shown
that loss of FLNA function prevents cells from acquiring con-
sistent directed polarity and reduces motility in the SVZ and
the intermediate zone (48). We used a FLNA dominant-
negative construct that lacks the actin-binding domain, and
therefore is thought to alter FLNA localization and actin-
dependent signaling. By 48 h after transfection in embryonic
day 16.5 (E16.5) mice, clusters of VZ progenitors expressing
the EGFP-DABD-FilaminA construct were seen lining the ven-
tricle, whereas progenitors transfected with the control EGFP
alone construct were observed migrating toward the cortical
plate (Supplementary Material, Fig. S4A). Approximately
80% of progenitors with inhibited FlnA function resided
within 50 mm of the neuroependyma (as compared with 60%
of control EGFP progenitors, P , 0.05), suggesting that some
transfected cells were unable to migrate from the ventricular
surface (Supplementary Material, Fig. S4B). Additionally,
virtually all of the EGFP-positive cells from both the experi-
mental and control animals stained for nestin and vimentin,
suggesting that most of these cells were neural progenitors
along the VZ, rather than post-mitotic neurons (Supplementary
Material, Fig. S4C). Taken in the context of the prior obser-
vations of a normal-appearing cortex in human PH brains,
these results suggest that FlnA can disrupt progenitor migration
from the ventricular surface, through a cell-autonomous
impairment in motility, and/or non-cell- autonomous disrup-
tion of the ventricular lining.
Prior studies had shown that loss of FlnA function in mice
leads to a disruption of E-cadherin localization along the
neuroepithelium (34). To speciﬁcally address whether inhibition
of FlnA in progenitors along the neuroepithelium contributes to
this disruption of the neuroependymal lining, and therefore
potentially impairs initial neuronal migration, we blocked FlnA
signaling, using the dominant-negative FLNA construct both in
vivo and in vitro.Again,weperformedin utero electroporations
of the developing mouse (E16.5) lateral ventricles and cortex
with the dominant-negative EGFP-DABD-FilaminA construct
(48). Forty-eight hours after electroporation with EGFP-
DABD-FilaminA, disruptions in the lateral ventricular lining
were observed with the neuroependymal markers a-and
b-catenin (Fig. 4A and B); effects were not observed in control
neuroependyma electroporated with EGFP alone (Fig. 4A and
B). These disruptions were most prominent in areas where
neuroependymal cells had FlnA signaling disrupted (as assayed
by EGFP-DABD-FilaminA expression), and where there were
speciﬁc and localized cell connections to the lateral ventricle
(Fig. 4A; higher magniﬁcation is lower left insert, for serial sec-
tions see Supplementary Material, Fig. S5). Our observations
suggested that disruption of FlnA signaling can result in the
loss of cell–cell contacts in the polarized neuroependymal
cells. To further address this possibility, we transfected the
FLNA dominant-negative construct into MDCK cells. When
b-catenin was used as a marker for the integrity of cell–cell con-
tacts, we ﬁnd that impaired FlnA function led to a partial loss of
b-catenin expression along the surface of the cell (small arrow-
heads, Fig. 4C). In addition, we transfected neural progenitors
in utero by electroporation of the dominant-negative FLNA
plasmid, subsequently dissociated them from the cortex, and
cultured them in vitro. Loss of FlnA function in these cells led
to impairments in cell spreading and adhesion, consistent with
the observed loss of cell– cell contact (Fig. 4D). The loss of
cell adhesion and spreading was not a result of increased cell
death within the FlnA-inhibited cells (Fig. 4E). Progenitors
expressing the dominant-negative FLNA construct exhibited a
more rounded appearance, but did not express the cell death
marker caspase 3. These ﬁndings show that loss of catenin
staining is not limited to transfected cells, suggesting that the
phenotype is not entirely cell-autonomous. Additionally, the
current studies suggest that loss of FlnA function in progenitors
along the neuroepithelium is sufﬁcient to reproduce the loss of
adhesion seen in FlnA null mice.
Loss of Big2 function disrupts the neuroependyma
and leads to PH nodule formation
Mutation of ARFGEF2 (which codes for BIG2) causes an
autosomal recessive form of PH in humans (37). Big2 has
been shown to be expressed at its highest levels along the ven-
tricular lining (47), and both ARFs and GEFs are expressed
within the brain during development. Moreover, BFA has
been shown to bind the Sec7 domain of Big2 GEF, and to
502 Human Molecular Genetics, 2009, Vol. 18, No. 3
Figure 3. Disruption of the neuroependyma lining along the periventricular nodules in PH brains. (A) Schematic demonstrates the region of impaired neuroe-
pithelial integrity in the neuronal nodules. (B–F) Photomicrographs of tissues from control and PH cases (2-month-old female with PH), following immunostain-
ing for FLNA (B), b-catenin (C), a-catenin (D), BIG-2 (E), vimentin (F) and S100B (G) shows loss of the neuroependymal lining along the heterotopia.
Moreover, in areas within PH brains, where the ependyma is intact, there is no disruption of radial glial differentiation as assessed by S100B staining(G).
Finally, both FLNA and BIG2 (the protein encoding the gene shown to cause the autosomal recessive form of PH) (37) expression patterns are notably conﬁned
to the neuroepithelial lining (B and E). (H) Fluorescent photomicrographs of FLNA and b-catenin staining along the neuroependymal lining of the ventricular
zone in a 37-week-old GA male with PH and a FLNA mutation demonstrating the disruption along the ventricular zone.
Human Molecular Genetics, 2009, Vol. 18, No. 3 503
inhibit ARF activity (49). We therefore evaluated in a murine
model, whether loss of Big2/ARF activity within neural pro-
genitors in vivo can disrupt the neuroepithelial lining in a
manner similar to what occurs with loss of FlnA function. A
single injection of BFA (20 –40 m
M) was performed in E16.5
mice or three serial intraventricular injections were performed
in post-natal day P0 mice with subsequent injections on P1 and
P2 mice. The inhibition of GEF function led to ventriculome-
galy and heterotopic nodule formation along the ventricular
lining (Fig. 5A, Supplementary Material, Fig. S6). The ventri-
cular lining appeared to be disrupted, with an aberrant distri-
bution of neurons along the ventricular surface. However,
injections in later-born P0 animals led to fewer nodules and
fewer mis-sited neurons along the lining, despite multiple
BFA injections. This observation likely reﬂects the end of
corticogenesis in the later-age mice. To more closely assess
whether these nodules form through a disruption of the ventri-
cular lining, we examined the integrity of the neuroepithelium
at time points more closely following BFA injection into
embryonic mice. Two hours after Big2/ARF inhibition, the
neuroependymal lining of the VZ was not uniformly intact,
as indicated by the discontinuous expression of phalloidin
and N-cadherin (Fig. 5B), suggestive of an abnormality in
the adherens junctions of the neuroependymal cells. By 7 h
after Big2/ARF inhibition, the neuroependyma was clearly
disrupted, as evidenced by the b-catenin staining pattern
Figure 4. Loss of FlnA function disrupts neuroepithelial cell contacts along the murine ventricular lining. (A) The neuroependymal lining (arrowheads), as
assessed by b-catenin staining, appears disrupted 48 h after in utero electroporation of the dominant-negative EGFP-DABD-FilaminA construct, by comparison
to an EGFP control construct, via confocal microscopy. Electroporation is performed on embryonic day 16.5 (E16.5) embryos and immunostaining is performed
in the E18.5 embryo. The number of GFP-positive cells along the lining with co-localized surface expression of b-catenin is quantiﬁed relative to the total
number of GFP-positive cells, following transfection of either control or EGFP-DABD-FilaminA construct. This change in b-catenin distribution and alteration
in cell contacts are quantiﬁed below (Student’s t-test:
P , 0.02; control n ¼ 10, experimental n ¼ 5). (B) The neuroependymal lining, as assessed by a-catenin,
is also disrupted 48 h after in utero electroporation of the dominant-negative EGFP-DABD-FilaminA construct. Electroporation is performed on E16.5 embryos
and immunostaining is performed in the E18.5 embryo. (C) MDCK cell cultures transfected with the dominant-negative EGFP-DABD-FilaminA similarly leads
to a partial loss of cell– cell contacts (b-catenin, small arrowheads) and alterations in b-catenin distribution as compared to cells transfected with EGFP alone.
Cultures are maintained for 48 h after transfection prior to immunostaining with the cell adhesion marker, b-catenin. The number of transfected cells with dis-
rupted cell surface expression of b-catenin was quantiﬁed in both control and EGFP-DABD-FilaminA positive cells. The alterations in cell contacts are sum-
marized to the right (Student’s t-test:
P , 0.02; control n 3, experimental n 3 independent experiments). (D) Fluorescent photomicrograph of neural
precursors 2 days after transfection either with control EGFP or dominant-negative EGFP-DABD-FilaminA constructs, followed by staining for the actin cytos-
keleton with phalloidin (rhodamine). Inhibition of FLNA leads to a more rounded appearance in the precursor cells and overall loss in cell membrane spreading,
consistent with a disruption in cell adhesion. Cell areas of transfected cells are measured using NIH ImageJ software (Student’s t-test:
P , 0.05; control n 3,
experimental n 3 independent experiments). (E) Fluorescent photomicrograph of neural precursors 2 days after transfection with control EGFP or dominant-
negative EGFP-DABD-FilaminA constructs, followed by staining for the apoptotic cell death marker caspase3. The rounded appearance of the neural cells fol-
lowing inhibition of FLNA is not due to increased cell death (scale bar ¼ 25 mm).
504 Human Molecular Genetics, 2009, Vol. 18, No. 3
Figure 5. Inhibition of Arfgef2/Big2 interrupts the integrity of the neuroependymal lining and leads to periventricular nodule formation in mice. (A) Periven-
tricular heterotopic nodules (PH1 and PH2) and enlarged ventricles (LV) are seen on Cresyl violet stained tissue in a 2-week-old mouse following early post-
natal intraventricular injections of 40 m
M BFA. Heterotopic nodules (PH1) were seen just below the ventricular surface. Clusters of cells (PH2, arrowheads) also
extended beyond the neuroependymal lining to lie directly along the LV. Immunostaining for these ectopically localized cells shows that they express the neur-
onal marker NeuN (below). To the right, the higher magniﬁcation photomicrographs of PH1 and PH2 show neurons ectopically positioned along the ventricle.
(B) Two hours after intraventricular injection of BFA into E16.5 mice, phalloidin and N-cadherin staining along the ventricular zone neuroependyma is discon-
tinuous. (C) Within 7 h after intraventricular injection into E16.5 mice, BFA disrupts the continuity of b-catenin staining along the ventricular neuroepithelium.
(D) Through inhibition of Big2, BFA impairs transport of adhesion molecules such as b-catenin from the Golgi apparatus to the cell surface in MDCK cells. This
impairment in vesicle transport likely contributes to heterotopia formation. (E) After exposure to BFA for 7 h, the trafﬁcking of b-catenin from the Golgi
complex to the cell membrane is disrupted.
Human Molecular Genetics, 2009, Vol. 18, No. 3 505
(Fig. 5C). Inhibition of Big2/ARF by BFA in vitro suggested a
disruption in the vesicle trafﬁcking of adhesion molecules
such as b-catenin in MDCK cells (Fig. 5D). In BFA-treated
MDCK cells, b-catenin accumulated in the Golgi apparatus,
rather than being transported to the cell surface (Fig. 5E).
As was seen for altered FlnA function, the loss of Big2 func-
tion impaired the targeting of adhesion molecules; this impair-
ment could provide an underlying basis for disruption of the
integrity of the neuroepithelium along the VZ.
Loss of alpha-SNAP function leads to denudation of the
neuroependyma along the VZ, and induction of
While FLNA has been implicated in cell motility, this actin-
binding protein also associates with the Golgi membrane and
regulates endosomal trafﬁcking (50,51). BIG2 directs ARF
activation, and ARF proteins are major regulators of vesicle
formation in intracellular trafﬁcking (52). Impairments in
vesicle transport could disrupt delivery of various adherens
junction proteins within both the neuroepithelium/immature
ependyma and neural progenitors of the SVZ, thereby compro-
mising the integrity of the neuroependyma. Prior reports have
described neuroependymal denudation within the hyh mutant
mouse (53–55), which harbors a mutation in the vesicle
fusion-related gene Napa (53,56). Prior studies have also
shown that polarity markers such as E-cadherin, b-catenin
and atypical protein kinase C (aPKC) were abnormally distri-
buted along the neuroepithelial lining (57). We therefore
examined whether this mutant mouse develops heterotopic
nodules. H-E stained, coronal brain sections from the mutant
hyh mouse at various ages showed an impairment of the integ-
rity of the ventricular lining followed a clear temporal and
spatial pattern. As early as E12.5, the neuroependyma of the
fourth ventricle was disrupted, while that of the lateral ventri-
cles was still unaffected (Fig. 6A and B). At E14.5, a rufﬂing
and disintegration of the neuroependyma lining the ganglionic
eminences was seen, leading to a protrusion of cells from the
SVZ into the ventricular lumen (Fig. 6C and D). At later ages
(E16.5), the disorganization of the VZ progressed rostrally,
involving the neuroependyma lining of the septum (Fig. 6E
and F). Interestingly, alpha-SNAP immunostaining in wild-
type embryos showed this protein to be preferentially
expressed in cells along the immature ependyma of the gangli-
onic eminences and the cerebral cortex (Fig. 6G– J). More-
over, immunostaining for caveolin-1, a membrane-associated
protein that is highly expressed in the neuroepithelium (58),
revealed disruption of the neuroepithelial architecture
(Fig. 7, cf A– B and E– F). We then analyzed the phenotype
of the cells protruding into the ventricular lumen and found
them to express neuronal markers, likely corresponding to
neuronal progenitors or neuroblasts (Fig. 7C and G). Scanning
electron microscopy clearly revealed denudation of the
neuroepithelium, with progenitor cells and macrophages
becoming exposed to the lateral ventricle cavities (Fig. 7D
and H). Denudation of neuroepithelium/ependyma and altera-
tions of the SVZ have been shown to occur in human fetuses
with a moderate communicating hydrocephalus (58,59).
With continued development in the mouse, the multiple
areas of neuroepithelium/ependyma denudation give rise to
nodules along the lateral ventricles (Fig. 8A –C; Supplemen-
tary Material, Fig. S7). Thus, in post-natal mutant hyh mice,
heterotopic nodular neurons were frequently observed in
the vicinity of the ventricular walls, where the ependymal
lining was missing (Fig. 8C –E). In contrast, nodules were
never seen below those regions of the ventricular walls that
retained the ependymal lining. Because nodules were distinct
during the early post-natal period, and frequently found
near the SVZ, where a small degree of neurogenesis continues
post-natally (Fig. 8C), we studied whether PH neurons
originate post-natally from over-proliferation of cells in the
SVZ, or whether they correspond to post-mitotic neurons
born at embryonic stages and spatially arrested near VZ.
We used cumulative BrdU (5-bromo-2
during a 3-day period (P3 –P6). Numerous cells of the
SVZ were found to be labeled with BrdU, whereas those
of the nodules were not, indicating that these cells
corresponded to post-mitotic neurons born at embryonic
stages (Fig. 8F and G).
A mutation in either of two genes (FLNA or ARFGEF2) has
been shown to cause PH in humans (27,28,38). While these
two genes appear quite disparate functionally, the similarities
in the radiographic characteristics of bilateral heterotopic
nodules, between cases of PH arising from FLNA mutation
and cases arising from ARFGEF2 mutation, suggest that the
two genes have a common ﬁnal effector, in giving rise to aber-
rant cortical development. Through examination of post-
mortem human PH brains (including a familial male case of
PH due to a FLNA mutation), we showed that the vast majority
of neurons migrated into the cerebral cortex. Detailed examin-
ation of the ependymal lining of the heterotopic nodules
revealed a signiﬁcant disruption in the integrity of the neuro-
ependyma. Experimental disruption of FlnA signaling in mice,
both in vivo and in vitro, resulted in a failure to initiate
migration, an altered integrity of the ependymal lining and a
loss of cell adhesion and cell– cell contacts. Inhibition of
Arfgef2/Big2 induced by BFA caused a similar disruption in
the neuroepithelial lining and led to formation of periventricu-
lar nodules in mice. Finally, we described the formation of
nodules in the hyh mouse: this mouse’s mutation in the
Napa gene disrupts SNARE receptor (SNARE)-mediated
vesicle transport and membrane fusion. Because alpha-SNAP
is a protein involved in vesicle fusion, it should be widely
expressed in the body. However, evidence indicates that it is
preferentially expressed in the CNS and at early developmen-
tal stages (56); within the CNS, alpha-SNAP is most highly
expressed in the VZ and SVZ (present report). This expression
pattern would explain why the alpha-SNAP mutation is princi-
pally expressed spatially in these zones, and at certain stages
of development. Overall, our results suggest that PH, while
linked to aberrations in neuronal motility, also can arise
from a disruption in the neuroependyma, where loss of
FlnA, Arfgef2/Big2 and Napa/alpha-SNAP functions alter
vesicle trafﬁcking and the adhesion of neural progenitors.
Genetic studies and clinical observations associated with
X-linked PH suggest that the role of FLNA in cell motility
506 Human Molecular Genetics, 2009, Vol. 18, No. 3
is not entirely responsible for periventricular nodule for-
mation. For example, random X-inactivation would imply
that 50% of the neurons born would not express the
normal ﬁlamin protein. However, in most reported PH cases
and in the post-mortem tissues examined here, the overlying
cortex is relatively normal, and only a small proportion of
Figure 6. Loss of alpha-SNAP function in hyh mice disrupts the integrity of the neuroepithelium (ventricular zone). Coronal sections, stained with
hematoxylin-eosin, through the brain of wild-type (wt) and mutant hyh (hyh) mice, at E12.5, E14.5 and E16.5. A caudal (fourth ventricle) to rostral (lateral
ventricles) progressive disruption of the ventricular zone (VZ) lining the ventricular system is shown. (A and B) At E12.5, the neuroepithelium of the ganglionic
eminence (GE) appears intact, while neuroepithelium denudation has already started at the fourth ventricle in the hyh mouse (inserts in lower left and right
corners). (C and D) At E14.5, loss of neuroepithelium occurs along the caudal GE in the hyh mouse (arrows, compare inset images in C and D). (E and F)
By E16.5, a breakdown of neuroepithelium and disorganization of the subventricular zone (SVZ) reaches the rostral horns of lateral ventricles in the hyh
mouse (arrows, compare inset images in E and F). In the hyh mouse, denudation of the neuroepithelium leads to protrusion of progenitor cells from the
SVZ into the ventricular lumen (top inset imagess in D and F). (G –J) Immunolocalization of alpha-SNAP in the GE in wild-type tissue at E14.5 (G– H)
and the cerebral cortex at E16.5 (I – J). (G and I) Alpha-SNAP immunoreactivity is mainly found in the cells of the VZ (VZ, arrows). (H and J) Differential
interference contrast (DIC, Nomarski optics) imaging of the same area is shown in G and I, respectively. LV, lateral ventricle. Scale bars: 250 mm (A– F);
500 mm (low magniﬁcation insets in A–F); 50 mm (high magniﬁcation insets in A– F and G –J).
Human Molecular Genetics, 2009, Vol. 18, No. 3 507
Figure 7. Loss of alpha-SNAP function in hyh mice disrupts the progenitor cell population along the neuroependyma. (A–D) Wild-type mice; (E– H) hyh mutant
mice. (A) Sagittal, hematoxylin– eosin stained section through the CNS of an E14.5 wild-type mouse. Boxed area is shown in B. (B) Higher magniﬁcation image
of the area of the ganglionic eminence (GE) shown in A. The integrity of the ventricular zone (VZ)/neuroepithelium and subventricular zone (SVZ) is clearly
seen. Inset: caveolin-1, functional marker, also reveals the integrity of the VZ (arrow). (C) Section adjacent to that shown in (B), immunostained for
b-III-tubulin. The cells of the VZ are not immunoreactive, while those of the SVZ express the neuronal marker. (D) Scanning electron micrograph of the ven-
tricular surface of the GE of a wild-type E18.5 embryo, showing a mosaic-like arrangement of cells, most of which are monociliated (stem cells/radial glia)
(arrows) with few multiciliated cells (immature ependyma). (E) Sagittal hematoxylin–eosin stained section through the CNS of a mutant hyh E14.5 mouse.
Boxed area framed is shown in (F). (F) Higher-magniﬁcation image of the area of the GE shown in (F). Denudation of the neuroepithelium and exposure of
the cells of the SVZ to the ventricular lumen are evident (arrows). A few patches of VZ still remain intact. Inset: Disorganization of the VZ and SVZ (asterisk)
can also be shown by immunostaining for caveolin-1. Arrow points to a patch of VZ. (G) Section adjacent to that shown in (F), immunostained for b-III-tubulin.
Immunoreactive cells of the SVZ are fully exposed to the ventricular cavity (arrows). VZ indicates an undetached patch of neuroepithelium. (H) Scanning elec-
tron micrograph of the ventricular surface of the GE of a mutant hyh (E18.5) embryo, revealing denudation of the neuroepithelium with progenitor cells (PC)
lining the VZ with associated macrophages (M). Scale bars: 500 mm (A and E); 50 mm (B, D, F and G); 25 mm (insets in B and F); 10 mm (D and H).
508 Human Molecular Genetics, 2009, Vol. 18, No. 3
the neurons constitute the nodules (42,60). Moreover, recent
genetic analysis has identiﬁed FLNA mutations in several
male patients, many of whom were not somatic mosaics for
FLNA mutations (as was the case for the 37-week GA male
included in this study), implying that—despite the fact that
all neurons bear the FLNA mutation—the neurons either
migrate completely into the cortex or do not migrate at all
(but never halfway) (28,39,61). Furthermore, autosomal reces-
sive mutations in ARFGEF2 result in PH with radiographic
ﬁndings virtually identical to these seen in PH due to a
FLNA mutation, except for the presence of the severe micro-
cephaly associated with ARFGEF2 mutation (37). In
ARFGEF-related PH, all of the neurons necessarily harbor
homozygous mutations in ARFGEF2 , but still form hetero-
topic nodules and a relatively normal-appearing cortex,
suggesting that the periventricular nodules are unlikely to
be due to motility impairments alone. Finally, the recessive
nature of the disorder suggests that PH is not a cell-auton-
omous process, given that many neurons migrate appropriately
even while others do not.
Several aspects of our current ﬁndings suggest that PH does
not arise solely from a cell-autonomous motility defect. Most
neurons actually migrate into the developing cerebral cortex
and take up their proper ﬁnal positions in the appropriate
Figure 8. Loss of alpha-SNAP function in hyh mice leads to PH formation. (A) Hematoxylin-eosin (H/E) stained coronal section through the brain of PN14 (P14)
hyh mutant mouse, displaying nodular periventricular heterotopias (arrowheads) below the ependymal-denuded ventricular walls. Boxed area is shown in(C).
CC, cerebral cortex; HI, hippocampus; LV, lateral ventricle. (B) Adjacent section to that of (A) immunostained for GLUT-1. The ependyma lining the hippo-
campus (HI) is preserved and GLUT-1 immunoreactive (arrow). The dorsal and lateral walls of the ventricle are denuded (asterisks). (C) Higher-magniﬁcation
image of the area boxed in (A). Nodular periventricular heterotopias (PH1 and PH2, oval regions delineated by dashed lines) are seen adjacent to the ependymal-
denuded ventricular wall (D and E) and the subventricular zone of the lateral wall. (E) Periventricular nodules are formed by cells with large nuclei and pro-
minent nucleoli. The same section shown in (A, C was bleached and used for toluidine staining (E), bleached again and used for immunostaining (D). The two
rectangular areas in C are shown in (D) and (E), respectively. (D) Immunostaining with a neuronal marker (b-III-tubulin) shows that the cells forming the peri-
ventricular nodules (PH1) in C are indeed neurons (arrows). (E) Similarly, toluidine blue staining demonstrates that periventricular nodules (PH2) in (C) displays
a neuronal phenotype. (F) Frontal section through the brain of a hyh mutant mouse injected with BrdU from P4 (PN4) to P6 (PN6) and sacriﬁced 3 h after the last
BrdU injection on P6. PH, periventricular heterotopia; SVZ, subventricular zone; CC, cerebral cortex. (G) Higher-magniﬁcation image of a portion of (F)
showing that cells within the nodule (PH) do not proliferate post-natally, while those of the SVZ continue to proliferate post-natally and are BrdU-labeled
(inset). Scale bars: 200 mm (A, B and F); 50 mm (C and G); 10 mm (D, E, inset in G).
Human Molecular Genetics, 2009, Vol. 18, No. 3 509
cortical layers, as evidenced by the precise lamination seen
with FOXP1 and CUX1 immunostaining. The lack of
FOXP1 neuronal expression in the nodules suggests that the
earliest-born neurons were able to migrate from the VZ and
into the appropriate lower layers of the cerebral cortex (as
indicated by the expression of FOXP1 in the deep layers of
the cortex). The increased proportion of CUX1-positive
neurons in the nodules, however, would argue that the later-
born neurons were the ones that were predominantly affected.
This preponderance of later-born neurons in the nodules is not
necessarily consistent with PH’s being a cell-autonomous cell
motility problem; if that were the case, the migration of all
neuronal populations would be expected to adversely affected.
Rather, a loss of ependymal integrity, which occurs later in
development, would be a more likely reason why later-born
neurons are affected. GABAergic neurons that arise from the
ganglionic eminences and undergo long-distance migration
to the VZ are found in the nodules suggesting that some
migration of this neuronal population occurred appropriately.
Finally, recent studies show that the mutant FlnA mouse,
while embryonic lethal, does not impair neuronal migration
during embryogenesis (35,36). Taken in sum, our data are
most consistent with the idea that FLNA mutations cause
PH, secondary to impairments in neuronal motility
(cell-autonomous), but also PH can arise from a disruption
in neural progenitors along the VZ, a disruption that inﬂuences
the migration of later-born neurons (non-cell-autonomous).
Previous studies have suggested that PH arises from an
impairment of the initial migration of neurons. Inhibition of
FlnA signaling by dominant-negative FlnA transfection was
shown, in prior work, as well as here, to disrupt the initial
migration and the morphology of migratory neuroblasts in
the SVZ (48). Furthermore, FilaminA-interacting protein
(FILIP) has been suggested to be important, in allowing
neurons to exit the VZ (48). Such observations, however, do
not necessarily explain the preponderance of later-born
neurons in the nodules, unless both FILIP and FLNA are
more critical for the decision by later-born migratory
neurons to exit the SVZ. Later-born post-mitotic neurons do
migrate longer distances and must migrate past earlier-born
neurons as they enter the more superﬁcial layers of cortex,
suggesting that additional cues are required by the later-born
neurons. Our expression data show that both FlnA and Big2
proteins are highly expressed in, and strongly restricted to
the neuroependymal lining; they are not highly expressed in
the SVZ. Loss of FLNA function has additional effects; it
can cause aortic rupture, gut dysmotility and increased skin
elasticity (27,62,63), with high FLNA expression restricted
to the lining of the blood vessel, intestines and skin, respect-
ively (Supplementary Material, Fig. S8). Defects in the
linings of these organs could in part account for the above
Several observations suggest that PH does not result from
an over-production of neurons. The brains of individuals
with PH that arises from ARFGEF2 mutations are microcepha-
lic, and Big2 inhibition in vitro leads to diminished prolifer-
ation of neurons (37). A recent case study of PH suggested
that microcephaly in males can be associated with FLNA
mutations (64). Neural precursors (neurospheres) and ﬁbro-
blasts obtained from Case 3 failed to demonstrate any increase
in proliferation (data not shown). Similarly, BrdU labeling of
proliferating cells in neurospheres obtained from hyh mutant
mice showed no difference from wild-type neurospheres
(data not shown). The hyh mice showed a progressive loss
of proliferative progenitor cells during development (57).
Direct inhibition of FlnA signaling in HEK293 cells by
dominant-negative over-expression did not produce any
increase in cell number (data not shown). Taken together,
these observations suggest that nodule formation in the PH
brain is not a direct consequence of over-proliferation of
neurons along the VZ during cerebral cortical development.
However, studies have suggested that endothelial cells can
stimulate their own self renewal, and can expand neurogenesis
in NSCs (65). Thus, it remains to be determined whether the
loss of blood brain barrier integrity along the VZ, or the endo-
thelial vasculature, contributes to an increase in neural pro-
genitor proliferation, and periventricular nodule formation.
Disruption of the neuroepithelial lining, in giving rise to PH,
mirrors the loss of structural integrity along the pial surface of
the brain that produces cobblestone lissencephaly (66–68). In
cobblestone lissencephaly, neurons have no inherent motility
defect; rather, they migrate beyond the marginal zone into the
leptomeninges and through the external basement membrane
during cortical development. The loss of integrity of the
molecular layer leads to nodules composed of neurons, on the
outermost surface of the brain (thought to resemble
cobblestones). Four genes, fukutin, POMGnT1, POMT2 and
POMT1, have been associated with cobblestone lissencephaly
(69–71); all are implicated in the glycosylation of dystro-
glycans. Hypoglycosylation of dystroglycan abolishes binding
activity for such ligands as laminin, neurexin and agrin, and
thereby compromises the integrity of the dystrophin-associated
extracellular matrix adhesion complex (72). In a fashion akin to
neuronal migration, neural progenitors perform interkinetic
nuclear migration in the VZ, with cells in synthesis (S) phase
positioned in the upper half of the epithelium away from the
ventricular surface (73– 75). Cells transitioning from S to
growth (G) 2 phase have nuclei that translocate in the cell
toward the ventricular surface, and cells in mitosis (M) lie
adjacent to the ventricular surface. Following M phase, cells
re-entering G1 phase translocate away from the neuroepithe-
lium, while post-mitotic neurons generally migrate toward
the pial surface and cortical plate. Loss of the neuroepithelial
integrity along the ventricular surface could conceivably
disrupt cells exiting M phase, causing them to ‘over-migrate’
or to protrude into the ventricular lumen, giving rise to
periventricular nodules. This possibility would be consistent
with the observed loss of neuroependymal integrity in the
human pathological cases of PH, in the hyh mutant mouse
and in the mouse models in which inhibition of FlnA and
Big2 occurred. Thus, in some ways, PH reﬂects a pathological
process similar to that seen in cobblestone lissencephaly; the
difference is merely that in PH the inner neuroependymal
lining is affected, rather than outer surface of the brain.
Several lines of evidence suggest that PH results from a dis-
ruption of vesicle trafﬁcking. First, a number of peripheral
membrane proteins, including FLNA, associate dynamically
with Golgi membranes during the budding and trafﬁcking of
transport vesicles in eukaryotic cells (50). FLNA has been
shown to regulate furin sorting in the trans-Golgi network/
510 Human Molecular Genetics, 2009, Vol. 18, No. 3
endosomal system (51). Thus, while FLNA regulation of actin
may directly alter the cytoskeleton required for migration, an
additional role for FLNA, in vesicle trafﬁcking, would not
be surprising, given that the actin cytoskeleton is required
for vesicle budding and vesicle movement (76 –78). Second,
other genes implicated in periventricular nodule formation
appear to be involved in vesicle trafﬁcking. ARFGEF2 regu-
lates the ARFs, which bind to vesicle coat proteins and
adaptors. Similarly, the studies reported in the present paper
suggest that mutations in Napa also result in PH in the
hyh mouse. Alpha-SNAP is involved in SNARE-mediated
vesicle fusion in many cellular contexts (79). Finally,
Mekk4, while it has not been directly linked to vesicle trafﬁck-
ing, has been shown to interact with FlnA, and to regulate
FlnA expression (86). It remains to be seen whether each of
these genes interact directly within the same pathway.
Once we better understand the functions of the genes that
regulate the interaction between the actin cytoskeleton and
Golgi apparatus (and consequently vesicle transport),
during brain development, we will begin to start to form a
clearer picture of the causal mechanisms of human PH,
and the associated CNS phenotype. Heterotopic nodules are
the primary feature seen in this disorder, and disruption in
the transport or recycling of adherens junction molecules
could explain a breakdown in the neuroepithelial lining. PH,
however, is also associated with a thinning of the cortex and
in extreme cases, microcephaly. Vesicle transport (via the
recycling endosome) is responsible for the delivery of speciﬁc
lipids and proteins to the cleavage furrow, and is crucial
for cell abscission. Disruption of this process would explain
the altered rates of symmetric and asymmetric division that
lead to changes in neural precursor fate and a thinner cortex
in PH. Finally, humans with PH show an elevated incidence
of dyslexia and psychiatric disorders (80,81), suggesting
problems with synaptic connectivity. Impairments in actin-
based vesicle trafﬁcking could also disrupt these contacts;
disruption of both FLNA and Big2 has already been shown
to disrupt recycling of surface receptors and neurite exten-
sion, necessary for dendritic arborization. Further studies
are needed to determine whether this common function in
vesicle trafﬁcking leads to the multiple observed phenotypes
MATERIALS AND METHODS
Human tissue, ethical and licensing considerations
The current studies have been approved by the Institutional
Review Board (IRB) at the Beth Israel Deaconess Medical
Center and Brigham and Women’s Hospital. De-identiﬁed
human discarded tissue was obtained from pathological
samples during autopsy. The diagnosis of PH in the
37-week-old GA male (Case 4) and 2-month-old female
(Case 3) was made prior to autopsy by prenatal screening
through ultrasound and/or conﬁrmed by MR imaging. Conﬁr-
mation of the diagnosis of PH in the adults cases (Cases 1
and 2) were made at autopsy.
Neuropathological examination was performed on four brains
with PH: a 37-week GA male (Case 4), a 2-month-old female
(Case 3), a 27-year-old female (Case 3) and an 83-year-old
male (Case 1).
The 37-week GA male (Case 4) was an archival case (ﬁxed
in formalin and stored as a parafﬁn block); he was born by
induction at 36 weeks gestation, and died at 7 days of life,
likely from vascular complications. Details have previously
been described (39), but the child had PH and came from a
family with multiple affected members (including the
mother and aunt); he had a documented 8-bp deletion in the
intron/exon boundary of exon 25 of the FLNA gene (39).
Macroscopically, the brain was small (brain weight 290.5 g;
expected 350– 400 g by CDC growth chart), and was notable
for the presence of multiple nodules of heterotopic gray
matter, located bilaterally in the angles of the lateral ventri-
cles. The majority of the cortex showed a normal six-layer
pattern. However, microscopic examination did reveal a
small area of unlayered polymicrogyria in one insula, as
well as abnormal layering in the inferior portion of the tem-
poral lobes. Additional extra-CNS abnormalities, often seen
in severe cases of PH arising from FLNA mutations, included
atrial and ventricular septal defects with a PDA, thrombocyto-
penia and a malrotated and shortened gut.
The 2-month-old female (Case 3) with PH was born by Cae-
sarian section after a 40-week gestation. At birth, the infant
had an irregular breathing pattern and was placed on a respir-
ator. The infant was noted as having dysmorphic features.
Laboratory workup was notable for a mild anemia. The
subject had a normal karotype (46,XX). Mitochondrial dys-
function was excluded by the presence of normal activity of
the electron transport chain complexes I—IV. She was diag-
nosed with a ventricular septal defect by an echocardiogram.
A brain MRI revealed bilateral subependymal nodular hetero-
topic gray matter (bilateral PH) and aqueductal stenosis
(Fig. 1). During her hospital stay, the infant was observed
having at least one tonic-clonic seizure, before being treated
with phenobarbital. The subject died at 2 months of age
from respiratory failure. Post-mortem brain tissue was
obtained immediately following autopsy for histological
characterization (frozen tissue), and for generation of both
neural progenitors (neurospheres) and skin ﬁbroblasts. No
FLNA mutation was detected, upon sequencing of only the
coding regions of the gene.
The PH observed in the remaining two adult cases was an
incidental ﬁnding at autopsy. The 27-year-old female (Case
2) died from acetaminophen overdose. The 83-year-old male
(Case 1) also died from causes unrelated to PH. No sequencing
for FLNA mutations was performed on these archival samples
(ﬁxed in formalin and stored as a parafﬁn block), due to poor
The hyh mutant mice (hydrocephalus with hop gait)
(B6C3Fe-a/a-hyh/J were obtained from The Jackson Labora-
tory (Bar Harbor, ME, USA) and maintained at Austral de
Chile, Valdivia, Chile. Housing, handling, care and processing
Human Molecular Genetics, 2009, Vol. 18, No. 3 511
of animals were carried out following the regulations approved
by the council of the American Physiological Society. Mice
were fed ad libitum and were maintained under a constant
12 h light/12 h dark photoperiod, and a constant room temp-
erature of 258C. Heterozygous females were caged together
with heterozygous males, and were separated when the pre-
sence of a vaginal plug was evident, designated as E0.5.
Embryos (E12.5– E18.5) were obtained from anesthetized
pregnant dams. Mutant embryos were identiﬁed by the
absence of the ventral neuroepithelial layer under microscopic
analysis, and by genotyping using PCR.
Immunohistochemistry of human and mouse tissues
Immunohistochemistry was performed on tissue sections
using standard techniques. In brief, tissue sections were
de-parafﬁnized in serial ethanol washes (if stored in parafﬁn),
placed in blocking solution with phosphate-buffered saline
(PBS) containing 10% fetal calf serum, 5% horse serum and
5% goat serum or 10% donkey serum and incubated overnight
in the appropriate antibody at 48C. Antibody staining assessed
for neuronal proliferation (Ki-67, courtesy BWH pathology;
phospho-histone H3: 1:200 dilution of stock, Upstate Biotech-
nology), neuronal migration [FOXP1, courtesy A. Banham,
using previously described methods (43); Cux-1, courtesy
Marta Nieto (46)] and neuronal differentiation (Calbindin,
Sigma; c-Neu, Santa Cruz Biotechnology; TLE-1, Abcam;
TBR-1, courtesy Dr Hevner; NSE, Zymed; Neu-N, courtesy
BWH pathology; neuroﬁlament; Sternberger Monoclonal;
GAD, GABA and glutamate, Sigma). To assess the neuro-
ependymal lining, we used neuroepithelial marker antibodies
[a- and b-catenin, Transduction laboratories, 1:250 dilution
of stock; S100B, Abcam, 1:1000; vimentin, Zymed, stock;
FLNA, Novocastra; BIG2, 1:200, using previously described
methods (37)]. FLNA staining was also performed on adult
parafﬁn-embedded human sections from the skin, aorta and
gut. Tissue sections were processed through standard ﬂuor-
escent secondary antibodies (Hoechst 33342 dye, Molecular
Probes; CY3, Jackson Immunoresearch Laboratories and
FITC, Sigma). Cux-1 staining could not be performed on the
archival parafﬁn-embedded tissue samples, since frozen sec-
tions are required for such staining.
For the mouse studies, embryos were sacriﬁced at various
ages and ﬁxed by immersion in 4% paraformaldehyde in
PBS at 48C or Bouin’s ﬁxative solution at room temperature.
Adult tissue was collected after intracardiac perfusion with
PBS followed by 4% paraformaldehyde in PBS or Bouin’s
ﬁxative solution. Tissues were processed for parafﬁn embed-
ding, for vibratome sectioning or for cryostat sectioning of
frozen sections. Immunohistochemistry was performed on
sections as described earlier. Additional antibodies used
included alpha-SNAP-speciﬁc antibody (clone 4E4) (1:50)
(Exalpha Biologicals); neuronal marker beta-III-tubulin
(G7121) (1:6000) (Promega); caveolin-1 (N-20) (1:250)
(Santa Cruz Biotechnology) and GLUT-1 (kindly provided
by Coralia I. Ribas, Memorial Sloan-Kettering Cancer
Center, NY, USA). To detect alpha-SNAP signal, we incu-
bated samples sequentially with a biotinylated secondary anti-
body (Vector Laboratories) and streptavidin Alexa Flour-488
(Molecular Probes) for 30 min each; binding was evaluated
on an Olympus Fluoview FV1000 laser scanning confocal
BrdU (Sigma) was used to label cells undergoing proliferation,
around the end of the ﬁrst post-natal week. Wild-type and hyh
mutant mice were injected i.p. with three daily doses of BrdU
(100 mg/kg) for three consecutive days (P4 – P6) and were
sacriﬁced on P6, 3 h after the last injection. Under anesthesia,
the mice were transcardially perfused with heparinized
buffered saline, followed by Bouin’s ﬁxative. The brains
were removed and post-ﬁxed for 48 h in the same ﬁxative.
Samples were embedded in parafﬁn and processed for BrdU
immunohistochemistry. Brieﬂy, sections were sequentially
incubated with: (i) a monoclonal antibody against BrdU
(G3G4; Developmental Studies Hybridoma Bank) at 1:50 for
18 h; (ii) a biotinylated secondary antibody (Vector) for
30 min and (iii) Vectastain Elite ABC reagent (avidin DH:
biotinylated horseradish peroxidase H; Vector) for 30 min.
The reaction product was detected with 3.3
Scanning electron microscopy
Wild-type and hyh mutant mice at E18.5 were anesthetized,
and their brains were removed and ﬁxed by immersion in
4% paraformaldehyde, 2% glutaraldehyde, 2% acrolein in
M phosphate buffer, pH 7.4. The blocks of tissue were
dissected into small pieces containing different ventricular
regions and were post-ﬁxed in the same ﬁxative overnight,
at 48C. After dehydration and critical point drying, the
blocks of tissues were coated with gold and visualized in a
scanning electron microscope, using a secondary electron
The total number of cells labeled by CUX1 or FOXP1 was
quantiﬁed within random ﬁelds represented within the hetero-
topic nodules. The percentage of cells stained for each marker
was calculated by dividing the number of positive labeled cells
by the total number of cells. Four ﬁelds were quantiﬁed within
the heterotopic nodules.
In utero electroporations and intraventricular injections
Electroporation procedures using the FLNA dominant-
negative construct (EGFP-DABD-FilaminA) followed pre-
viously published methods (48). In brief, E16.5-pregnant
dams were deeply anesthetized with Avertin. The skin over-
lying the uterus was shaved, sterilized and draped, and
incised to reveal the embryos. A 2-ml DNA/EGFP mixture
(1:3 mg/ml DNA:GFP ratio) was injected into the ventricle
with a Hamilton syringe. A 50 mV, 100-ms duration pulse
was applied, using electroporation prongs, across the surface
of the embryo’s head. For intraventricular injections of
BFA (20 –40 m
M), 1–2 ml injections into E16.5 mice were
performed using a pulled glass micropipette. A 1–2 ml intra-
ventricular injection of BFA was performed in P0 mice
512 Human Molecular Genetics, 2009, Vol. 18, No. 3
consecutively over 3 days. Following electroporation or intra-
ventricular injections, the maternal wound was sutured, and
the pregnant dam was allowed to recover, and was returned
to normal care.
Histological analysis of neuroependymal lining
After survival times of 2– 3 days, pregnant mice were deeply
anesthetized with sodium pentobarbital and cervically
dislocated. The embryos were harvested, decapitated and
drop-ﬁxed in 4% paraformaldehyde in 0.1
M PBS overnight.
The brains were blocked for coronal sectioning through
regions of positive GFP expression, and serial sections
50 mm thick were cut on a vibrating microtome. Alternate
sections were processed and mounted for ﬂuorescence and
cresyl violet staining, for assessment of cellular morphology,
migration of GFP-positive cells from the ventricle and
integrity of the neuroependymal lining. Fluorescence sections
were air-dried on gelatin-coated slides, and were subsequently
either dipped brieﬂy in Histoclear (National Diagnostics) and
permanently mounted in Fluoromount (Gurr), or else directly
mounted in mineral oil on slides. For the BFA injections,
mice were allowed to survive for upwards of 4 weeks prior
to sacriﬁce and histological analyses. Histological analysis
was performed using a microscope with epiﬂuorescence and
high numerical aperture optics (Zeiss or Olympus micro-
scope), or by confocal microscopy (Harvard Medical School
Cell Biology Microscopy Core).
MDCK cultures were maintained according to previously
published methods (37). For transfection using the dominant-
negative FLNA (48), FuGene (Roche) was allowed to equili-
brate to room temperature and then 3 ml of FuGene was
added to 100 ml of Dulbecco’s modiﬁed eagle medium
(DMEM) without serum. One microgram of the DNA con-
struct was added to each well, allowed to incubate for
15 min and washed. After 6 –12 h, medium was removed,
and was replaced with DMEM culture medium containing
10% fetal calf serum and 1% penicillin– streptomycin.
Twelve to 24 h after transfection, the cultures were ﬁxed,
immunostained for the desired markers and examined by con-
focal microscopy or routine ﬂuorescence (Harvard Medical
School Cell Biology Microscopy Core).
Western blot analysis
Protein was extracted from the heterotopic nodules and the
cortex by slight modiﬁcations of previously described
methods, or from brains of mice from various developmental
stages (82), solubilized in lysis buffer, separated on a 7.5%
SDS–PAGE gel and transferred onto PVDF membrane.
Protein from the human brain was probed with anti-ﬁlamin
(Novacastra) antibodies, and protein from the mouse brain
was probed with anti-pan-Arf (Afﬁnity Bioreagents) or
anti-Big2 antibodies (37), and detected by enhanced chemi-
Tissue dissociation and isolation
Methods for VZ dissection and dissociation follow general
guidelines used previously in murine cortical cell dissociations
(83,84) and in human neurosphere isolation (85). In brief, the
brains were cut into coronal sections, and samples were dis-
sected to obtain tissue along the periventricular zone within
the frontal cortex; tissue was then minced and washed in
cold Hanks-buffered saline solution (HBSS), and placed in
trypsin solution at 378C for 30 min. The sample was then
passed through a cell strainer, to isolate single cells, and was
washed in (DMEM) with 10% fetal calf serum, to inactivate
the trypsin. The dissociated cells were spun down, the
medium was aspirated and the cells were placed in neuro-
sphere medium (Cambrex bullet kit) containing EGF, FGF,
neural cell survival factor, gentamicin and amphotericin B,
for expansion. The cultures were maintained in a 378C/5%
incubator, and medium was aspirated and renewed on a
weekly basis. Individual neurospheres could be dissociated
and re-expanded to ensure a clonal population.
Supplementary Material is available at HMG online.
We wish to thank Dr Sato for kindly providing the dominant-
negative FLNA construct. The monoclonal antibody against
BrdU was obtained from the Developmental Studies Hybri-
doma Bank at the University of Iowa, Department of Biologi-
cal Sciences, Iowa City, IA 52242. The authors wish to thank
Dr Adriana Verschoor (Wadsworth Center) for critical reading
and input on our manuscript.
Conﬂict of Interest statement. None declared.
This work was supported by grants to R.J.F. from the NIMH
(1K01MH71801) and a Young Investigator Award from
Cure Autism Now; to V.L.S. from Julian and Carol Cohen,
Alzheimer’s Association, Harvard Center for Neurodegenera-
tion, and 1R21HD054347; to E.M.R. from Fondecyt, Chile
(Fondecyt 1070241) and to L.F.B. from DID-UACh
(DID-2005-12) and CONICYT, Chile. V.L.S. is a Beckman
Young Investigator and Doris Duke Clinical Scientist Devel-
opment Award Recipient. C.A.W. is an Investigator of the
Howard Hughes Medical Institute. A.H.B. and P.J.B. are sup-
ported by the Leukaemia Research Fund. We are very grateful
to Genaro Alvial and Ricardo Silva (Univerisdad Austral de
Chile) for their technical assistance.
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