1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard
Medical School, 44 Binney Street, Boston, MA 0215, USA
2 Department of Pathology, Division of Neuropathology, Brigham and Women's
Hospital, 75 Francis Street, Boston, MA 02115, USA
3 Division of Neuropathology, Children's Hospital Boston, 300 Longwood Avenue,
Boston, MA 02115, USA
4 GTC Biotherapeutics, 5 Mountain Rd, Framingham, MA 01701-9322, USA
5 Department of Neurobiology, Pharmacology and Physiology, University of
Chicago, Chicago, IL 60637, USA
6 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02138, USA
7 Division of Newborn Medicine, Children's Hospital Boston, 300 Longwood Avenue,
Boston, MA 02115, USA
* Author for correspondence (e-mail: david_rowitch{at}dfci.harvard.edu)
Accepted 3 February 2003
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SUMMARY |
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Key words: Emx2, Wnt1, Dysplasia, Cerebral, Cortex, Telencephalon, Marginal zone, Subplate, Preplate, Cortical hem, Organizer, Transgenic, Leptomeningeal, Glioneuronal, Heterotopia
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INTRODUCTION |
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The term cortical dysplasia is used to describe a heterogeneous group of
CNS malformations in which the organization of the cortical plate is
disrupted, typically owing to abnormal cellular migration
(Friede, 1989;
Gleeson and Walsh, 2000
).
Marginal zone heterotopias (MZH) or leptomeningeal glioneuronal heterotopias
(LGH) are one form of dysplasia in which ectopic nests of glial and neuronal
cells are observed in the cortical MZ or overlying leptomeninges, respectively
(Greenfield et al., 2002
).
These lesions are frequently seen in association with other features of
dysplasia as well as other CNS malformations and congenital syndromes such as
Trisomy 13 or type II lissencephalies (Walker-Warburg syndrome)
(Friede, 1989
;
Greenfield et al., 2002
;
Hirano et al., 1992
). They are
among the most common neuropathological malformations found in human brains
with cortical dysplasia (Mischel et al.,
1995
). Although their clinical significance is not fully
understood, they are presumed foci of seizure activity because of their
cortical location, neuronal composition and high incidence in individuals with
epilepsy (Mischel et al.,
1995
).
Insights into the genetic and molecular causes of cortical dysplasia are
beginning to emerge from general investigation of cortical development in mice
and humans (Gleeson and Walsh,
2000). In particular, certain transcription factors have been
described with important roles in MZ and SP development. Mutation of
Lmx1a, a LIM-homeodomain factor that is expressed in the dorsomedial
telencephalon, results in diffuse and focal abnormalities of the MZ
(Costa et al., 2001
). Second,
function of the homeodomain protein EMX2 is required for several aspects of
cortical development including regulation of neural precursor cell
proliferation (Galli et al.,
2002
; Heins et al.,
2001
; Tole et al.,
2000
) and lamination of the cortical plate
(Galli et al., 2002
;
Heins et al., 2001
;
Mallamaci et al., 2000a
;
Tole et al., 2000
), as well as
formation of preplate derivatives. Loss-of Emx2 function or
Emx1/2 function is associated with a decrease or absence,
respectively, of Cajal-Retzius and SP cells within regions of the cortex
(Bishop et al., 2003
;
Mallamaci et al., 2000a
;
Shinozaki et al., 2002
).
Additionally, defects in tangential neuronal migration and axon pathfinding
from the ventral forebrain to the cortex of Emx-null animals have
been described (Bishop et al.,
2003
; Shinozaki et al.,
2002
).
Despite its pivotal roles during brain development, downstream
transcriptional targets of Emx2 have yet to be identified. Emx2 is
expressed in the cortical ventricular zone and cortical hem, which is located
adjacent to the dorsal midline of the embryonic telencephalon. The cortical
hem is generally considered to be an organizing center and source of secreted
molecules with important roles in the specification and proliferation of
forebrain cell types (Furuta et al.,
1997; Grove et al.,
1998
). Numerous Wnt genes are expressed in overlapping patterns
within the cortical hem (Grove et al.,
1998
), and Wnt3a has been shown to be essential for
proliferation and expansion of hippocampal precursor cells in this region
(Lee et al., 2000
). Although
expression of Wnt1 is observed throughout most of the dorsal midline
of the CNS it is excluded from the telencephalon and cortical hem. We have
previously proposed that this aspect of Wnt1 regulation relies on
transcriptional repression, because deletion of certain Wnt1
cis-acting regulatory sequences results in ectopic reporter gene expression in
the telencephalon of transgenic mice
(Rowitch et al., 1997
).
Mutation of a single homeodomain core-binding site (HBS1) within the 5.5 kb
Wnt1 enhancer results in ectopic dorsomedial telencephalic reporter
gene expression (Iler et al.,
1995
; Rowitch et al.,
1998
) in a pattern reminiscent of Emx2
(Boncinelli et al., 1993
). EMX2
as well other homeodomain proteins have been shown to bind HBS1 in vitro
(Iler et al., 1995
), and in a
previous analysis of Emx2-null embryos, weak ectopic expression of
Wnt1 in the 10.5 dpc telencephalon was observed
(Yoshida et al., 1997
). Taken
together, these experiments are consistent with a model in which EMX2
functions to repress Wnt1 expression in the developing telencephalic
organizer regions (Rowitch et al.,
1997
). However, further experiments to demonstrate functional
consequences of ectopic Wnt1 expression in the cortex and functional
or phenotypic correlation between this and Emx2-null animals are
required to assess this putative interaction and establish its possible
significance for normal development.
We report the presence of marginal zone and leptomeningeal heterotopias in Emx2/ mice. Our data suggest that this lesion and other abnormalities of the superficial cortical plate (layer I) in Emx2/ mice result from a failure to repress Wnt1. First, we observed striking ectopic expression of Wnt1 in the cortical hem and medial cortical ventricular zone of Emx2/ mice but not heterozygous littermates. Second, Wnt1 gain-of-function analysis produced heterotopias identical to those in Emx2/ mice. Finally, we observed diffuse abnormalities of the early-born Calretinin- and reelin-positive cells and also the plexiform network of cellular processes derived from these cells within the marginal zone and subplate, similar to those described for Emx-null animals. These findings indicate that EMX2 acts to repress Wnt1 in the developing telencephalon. Furthermore, they suggest that ectopic activation of Wnt1 signaling is sufficient to cause anomalous formation of the marginal zone and subplate, as well as defects in neural migration and heterotopias in Emx2/ mice.
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MATERIALS AND METHODS |
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Generation of transgenic mice, animal husbandry and genotyping
Four independent founder lines of transgenic mice that expressed the
Wnt-lacZ-HBS reporter construct were generated by pronuclear
injection of one-cell BL6CBAF1/J embryos as described
(Rowitch et al., 1998
).
Genotyping was by Southern blot with a probe directed against Wnt1
(details supplied upon request). Of 12 independent Wnt1
HBS
transgenic founders generated, one was analyzed at 13.5 dpc, and three at 18.5
dpc-PN1. One stable Wnt1
HBS transgenic line was followed and
could be maintained in a hemizygous state. Homozygous offspring (n=4)
were generated at 12.5 dpc for analysis. This line was subsequently lost
because of infection with MHV. Extent Emx2 mutant mice (generously
provided by Prof. Peter Gruss, Goettingen, Germany) on a mixed C129Sv/J-C57Bl6
genetic background were generated and genotyped as previously described
(Pellegrini et al., 1996
).
Matings of transgenic mice were timed and embryos harvested such that noon the
day of vaginal plug discovery was considered to be 0.5 dpc.
Human heterotopias
Surgical biopsy and autopsy material containing leptomeningeal glioneuronal
heterotopias was identified by review of the case records and archival
material in the Neuropathology Division of Children's Hospital, Boston. Four
appropriate cases were identified and analyzed by Hematoxylin and Eosin
analysis in addition to immunohistochemistry for glial (GFAP) and neuronal
markers (NeuN, synaptophysin).
Histopathology
Mouse embryos and brains were fixed in freshly made 4% paraformaldehyde/PBS
overnight at 4°C. Tissue for frozen sections was then impregnated in 50%
Sucrose/PBS overnight at 4°C, frozen in OCT, and then sectioned at 16
µm. Tissue for paraffin wax-embedded sections was dehydrated, embedded and
sectioned at 5 µm according to standard protocols. Human neuropathological
specimens were fixed in 10% buffered formalin, according to standard clinical
protocols, and then embedded in paraffin wax for sectioning. Sections were
stained with Hematoxylin and Eosin and photomicrographs were taken digitally
using a Zeiss Axioskop and Axiocam imaging system.
Immunohistochemistry and whole-mount ß-galactosidase
staining
Paraffin wax embedded and frozen sections were subjected to heat antigen
retrieval at 99°C in 10 mM sodium citrate buffer for 20 minutes for all
antibodies. Staining was performed using the HRP/DAB based Envision+ staining
system (DAKO) according to the manufacturers specifications with the only
modification being increased incubation time for primary antibodies
(overnight, 4°C) and secondary antibodies (1 hour, room temperature).
Primary antibodies were Calretinin (Zymed #18-0211), GFAP (DAKO #Z0334),
Nestin (BD #611658), NeuN (Chemicon #MAB377) and laminin (DAKO). Staining for
ß-galactosidase activity in transgenic embryos was performed as
previously described (Whiting et al.,
1991).
RNA in situ hybridization
Frozen and paraffin wax-embedded section RNA in situ hybridization was
performed as previously described (Lu et
al., 2001). Protocol modifications specific to use with paraffin
wax-embedded sections included pretreatment of sections with 0.2 N HCl for 10
minutes at room temperature followed by incubation in 2x SSC for 10
minutes at 70°C. Sections were then treated with Proteinase K (50
µg/ml) for 10 minutes at 37°C. After hybridization, slides were treated
with RNaseA (20 µg/ml) for 15 minutes at 37°C, then washed and
developed according to standard protocols. The probes used were for the genes
Wnt1 (Parr et al.,
1993
), Emx2 and Wnt2b
(Tole et al., 2000
),
Bmp4 (Lee et al.,
2000
), reelin (Alcantara et
al., 1998
) and Gfap (modified construct from A. Ruiz i
Altaba).
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RESULTS |
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The distribution and morphological appearance of the marginal zone
heterotopias were strikingly similar to human heterotopias we identified in
surgical and autopsy tissue from individuals with cortical dysplasia
(Fig. 1G-I). The lesions most
closely resembled those referred to in humans as leptomeningeal glioneuronal
heterotopias (nodular heterotopias or `brain warts')
(Friede, 1989;
Greenfield et al., 2002
).
Comparison of the histological features of representative lesions from a
36-week-old infant and lesions from an 18.5 dpc
Emx2/ mouse, demonstrated the shared
findings of frontoparietal regional restriction, extension of cells into the
overlying meninges, frequent association with a central penetrating blood
vessel and a polypoid shape (Fig.
1F,G) (Ellison,
1998
; Friede,
1989
; Greenfield et al.,
2002
; Hirano et al.,
1992
; Iida et al.,
1994
). Serial sectioning of the lesions confirmed that nearly all
the lesions involved the subarachnoid space or leptomeninges. The more
differentiated appearance of the human lesions was probably due to the older
developmental age of the individual relative to the mouse. Both human and
mouse lesions contained NeuN-positive neurons as well as GFAP-expressing glia
by immunohistochemical and in situ hybridization analysis, confirming shared
features within the heterotopic tissue
(Fig. 1H-K). We conclude that
formation of marginal zone and leptomeningeal glioneuronal heterotopias
comprises a novel neuropathological feature in Emx2-null mice, and
that such lesions are comparable to human LGH.
Loss of Emx2 function results in strong ectopic expression
of Wnt1 in the dorsomedial telencephalon
The findings above and previous observations indicate critical roles for
EMX2 during cortical development and highlight its possible relevance in the
understanding of human LGH. In order to investigate mechanisms underlying
Emx2 function, we focused on possible transcriptional targets of EMX2
during brain development. As we have previously proposed, one candidate locus
for repression by EMX2 is Wnt1
(Iler et al., 1995;
Rowitch et al., 1997
;
Rowitch et al., 1998
).
However, it was unclear whether ectopic expression of Wnt1 occurred
in Emx2/ embryos at times associated with
formation of preplate derivatives (
11-12.5 dpc)
(Monuki and Walsh, 2001
). To
investigate this, we performed whole-mount in situ hybridization on wild-type
and Emx2-null embryos at 12.5 dpc. Wnt1 is normally
expressed along the dorsal midline of the CNS with a rostral limit in the
rostral diencephalon (Fig. 2A).
In contrast to the wild-type pattern, Emx2/
embryos also showed strong ectopic expression of Wnt1 in the
dorsomedial telencephalon and the rostral diencephalon that peaked at 12.5 dpc
(Fig. 2B). Ectopic expression
was barely detectable at 14.5 dpc and was absent at 18.5 dpc (data not
shown).
|
A homeodomain DNA-binding site in the Wnt1 enhancer
regulates Wnt1 expression within the Emx2 domain
The findings above suggested that EMX2 activity was necessary for
repression of Wnt1 expression in the telencephalon during the time of
formation of the preplate and its derivatives. Indeed, previous work has
suggested that EMX2 and other homeodomain proteins (e.g. DLX2, MSX1) can
directly bind the Wnt1 enhancer via a core-binding site (HBS1)
(Iler et al., 1995) that is
conserved at the pufferfish wnt1 locus
(Rowitch et al., 1998
).
Additionally, mutation or deletion of HBS1 resulted in ectopic expression of a
reporter transgene in the dorsomedial telencephalon
(Iler et al., 1995
;
Rowitch et al., 1998
). To test
whether ectopic expression of Wnt1 was sufficient to recapitulate
aspects of the Emx2 cortical phenotype, we first over expressed
Wnt1 using a strong 1.1 kb enhancer fragment that carried a mutation
of HBS1 (Fig. 3A)
(Iler et al., 1995
). However,
preliminary analysis indicated that this resulted in defects of neural tube
closure in the midbrain region (data not shown), most probably because of the
proliferative effects of Wnt1 and excessive neural overgrowth, as
previously observed (Danielian and McMahon,
1996
; Dickinson et al.,
1994
). To address this problem, we characterized the expression of
additional reporter transgene constructs with various deletions within the 1.1
kb Wnt1 enhancer (Rowitch et al.,
1998
). An enhancer fragment with deletion of HBS1 and 300 bp of
surrounding sequence (Fig. 3B,
referred to hereafter as
HBS) drove strong ectopic expression of the
lacZ reporter gene in the telencephalon
(Fig. 4A-D). Yet, in comparison
to previous results with the full-length 1.1 kb enhancer
(Iler et al., 1995
;
Rowitch et al., 1998
),
ß-galactosidase activity was relatively weak in regions of endogenous
Wnt1 expression, such as the roofplate and midbrain-hindbrain region
(Fig. 4B). Ectopic
telencephalic expression of the
HBS reporter transgene was first
detectable at
10-somites (8.75-9 dpc,
Fig. 4A) and was maintained at
least until 16.5 dpc (Fig. 4C).
These studies suggested that
HBS DNA regulatory sequences were suitable
to drive transgene expression within the dorsomedial telencephalon while
avoiding adverse consequences of Wnt1 overexpression in its
endogenous domain (e.g. neural tube defects).
|
|
Analysis of Wnt1 mRNA transcripts in situ showed that the
expression pattern of the Wnt1-HBS-transgene in the
dorsomedial telencephalon of animals derived from the stable line closely
resembled that of Emx2 (compare
Fig. 4E with
Fig. 4G,H). At 12.5 dpc, the
transgene was most strongly expressed in the ventricular zone neuroepithelium
of the cortical hem region adjacent to the choroid plexus primordium (cpp,
Fig. 4G). Moreover, we observed
an apparent gradient of transgene expression within the adjacent cortical
ventricular zone that was stronger in dorsomedial regions than dorsolateral
regions of the telencephalon (Fig.
4G,H) similar to the expression gradient of endogenous
Emx2 (Fig. 4E).
Ectopic expression was reduced but maintained within the medial most cortical
regions within the ventricular zone and subplate at 18.5 dpc (data not shown).
Even so, Wnt1 expression was not seen within the marginal zone at
early or late stages in the transgenic mice.
Ectopic expression of Wnt1 in the dorsomedial telencephalon
does not alter cortical patterning
During forebrain development, organizing centers are important determinants
of regional specialization. One such organizing center, termed the cortical
hem, is markedly reduced in size in Emx2-null mice
(Muzio et al., 2002). The
finding of ectopic expression of Wnt1 in the cortical hem of
Wnt1-Tg mice initially suggested the possibility of early patterning
abnormalities. However, histopathological analysis of Wnt1-Tg and
littermate controls at 12.5 dpc showed only subtle differences in the
delineation of the choroid plexus primordium from the adjacent thalamic
eminence and cortical hem in transgenic animals, and no reduction in size of
the cortical hem (Fig. 5E,F).
The most medial part of the cortical primordium (hippocampal anlagen) was also
examined just before birth with a range of hippocampal field-specific gene
expression markers as previously described
(Tole et al., 2000
). Although
this region was sometimes significantly larger than normal in Wnt1-Tg
mice, hippocampal field specification was comparable with that in littermate
controls (data not shown).
|
Ectopic expression of Wnt1 results in diffuse abnormalities
in the marginal zone and subplate
To investigate phenotypic similarities between Emx2 null and
Wnt1-Tg mice further, we focused on development of the marginal zone
and subplate, which show a marked reduction in the number of reelin-positive
CR cells and Calretinin-positive cells within the MZ and SP of
Emx2-null mice (Fig.
6A,B) (Mallamaci et al.,
2000a; Shinozaki et al.,
2002
; Yoshida et al.,
1997
). As shown (Fig.
6C), analysis of the anterolateral cortex of Wnt1-Tg mice
at 18.5 dpc revealed patchy areas containing markedly reduced numbers of
reelin-positive CR cells. Scattered ectopic reelin-positive cells were seen in
the cortical plate, marginal zone and subplate of both
Emx2/ and Wnt1- Tg mice. These
cells lacked normal Cajal-Retzius morphology and were extremely small,
possibly representing reelin-positive cortical interneurons or abnormally
differentiated Cajal-Retzius-like cells
(Fig. 6B,C, arrowheads).
|
Wnt1-Tg mice develop leptomeningeal glioneuronal
heterotopias
Having observed abnormal formation of preplate derivatives, we next
examined Wnt1-Tg mice for other features of cortical dysplasia.
Histological analysis at 18.5 dpc revealed that Wnt1-Tg founder mice
had well-formed LGH (Fig.
7A-C). Approximately two to four lesions/brain were observed in
two out of three independent founder animals examined at 18.5dpc-PN1.
Moreover, the lesions occurred in an anterior and dorsal distribution similar
to that seen in Emx2/ mice.
Immunohistochemistry confirmed the presence of numerous NeuN-positive neurons
(Fig. 7D), as well as
calretinin-positive neurons and processes
(Fig. 7E,F) within the lesions
from both mice.
|
Ectopic expression of Wnt1 causes heterotopias and
abnormalities of preplate derivatives early in cortical development
Ectopic Wnt1 expression in the telencephalon of
Emx2/ mice showed a peak at 12.5 dpc, and
was undetectable by 18.5 dpc (Fig.
2 and data not shown). This suggested that heterotopias might form
contemporaneously with splitting of the preplate and the onset of radial
neuronal migration. To assess timing further, we analyzed effects of ectopic
Wnt1 expression in an additional transgenic founder at 13.5 dpc.
Similar to findings at 18.5 dpc, we observed a severe reduction in the number
of calretinin- and reelin-positive cells and processes within the anterior to
mid-cortical fields in the marginal zone and subplate of 13.5 dpc
Wnt1-Tg animals (Fig.
8A,B). Posterior and medial cortical regions showed a relatively
normal number and distribution of calretinin and reelin-positive cells within
the marginal zone although the processes of cells within the subplate were
variably reduced within these areas (Fig.
8B, parts i-l).
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DISCUSSION |
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Because the heterotopias in Emx2/ mice at
late stages are histopathologically identical to MZH and LGH found in humans
with cortical dysplasia, we propose that
Emx2/ mice may be useful as a model to
better understand this disorder. Our analysis indicates that both human and
mouse lesions have a morphology suggestive of overmigration of radially
migrating cells into the marginal zone and leptomeninges. In addition, the
cortical plate below the lesion is commonly disrupted into deeper layers,
sometimes through to the subplate or subcortical regions. Although we were not
able to obtain early human lesions to confirm a similar time of onset to the
mouse (i.e. prior to 13.5 dpc), mature large heterotopias have been
histologically identified in human fetal autopsy specimens as early as 18 and
20 weeks gestation, implying that their onset could coincide with that of the
mouse upon evaluation of earlier stage human embryos
(Iida et al., 1994).
As MZH and LGH are seen in association with a wide range of CNS disorders
and functional mutations in mice and humans, the genetic basis of these
lesions is likely to be complex (Costa et
al., 2001; Halfter et al.,
2002
; Moore et al.,
2002
). Although EMX2 and WNT1 might play a
direct role in human heterotopia formation or subplate abnormalities, no such
role has been described previously in the literature. Alterations of Wnt
pathway signaling have been described within human cortical resections
containing focal intra-cortical dysplasias, but the presence of heterotopias
was not addressed in this study (Cotter et
al., 1999
). Loss-of-function mutations in EMX2 have been
described in individuals with the radiological diagnosis of schizencephaly, a
rare cortical dysplasia syndrome (Brunelli
et al., 1996
; Faiella et al.,
1997
; Granata et al.,
1997
). However, further work will be required to determine whether
EMX2 loss in these individuals is associated with heterotopias or
MZ/SP abnormalities as the neuroimaging techniques used to identify the
individuals lacked the resolution to detect such microscopic lesions and the
neuropathological findings in these cases have not been described.
Emx2 function is required for repression of Wnt1
within the dorsomedial telencephalon
Several lines of evidence support the proposal that EMX2 is a direct
repressor of Wnt1 expression during normal development of the
telencephalon. We have previously shown that EMX2 binds the highly conserved
HBS1-binding site in the Wnt1 enhancer, and that mutation or deletion
of HBS1 results in ectopic reporter transgene expression in the dorsomedial
telencephalon (Iler et al.,
1995; Rowitch et al.,
1998
). Second, Yoshida et al.
(Yoshida et al., 1997
) have
previously noted weak ectopic telencephalic expression of Wnt1 in
Emx2/ mice at 10.5 dpc. By contrast, our
results establish robust expression of Wnt1 in the cortical hem
organizer region and dorsal cortical ventricular zone at 12.5 dpc, extending
the observations of Yoshida et al.
(Yoshida et al., 1997
) and
raising the possibility of phenotypic consequences of ectopic Wnt1
expression at a critical stage in telencephalic development. Indeed, we
confirmed by histological and expression analysis that the cortex of
Wnt1-Tg mice was a partial phenocopy of
Emx2/ cortical dysplasia, sharing profound
abnormalities of the preplate and their derivative cell types. Finally, we
identified LGH in the cortex of Emx2/ mice
and verified that precise ectopic expression of Wnt1 within the
Emx2 domain was sufficient for LGH formation. Together, these
findings strongly support a model in which EMX2 directly binds the
Wnt1 enhancer and represses expression of Wnt1 in the
developing telencephalon (Fig.
8).
Previous work has shown that EMX2 regulates several distinct aspects of
cortical development. First, EMX2 activity regulates proliferation of neural
precursors of the developing telencephalon and neural stem cells
(Galli et al., 2002;
Heins et al., 2001
), which may
account in part for the small size of the cortex and cortical hem in the
Emx2-null mice. Additionally, Emx2 function is necessary for
proper development, but not specification, of the hippocampus
(Tole et al., 2000
). A similar
phenotype is observed in Wnt3a mutants
(Lee et al., 2000
). Thus, one
possibility is that EMX2 serves as a `competence factor' that is necessary for
neural precursors to respond to a growth signal, while Wnt3a and other Wnt
proteins may act principally as mitogens within the developing telencephalon.
We noted that the hippocampus and cortical hem are normally formed in the
majority in Wnt1-Tg animals, indicating that hypoplasia of these
structures in Emx2-null mice is unrelated to ectopic expression of
Wnt1. Indeed, the overall size of Wnt1-Tg brain was
typically larger than normal (megalencephalic), consistent with the known
mitogenic effects of Wnt1 and ß-catenin signaling on a wild-type
genetic background (Chenn and Walsh,
2002
; Danielian and McMahon,
1996
; Dickinson et al.,
1994
; Megason and McMahon,
2002
). Ectopic expression of Wnt1 alone also did not
reproduce the diffuse architectural abnormalities of cortical plate lamination
and radial glial morphology present in the cortex of
Emx2/ mice, most probably because of
variability of gene dosage and/or non-uniform (variegated) expression commonly
observed in the analysis of founder transgenic mice. Nevertheless, we observed
LGH and abnormal formation of subplate and marginal zone populations in a
total of 3/4 (75%) independent transgenic founders. In summary, our results
suggest that repression of Wnt1 by EMX2 is important in its capacity
as a determinant of cortical layer I and SP structure and function, but not
for its other roles in the regulation of neural precursor proliferation,
cortical lamination in layers II-VI or development of the hippocampus.
Factors underlying cortical dysplasia and heterotopia formation in
Emx2/ mice
The preplate (PP) forms as the initial wave of neural precursors leaves the
ventricular zone and migrates radially to the margin of the developing
telencephalon. The PP, MZ and SP then serve as a framework for the subsequent
influx of neurons from the ventricular zone, cortical hem and ventral
forebrain, potentially along tangential and radial processes of cells that
reside in these layers (Parnavelas,
2000). Because ectopic Wnt1 expression was never detected
in the PP or MZ of Emx2-null or Wnt1-Tg mice, it is likely
that it acts principally on the early ventricular zone progenitors of the
dorsomedial cortex. It is now known that cells from this region, especially
the cortical hem, appear much more migratory than originally assumed. Several
reports suggest that neurons from the hem migrate extensively into the
adjacent cortical primordium (Meyer et
al., 2002
; Monuki et al.,
2001
). As a result, ectopic Wnt1 is expressed early on in
the same progenitor cells that will later migrate into and throughout the
neocortex thereby creating the potential for very long-range functional
effects.
Although HBS Wnt1 regulatory sequences drove ectopic
expression in the dorsomedial telencephalon commencing at
10-somites
(8.75 dpc), we first observed heterotopias in Wnt1-Tg mice at 13.5
dpc, contemporaneous with PP maturation and the earliest formation of the MZ
and SP. This finding is consistent with a model in which heterotopias result
from defects in the developing MZ and SP or pre-existing defects in the PP. We
observed striking and diffuse deficiencies of reelin-expressing CR cells and
calretinin-expressing neurons as early as 13.5 dpc in the telencephalon of
Wnt1-Tg mice. Evidently, Wnt1 signaling inhibits development or
initial migration of preplate cells and their processes.
Another feature common to heterotopias of
Emx2/ and Wnt1-Tg mice is the novel
finding of abnormally located processes of radial glia outside the glia
limitans and limiting basement membrane of the leptomeninges. Such ectopically
placed processes imply the existence of focal defects in the glia limitans and
laminin 1-containing subpial basement membrane during the period of radial
migration of cells. In fact, mice with an unstable basement membrane, owing to
a mutation in laminin-g-1, develop defects in the basement membrane and glia
limitans at early stages (10-12 dpc) associated with numerous LGH
(Halfter et al., 2002).
Although we found defects in the glia limitans/basement membrane complex
associated with heterotopias in Emx2/ and
Wnt1-Tg mice using Nestin immunostaining at late stages (18.5 dpc),
we were unable to detect primary defects in the limiting plate in regions
overlying MZ heterotopias of Wnt1-Tg mice at early stages of radial
migration (13.5 dpc) by laminin 1, Nestin or TuJ1 immunostaining (Nestin and
TuJ1, data not shown). Together, these data suggest that positioning of the
radial glial foot process and formation of the pial basement membrane may be
linked to early events in formation of the marginal zone and subsequent
leptomeningeal heterotopia formation, but that such defects are not required
for the formation of heterotopias within the marginal zone.
EMX2-Wnt interactions during forebrain development
Regulatory interactions between Wnt signaling and Emx2 expression
in the telencephalon have recently been described
(Theil et al., 2002). However,
we did not observe abnormal expression of Emx2 in the telencephalon
of Wnt1-Tg mice (K.L., E.G. and D.R., unpublished), nor would one
expect regulation of Emx2 directed by Wnt1 given their
non-overlapping patterns of expression during normal development. Because
Emx2 expression is unaltered in the
HBS Wnt1
transgenic mice in precisely the same regions where we see ectopic
Wnt1 expression, it is evident that EMX2 cannot repress expression
from the
HBS Wnt1 enhancer construct. Similar observations
apply to analysis of lacZ reporter constructs with mutated or deleted
HBS sequences (Iler et al.,
1995
; Rowitch et al.,
1998
). Although we do not have evidence for direct EMX2
interaction/repression of Wnt1 at the HBS sequences in vivo, previous
work suggests this to be the case. Within the entire 5.5 kb Wnt1
enhancer, which has been shown to be necessary and sufficient for regulation
of Wnt1 in vivo (Danielian et
al., 1997
; Echelard et al.,
1994
), homeodomain proteins have been found to bind only the HBS1
and HBS2 sites located
5 kb downstream of the Wnt1 polyA
(Iler et al., 1995
;
Shang et al., 1994
). Together,
these observations suggest that regulation of Wnt1 by EMX2 is via
direct binding to HBS cis-acting regulatory sequences.
Because multiple Wnt genes are expressed in the developing forebrain (e.g.
Wnt2b, Wnt3a, Wnt5a, Wnt 7a, Wnt7b, Wnt 8a and Wnt8b), it is
not obvious why there should be a mechanism for repression of Wnt1.
It might be important to regulate the total dosage of Wnt protein during
development; alternatively, Wnt1 could have specific signaling
effects that do not overlap with other Wnt proteins. In this regard it is
interesting to note that ectopic expression of Wnt1 driven by
HBS1 regulatory sequences overlaps that of Wnt7a in the
cortical ventricular zone (Lee et al.,
2000
). Wnt family members can be divided into functional
classes based on downstream signaling via ß-catenin: Wnt1 and
Wnt3a signal via ß-catenin, in contrast to Wnt5a and
Wnt7a (see
http://www.stanford.edu/~rnusse/wntwindow.html).
Thus, it is possible that ectopic activation of ß-catenin signaling per
se affects the radial migration of neurons emigrating from the ventricular
zone and/or positioning of radial glia. Shinozaki et al. have recently shown
requirements for Emx1/2 function during tangential migration of MGE
precursors into the neocortex (Shinozaki
et al., 2002
). In particular, wild-type MGE precursors failed to
migrate into the Emx-null cortex, indicating a non-cell-autonomous
defect. Bishop et al. have suggested that Emx activity is critical for
afferent and efferent axonal projections in the neocortex
(Bishop et al., 2003
). Further
work will be required to establish whether aberrant expression of
ß-catenin or WNT1 protein is directly related to abnormal patterns of
neural migration, axon pathfinding and preplate development in Emx mutant
mice.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Alcantara, S., Ruiz, M., D'Arcangelo, G., Ezan, F., de Lecea,
L., Curran, T., Sotelo, C. and Soriano, E. (1998).
Regional and cellular patterns of reelin mRNA expression in the forebrain of
the developing and adult mouse. J. Neurosci.
18,7779
-7799.
Allendoerfer, K. L. and Shatz, C. J. (1994). The subplate, a transient neocortical structure: its role in the development of connections between thalamus and cortex. Annu. Rev. Neurosci. 17,185 -218.[CrossRef][Medline]
Bishop, K. M., Garel, S., Nakagawa, Y., Rubenstein, J. L. and O'Leary, D. D. (2003). Emx1 and Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, development of cortical efferents, and thalamocortical pathfinding. J. Comp. Neurol. 457,345 -360.[CrossRef][Medline]
Boncinelli, E., Gulisano, M. and Broccoli, V. (1993). Emx and Otx homeobox genes in the developing mouse brain. J. Neurobiol. 24,1356 -1366.[Medline]
Brunelli, S., Faiella, A., Capra, V., Nigro, V., Simeone, A., Cama, A. and Boncinelli, E. (1996). Germline mutations in the homeobox gene EMX2 in patients with severe schizencephaly. Nat. Genet. 12,94 -96.[Medline]
Chenn, A. and Walsh, C. A. (2002). Regulation
of cerebral cortical size by control of cell cycle exit in neural precursors.
Science 297,365
-369.
Costa, C., Harding, B. and Copp, A. J. (2001).
Neuronal migration defects in the Dreher (Lmx1a) mutant mouse: role of
disorders of the glial limiting membrane. Cereb.
Cortex 11,498
-505.
Cotter, D., Honavar, M., Lovestone, S., Raymond, L., Kerwin, R., Anderton, B. and Everall, I. (1999). Disturbance of Notch-1 and Wnt signalling proteins in neuroglial balloon cells and abnormal large neurons in focal cortical dysplasia in human cortex. Acta Neuropathol. (Berl) 98,465 -472.[CrossRef][Medline]
Danielian, P. S. and McMahon, A. P. (1996). Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 383,332 -334.[CrossRef][Medline]
Danielian, P. S., Echelard, Y., Vassileva, G. and McMahon, A. P. (1997). A 5.5-kb enhancer is both necessary and sufficient for regulation of Wnt-1 transcription in vivo. Dev. Biol. 192,300 -309.[CrossRef][Medline]
Dickinson, M. E., Krumlauf, R. and McMahon, A. P.
(1994). Evidence for a mitogenic effect of Wnt-1 in the
developing mammalian central nervous system.
Development 120,1453
-1471.
Echelard, Y., Vassileva, G. and McMahon, A. P.
(1994). Cis-acting regulatory sequences governing Wnt-1
expression in the developing mouse CNS. Development
120,2213
-2224.
Ellison, D. (1998). Neuropathology: A Reference Text of CNS Pathology. London, Chicago, IL: Mosby.
Faiella, A., Brunelli, S., Granata, T., D'Incerti, L., Cardini, R., Lenti, C., Battaglia, G. and Boncinelli, E. (1997). A number of schizencephaly patients including 2 brothers are heterozygous for germline mutations in the homeobox gene EMX2. Eur. J. Hum. Genet. 5,186 -190.[Medline]
Fairen, A., Morante-Oria, J. and Frassoni, C. (2002). The surface of the developing cerebral cortex: still special cells one century later. Prog. Brain Res. 136,281 -291.[Medline]
Friede, R. L. (1989). Developmental Neuropathology. Berlin, New York: Springer-Verlag.
Furuta, Y., Piston, D. W. and Hogan, B. L.
(1997). Bone morphogenetic proteins (BMPs) as regulators of
dorsal forebrain development. Development
124,2203
-2212.
Galli, R., Fiocco, R., de Filippis, L., Muzio, L., Gritti, A.,
Mercurio, S., Broccoli, V., Pellegrini, M., Mallamaci, A. and Vescovi,
A. L. (2002). Emx2 regulates the proliferation of stem cells
of the adult mammalian central nervous system.
Development 129,1633
-1644.
Gleeson, J. G. and Walsh, C. A. (2000). Neuronal migration disorders: from genetic diseases to developmental mechanisms. Trends Neurosci. 23,352 -359.[CrossRef][Medline]
Granata, T., Farina, L., Faiella, A., Cardini, R., D'Incerti, L., Boncinelli, E. and Battaglia, G. (1997). Familial schizencephaly associated with EMX2 mutation. Neurology 48,1403 -1406.[Abstract]
Greenfield, J. G., Graham, D. I. and Lantos, P. L. (2002). Greenfield's Neuropathology. London, New York: Arnold.
Grove, E. A., Tole, S., Limon, J., Yip, L. and Ragsdale, C.
W. (1998). The hem of the embryonic cerebral cortex is
defined by the expression of multiple Wnt genes and is compromised in
Gli3-deficient mice. Development
125,2315
-2325.
Halfter, W., Dong, S., Yip, Y. P., Willem, M. and Mayer, U.
(2002). A critical function of the pial basement membrane in
cortical histogenesis. J. Neurosci.
22,6029
-6040.
Heins, N., Cremisi, F., Malatesta, P., Gangemi, R. M., Corte, G., Price, J., Goudreau, G., Gruss, P. and Gotz, M. (2001). Emx2 promotes symmetric cell divisions and a multipotential fate in precursors from the cerebral cortex. Mol. Cell. Neurosci. 18,485 -502.[CrossRef][Medline]
Hirano, S., Houdou, S., Hasegawa, M., Kamei, A. and Takashima, S. (1992). Clinicopathologic studies on leptomeningeal glioneuronal heterotopia in congenital anomalies. Pediatr. Neurol. 8,441 -444.[CrossRef][Medline]
Iida, K., Hirano, S., Takashima, S. and Miyahara, S. (1994). Developmental study of leptomeningeal glioneuronal heterotopia. Pediatr. Neurol. 10,295 -298.[CrossRef][Medline]
Iler, N., Rowitch, D. H., Echelard, Y., McMahon, A. P. and Abate-Shen, C. (1995). A single homeodomain binding site restricts spatial expression of Wnt-1 in the developing brain. Mech. Dev. 53,87 -96.[CrossRef][Medline]
Lavdas, A. A., Grigoriou, M., Pachnis, V. and Parnavelas, J.
G. (1999). The medial ganglionic eminence gives rise to a
population of early neurons in the developing cerebral cortex. J.
Neurosci. 19,7881
-7888.
Lee, S. M., Tole, S., Grove, E. and McMahon, A. P.
(2000). A local Wnt-3a signal is required for development of the
mammalian hippocampus. Development
127,457
-467.
Lu, Q. R., Park, J. K., Noll, E., Chan, J. A., Alberta, J., Yuk,
D., Alzamora, M. G., Louis, D. N., Stiles, C. D., Rowitch, D. H. et
al. (2001). Oligodendrocyte lineage genes (OLIG) as molecular
markers for human glial brain tumors. Proc. Natl. Acad. Sci.
USA 98,10851
-10856.
Mallamaci, A., Mercurio, S., Muzio, L., Cecchi, C., Pardini, C.
L., Gruss, P. and Boncinelli, E. (2000a). The lack of
Emx2 causes impairment of Reelin signaling and defects of neuronal migration
in the developing cerebral cortex. J. Neurosci.
20,1109
-1118.
Mallamaci, A., Muzio, L., Chan, C. H., Parnavelas, J. and Boncinelli, E. (2000b). Area identity shifts in the early cerebral cortex of Emx2/ mutant mice. Nat. Neurosci. 3,679 -686.[CrossRef][Medline]
Megason, S. G. and McMahon, A. P. (2002). A
mitogen gradient of dorsal midline Wnts organizes growth in the CNS.
Development 129,2087
-2098.
Meyer, G., Soria, J. M., Martinez-Galan, J. R., Martin-Clemente, B. and Fairen, A. (1998). Different origins and developmental histories of transient neurons in the marginal zone of the fetal and neonatal rat cortex. J. Comp. Neurol. 397,493 -518.[CrossRef][Medline]
Meyer, G., Perez-Garcia, C. G., Abraham, H. and Caput, D.
(2002). Expression of p73 and Reelin in the developing human
cortex. J. Neurosci. 22,4973
-4986.
Mischel, P. S., Nguyen, L. P. and Vinters, H. V. (1995). Cerebral cortical dysplasia associated with pediatric epilepsy. Review of neuropathologic features and proposal for a grading system. J. Neuropathol. Exp. Neurol. 54,137 -153.[Medline]
Monuki, E. S., Porter, F. D. and Walsh, C. A. (2001). Patterning of the dorsal telencephalon and cerebral cortex by a roof plate-Lhx2 pathway. Neuron 32,591 -604.[Medline]
Monuki, E. S. and Walsh, C. A. (2001). Mechanisms of cerebral cortical patterning in mice and humans. Nat. Neurosci. Suppl. 4,1199 -1206.
Moore, S. A., Saito, F., Chen, J., Michele, D. E., Henry, M. D., Messing, A., Cohn, R. D., Ross-Barta, S. E., Westra, S., Williamson, R. A. et al. (2002). Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature 418,422 -425.[CrossRef][Medline]
Muzio, L., DiBenedetto, B., Stoykova, A., Boncinelli, E., Gruss,
P. and Mallamaci, A. (2002). Emx2 and Pax6 control
regionalization of the pre-neuronogenic cortical primordium. Cereb.
Cortex 12,129
-139.
Parnavelas, J. G. (2000). The origin and migration of cortical neurones: new vistas. Trends Neurosci. 23,126 -131.[CrossRef][Medline]
Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P.
(1993). Mouse Wnt genes exhibit discrete domains of expression in
the early embryonic CNS and limb buds. Development
119,247
-261.
Pellegrini, M., Mansouri, A., Simeone, A., Boncinelli, E. and
Gruss, P. (1996). Dentate gyrus formation requires Emx2.
Development 122,3893
-3898.
Rice, D. S. and Curran, T. (1999). Mutant mice
with scrambled brains: understanding the signaling pathways that control cell
positioning in the CNS. Genes Dev.
13,2758
-2773.
Rowitch, D. H., Danielian, P. S., Lee, S. M., Echelard, Y. and McMahon, A. P. (1997). Cell interactions in patterning the mammalian midbrain. Cold Spring Harb. Symp. Quant. Biol. 62,535 -544.[Medline]
Rowitch, D. H., Echelard, Y., Danielian, P. S., Gellner, K.,
Brenner, S. and McMahon, A. P. (1998). Identification
of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence
that governs Wnt-1 expression in the murine neural plate.
Development 125,2735
-2746.
Shang, Z., Ebright, Y. W., Iler, N., Pendergrast, P. S., Echelard, Y., McMahon, A. P., Ebright, R. H. and Abate, C. (1994). DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA. Proc. Natl. Acad. Sci. USA 91,118 -122.[Abstract]
Shinozaki, K., Miyagi, T., Yoshida, M., Miyata, T., Ogawa, M., Aizawa, S. and Suda, Y. (2002). Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex. Development 129,3479 -3492.[Medline]
Super, H., del Rio, J. A., Martinez, A., Perez-Sust, P. and
Soriano, E. (2000). Disruption of neuronal migration and
radial glia in the developing cerebral cortex following ablation of
Cajal-Retzius cells. Cereb. Cortex
10,602
-613.
Theil, T., Aydin, S., Koch, S., Grotewold, L. and Ruther, U.
(2002). Wnt and Bmp signalling cooperatively regulate graded Emx2
expression in the dorsal telencephalon. Development
129,3045
-3054.
Tole, S., Goudreau, G., Assimacopoulos, S. and Grove, E. A.
(2000). Emx2 is required for growth of the hippocampus but not
for hippocampal field specification. J. Neurosci.
20,2618
-2625.
Whiting, J., Marshall, H., Cook, M., Krumlauf, R., Rigby, P. W., Stott, D. and Allemann, R. K. (1991). Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression. Genes Dev. 5,2048 -2059.[Abstract]
Xie, Y., Skinner, E., Landry, C., Handley, V., Schonmann, V.,
Jacobs, E., Fisher, R. and Campagnoni, A. (2002).
Influence of the embryonic preplate on the organization of the cerebral
cortex: a targeted ablation model. J. Neurosci.
22,8981
-8991.
Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N.,
Kuratani, S. and Aizawa, S. (1997). Emx1 and Emx2 functions
in development of dorsal telencephalon. Development
124,101
-111.
Zecevic, N., Milosevic, A., Rakic, S. and Marin-Padilla, M. (1999). Early development and composition of the human primordial plexiform layer: an immunohistochemical study. J. Comp. Neurol. 412,241 -254.[CrossRef][Medline]