Max-Delbrueck-Center for Molecular Medicine, Berlin, 13092, Germany
Author for correspondence (e-mail:
willnow{at}mdc-berlin.de)
Accepted 10 November 2004
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SUMMARY |
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Key words: LDL receptor-related proteins, Bone morphogenic proteins, Sonic hedgehog, Yolk sac, Holoprosencephaly, Mouse, LRP2
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Introduction |
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LRP2, or megalin (herein referred to as megalin), is another member of the
LDL receptor gene family essential for brain development
(Saito et al., 1994;
Willnow et al., 1996
). At
mid-gestation, the receptor is expressed in the neuroepithelium of the embryo
as well as in the visceral endoderm of the yolk sac
(Willnow et al., 1996
;
Zheng et al., 1994
). Targeted
disruption of the respective gene in the mouse results in holoprosencephalic
syndrome and in perinatal lethality
(Willnow et al., 1996
).
However, the exact mechanism by which megalin contributes to forebrain
formation, and why holoprosencephaly (HPE) ensues in megalin-deficient mice,
is still unclear.
Holoprosencephaly is defined as a failure of the prosencephalon (forebrain)
to separate along the mid-sagittal axis into discrete hemispheres. This defect
is likely to be the result of defective patterning during development
involving improper specification of the rostral portion of the neural tube.
Many genetic pathways involved in neural tube patterning have been implicated
in the etiology of HPE (Wallis and Muenke,
1999). In particular, alterations in pathways that specify the
dorsoventral axis of the rostral neural tube may cause this syndrome. For
example, increased dorsal signaling through bone morphogenic proteins (BMPs),
members of the transforming growth factor ß superfamily, and through WNT
proteins, results in HPE (Golden et al.,
1999
; Wallis et al.,
1999
; Anderson et al.,
2002
; Lagutin et al.,
2003
). Also, loss of sonic hedgehog (SHH) expression, a factor
that specifies the ventral neural tube, leads to holoprosencephalic syndrome
in humans and in mice (Chiang et al.,
1996
; Roessler et al.,
1996
).
Concerning a role for megalin in forebrain development, some evidence
suggests involvement of the receptor in the SHH pathway
(McCarthy et al., 2002;
Farese and Herz, 1998
). Sonic
hedgehog is a secreted protein expressed in the notochord and the floorplate
that undergoes autocatalytic cleavage into a 19 kDa amino terminal (SHH-N) and
a 25 kDa carboxyl terminal (SHH-C) domain. During cleavage, a cholesterol
moiety becomes covalently attached to SHH-N, the active fragment that binds to
the receptor Patched (Ptch1) and activates downstream SHH target genes through
Smoothened (Smo). Cholesterol modification of SHH-N is crucial for proper
activity, perhaps by spatially restricting SHH activity
(Porter et al., 1996
).
Expression of megalin in the yolk sac and its ability to take up lipoproteins
suggests a possible function for the receptor in delivery of cholesterol-rich
lipoproteins from the maternal circulation to the embryo for SHH activation
(Willnow et al., 1992
;
Farese and Herz, 1998
).
Alternatively, expression of megalin in the neuroepithelium and its ability to
bind SHH indicates a more direct effect as a co-receptor in SHH signaling
(McCarthy et al., 2002
).
In the present study, we used mice in which the megalin gene was ubiquitously inactivated (including yolk sac and neuroepithelium) or conditional mutants with loss of megalin only in the neuroepithelium to examine the contribution of the receptor in these tissues to forebrain formation. Sustained expression of megalin in the yolk sac did not rescue mice from HPE, indicating that a receptor activity in the neuroepithelium is essential for normal forebrain development. In the neuroepithelium, megalin activity affects dorsal and ventral patterning of the forebrain, and promotes ventral cell fate specification.
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Materials and methods |
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Histological and immunohistological analysis
Embryos or yolk sac tissues were dissected from pregnant mice at designated
time points, fixed overnight in 4% paraformaldehyde in PBS at 4°C, and
embedded in paraffin. Routine paraffin sections were cut at 2-10 µm and
stained with hematoxylin and eosin (H+E). For immunohistology, sections were
incubated with sheep anti-megalin (1:50.000; provided by P. Verroust, INSERM,
Paris), mouse anti-ß III tubulin (TuJ1) (1:500; BabCO, Richmond, USA), or
rabbit anti-phospho-Smad1/5/8 (1:100; Cell Signaling Technology, Beverly, USA)
antisera followed by secondary peroxidase- (1:100; Dako, Hamburg, Germany),
Cy3- or Cy5-conjugated antibodies (1:400; Jackson ImmunoResearch Laboratories,
West Baltimore Pike, USA). Nuclei were counterstained with 1 µM YO-PRO-1
iodide (Molecular Probes, Eugene, USA).
In-situ hybridization
Embryos were dissected and fixed overnight in 4% paraformaldehyde in PBS at
4°C. Further processing of the embryos and whole-mount in-situ
hybridization was carried out essentially as described
(Hammes et al., 2001). The
localization of signals was studied in whole embryos or in plastic sections
thereof. Plasmids for generating in-situ probes were kindly provided by W.
Birchmeier [MDC, Berlin; Nkx2.1 (Titf1 Mouse Genome
Informatics), Olig2, Wnt1, Msx1, Fgf8], J. Huelsken (ISREC,
Epalinges; Ptch1, Bmp4, Wnt7, Gli2, Gli3), A. Joyner (Mount Sinai
Research Institute, Toronto; Gli1), J. L. R. Rubenstein (UCSF, San
Francisco; Dlx2), A. P. McMahon (Harvard University, Cambridge;
Shh), and B. Hogan [Vanderbilt University Medical School, Nashville;
Hnf3b (Foxa2 Mouse Genome Informatics)], or
generated from first strand mouse cDNA (Pax6, Pax2, Smo, Plp2).
Apoptosis and proliferation assays
Embryonic day 10.5 embryos were dissected, fixed, paraffin-embedded and
sectioned as described above. After de-waxing and rehydration, the sections
were incubated with 20 µg/ml proteinase K in 10 mmol/l Tris/HCl, pH 7.4 for
20 minutes at 37°C. Detection of apoptotic cells was performed using the
TUNEL reaction kit
(www.Roche.com)
following manufacturer's instructions. For detection of proliferation,
paraffin sections were incubated with rabbit anti-phosphohistone H3 antibody
(1:200; Upstate, Lake Placid) followed by secondary Cy3-conjugated sheep
anti-rabbit IgG (Sigma,
www.sigma.com),
and detection by immunofluorescence microscopy.
BIAcore analysis and cell culture experiments
Carrier-free preparations of recombinant human BMP4 and BMP5 were purchased
from Research and Development
(www.RnDSystems.com).
Recombinant His-tagged RAP was produced and purified from bacteria by standard
Ni2+ affinity chromatography. As a source for WNT1 we used
conditioned medium from Rat-2 cells infected with a retroviral Wnt1
expression construct that excrete large amounts of the protein (Rat-2/WNT1)
and compared it with medium from cells infected with the empty expression
vector (Rat-2/MV7) (cell lines kindly provided by A. M. C. Brown, Cornell
University, New York) (Giarre et al.,
1998). Interaction of megalin with BMP4, BMP5 and RAP (0.5-1
µmol/l) or WNT1 (20-fold concentrated medium) was tested by surface plasmon
resonance (SPR) as described before
(Hilpert et al., 1999
). For
cell uptake studies, BMP4 was iodinated according to the IODOGEN protocol
(specific activity 3.1x108 cpm/µg). Replicate monolayers
of rat choriocarcinoma (BN16) cells were incubated for 2 hours with 1.6 ng/ml
of 125I-BMP4 in the presence of buffer, 200 µmol/l chloroquine,
or 100 µg/ml RAP. Degradation of ligand was determined as the total amount
of 125I-labeled trichloroacetic acid-soluble material released into
the culture medium and expressed as picogram ligand per mg of total cell
protein (Hilpert et al.,
1999
).
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Results |
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Forebrain defects in mice with epiblast-specific megalin gene disruption
During early development, megalin is expressed in the visceral endoderm of
the yolk sac and in the neuroepithelium
(Fig. 2A,C). Conceivably, lack
of megalin expression in either the embryonic or the extra-embryonic tissues
(or both) may be responsible for the brain malformations in knockout mice. To
dissect the contributions of both tissues to the occurrence of forebrain
defects, we used conditional gene targeting to inactivate the megalin gene
specifically in the embryo proper and retain gene expression in the yolk sac.
To do so, we employed a mouse line carrying a megalin gene modified by
loxP sites (megalinlox/lox) that had been used
successfully before to achieve tissue-specific receptor gene inactivation
(Leheste et al., 2003).
megalinlox/lox animals were bred with mice carrying the
cre recombinase gene under control of the mesenchyme homeobox 2 gene promoter
(Meox2tm1(cre)Sor), directing Cre gene expression
to the epiblast (Tallquist and Soriano,
2000
). Consistent with this strategy, embryos homozygous for the
loxP-modified megalin gene and carrying the Cre transgene
(megalinlox/lox/Meox2tm1(cre)Sor)
lacked expression of the receptor in the neuroepithelium
(Fig. 2D) but not in the yolk
sac (Fig. 2B). Despite normal
expression of megalin in the visceral endoderm of the yolk sac,
megalinlox/lox/Meox2tm1(cre)Sor
embryos suffered from similar brain defects to
megalin/ embryos
(Fig. 2E,F), demonstrating that
megalin activity in the embryo proper is required for brain development.
|
Given the reduction in neuroepithelial wall thickness in knockout embryos (Fig. 1R), we first analyzed mitotic activity in this tissue. Decreased proliferation was observed in a restricted area of the rostral ventral neural tube, as evidenced by the number of nuclei that stained positive for phosphohistone (8.3±1.1 SD versus 22.7±2 SD mitotic cells per microscopic field; P=0.001; n=3 animals for each genotype) (Fig. 3B,E). Other areas of the neural tube were not affected (data not shown). Furthermore, TUNEL assays did not reveal any discernable difference in the number of apoptotic cells at E10.5 in the ventral neuroepithelium (5.0±0.5 SD versus 4.7±0.6 SD apoptotic nuclei per microscopic field, P=0.64) (Fig. 3C,F) or the dorsal neuroepithelium (not shown).
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So far our results had uncovered an obvious effect of megalin deficiency on the specification of ventral cell fate in the rostral ventral forebrain, probably due to locally restricted loss of Shh in the AEP. Consistent with normal expression of Shh in areas of the forebrain other than the AEP, the expression patterns of the receptor Ptch1 (Fig. 7E), the signaling component Smo (not shown), or global Shh target genes Hnf3b (Fig. 7F), Gli1, Gli2 or Gli3 (not shown) were unchanged in megalin knockout mice.
Altered dorsal telencephalic patterning in megalin/ mice
Given the loss of ventral Shh-dependent pathways in
megalin/ embryos, we also tested whether expression
of dorsal markers of the developing brain may be deranged in
receptor-deficient mice. Wnt1, a marker of early forebrain
development, is expressed in the dorsal midline of the di- and mesencephalon,
and in the isthmus of wild types at E10.5. No difference in expression
patterns of WNT1 (Fig. 7G) or
another WNT ligand, WNT7 (data not shown), was seen in knockouts, suggesting
normal dorsal patterning of the diencephalon. However, the situation was
different when evaluating dorsal markers also found in the telencephalon.
Pax6 is normally expressed in the dorsal part and ventral thalamus of
the diencephalon, and the eyes, as well as in the dorsal telencephalon
(Fig. 7H). By contrast, in
E10.5 megalin/ embryos Pax6 expression
significantly extended from dorsal areas into ventral areas of the
telencephalic neural tube, while expression in the optic vesicles was mostly
lost (Fig. 7H).
Enhanced Bmp4 signaling and aberrant Fgf8 expression in the dorsal midline of megalin/ mice
Fibroblast growth factor (Fgf) 8 and bone morphogenic protein (BMP) 4 are
morphogens that also play key roles in dorsoventral patterning of the rostral
head and that act in precise synergy with SHH to maintain normal
prosencephalic development (Schneider et
al., 2001; Ohkubo et al.,
2002
). Intriguingly, expression of both morphogens was also
significantly altered in mice lacking megalin.
At E10.5, the expression of Fgf8 was reduced in the rostral ventral telencephalon but extended from the commissural plate along the midline into more dorsal and caudal regions of the forebrain (Fig. 8A). Bmp4, demarcating dorsal midline structures, was also aberrantly expressed at E10.5. In wild types, Bmp4 expression was restricted to the dorsomedial part of the telencephalon and the dorsal midline of the most anterior diencephalon (Fig. 8B). In mutants, expression in the dorsomedial neuroepithelium of the tel- and diencephalon was significantly increased and extended along the midline into more caudal regions of the roof (Fig. 8B, arrowhead). Bmp4 overexpression in megalin-deficient embryos resulted in enhanced and ventrally expanded BMP4 signaling in the prosencephalon but not in other areas of the neural tube (such as the spinal cord), as shown by immunodetection of phosphorylated Smad proteins (P-Smad) (Fig. 8C). Similar changes in Bmp4 and Fgf8 signals were also seen in E10.5 embryos with epiblast-specific receptor gene defect (data not shown).
|
Megalin acts as an endocytic receptor for BMP4
Based on the distinct alterations in SHH and BMP4-dependent pathways in the
neural tube of megalin-deficient embryos, a direct involvement of this
receptor in the activity of either morphogen seems plausible. The ability of
megalin to bind SHH has been reported before
(McCarthy et al., 2002). To
also establish a potential role for this receptor in BMP4 signaling, we tested
the interaction of the receptor with BMP4 by surface plasmon resonance (SPR)
analysis. In this assay, BMP4 strongly bound to megalin, while the related
factor BMP5 or WNT1 did not (Fig.
9A), suggesting a possible function for megalin as BMP4 receptor
in the early forebrain. Exposure of megalin-expressing (BN16) cells to
radiolabeled BMP4 resulted in endocytic uptake and lysosomal degradation of
the protein (Fig. 9B). The
cellular catabolism of BMP4 was mediated by megalin, because uptake and
degradation of 125I-BMP4 was blocked completely by the megalin
antagonist receptor-associated protein (RAP) (Christensen et al., 1992;
Hilpert et al., 1999
) and by
chloroquine, an inhibitor of lysosomal degradation
(Fig. 9B). Taken together,
these findings indicated a possible function of megalin as BMP4 clearance
receptor in the neuroepithelium and increased BMP signaling in the forebrain
as a consequence of the megalin gene defect.
|
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Discussion |
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The role of megalin in forebrain development: yolk sac or neuroepithelium
Previously, deficiency for megalin in the yolk sac and/or in the
neuroepithelium was held responsible for the holoprosencephalic symptoms in
megalin/ mice
(Willnow et al., 1996;
Farese and Herz, 1998
;
McCarthy and Argraves, 2003
).
Using Cre-mediated conditional gene inactivation, we generated a
mouse line with loss of megalin in the embryo but not in the yolk sac.
Surprisingly, restored expression of megalin in the yolk sac did not prevent
forebrain malformation (Fig.
2F). The limited number of
megalinlox/lox/Meox2tm1(cre)Sor
embryos available (n=15) precluded a detailed comparison of the
phenotypes of mice with complete and with epiblast-specific megalin gene
deletion. Therefore, we cannot exclude the fact that some phenotypic
differences exist in forebrain abnormalities in these two lines that reflect
the influence of megalin in the yolk sac on neural tube formation. However,
similar alterations in Fgf8, Shh and Bmp4 expression in both
lines strongly suggest that megalin deficiency in the neuroepithelium is the
main cause of the observed forebrain defects.
Megalin deficiency impairs SHH-dependent ventral cell fate
While the role of megalin in the yolk sac still awaits elucidation,
experimental evidence in this study identified the contribution of this
receptor in the neuroepithelium to specification of dorsal and ventral
signaling pathways in the anterior neural tube. Axial patterning of the neural
tube is controlled by secreted factors that have distinct expression domains
in the rostral forebrain. In particular, an intricate balance of the
morphogens SHH and BMP4 is crucial for specifying dorsoventral forebrain
patterning (Sasai and De Robertis,
1997; Ohkubo et al.,
2002
).
Sonic hedgehog activity defines the ventral neural tube and establishes
ventral neuronal and oligodendroglial cell populations
(Marin and Rubenstein, 2001;
Nery et al., 2001
;
Rowitch et al., 1999
;
Lu et al., 2000
). Inactivation
of the Shh gene in the mouse causes defective axial patterning,
cyclopia and absence of ventral cell types
(Chiang et al., 1996
).
Surprisingly, lack of megalin results in loss of Shh expression
specifically in the AEP, whereas expression in further caudal regions of the
neural tube is not affected (Fig.
4). The identical phenotype is seen in other models of abnormal
dorsoventral patterning and HPE, in particular in those with increased BMP4
activity, as discussed below. Why some defects impair Shh activity
exclusively in the AEP is unclear at present, but region-specific differences
in the molecular mechanism of SHH action in the rostral forebrain compared
with more caudal regions, such as the spinal cord, have been clearly
established (Dale et al.,
1997
). The spatially restricted loss of SHH in the AEP may explain
why no overt changes in some components of the global Shh signaling
pathway (e.g. Gli1-2, Hnf3b) are detectable in
megalin/ embryos at E10.5, and why some defects in
Shh knockout mice (such as absence of distal limb structures) are not
shared by megalin-deficient animals.
In the AEP, the Shh signal peaks around E10.5, when it overlaps
with Olig2 and Nkx2.1, markers of oligodendroglial and
neuronal lineages, respectively (Fig.
7). Ectopic expression of Shh locally induces
differentiation of oligodendrocytes from ventral neuroepithelial precursors
(Nery et al., 2001), a process
blocked by anti-SHH antibodies (Orentas et
al., 1999
). Consistent with loss of Shh in the AEP,
megalin-deficient embryos suffer from impaired establishment of
neuroepithelial progenitors and from a dramatic reduction in oligendroglial
and interneuronal cell populations, as demonstrated by almost complete absence
of Olig2, Nkx2.1, Dlx2 and TuJ1-positive cells in the ventral (but
not in the dorsal) forebrain (Fig.
7). Potentially, lack of proliferation, increased cell death or
impaired differentiation may be held responsible for the absence of ventral
cell fate specification. Decreased mitotic activity can be detected in the AEP
of megalin/ embryos; thus, this effect clearly
contributes to forebrain anomalies in this mouse line. However, the absence of
proliferative defects in other areas of the rostral forebrain and the normal
expression of many ventral marker genes (e.g. Ptch1, Smo, Hnf3b, Gli1,
Gli2) strongly suggest that alterations in SHH-dependent differentiation
processes also play a causative role. This hypothesis is in agreement with
recent findings in which SHH signaling was disrupted in the ventral
telencephalon of mice at E9.5 using conditional targeting of the Smo
gene. Spatial and temporary specific ablation of ventral SHH activity resulted
in a complete absence of oligodendrocytes and interneurons in the ventral
forebrain, due to impaired differentiation rather than altered proliferation
or apoptosis of progenitor cells (Fuccillo
et al., 2004
).
Megalin deficiency increases BMP4 activity in the rostral dorsal neural tube
Whereas SHH specifies ventral cell fate, BMPs provide inductive signals for
dorsal cell types such as the astroglial lineage
(Liem et al., 1995;
Gross et al., 1996
;
Timmer et al., 2002
). They act
through suppression of cell proliferation and induction of apoptosis
(Furuta et al., 1997
), and
function as potent antagonists of the ventral neural tube by blocking SHH
action on dorsal cell types and by inhibiting ventral cell fate
(McMahon et al., 1998
;
Liem et al., 2000
). Consistent
with an opponent action of dorsal and ventral signaling pathways in the neural
tube, increased activity of BMPs suppresses expression of Shh in the
rostral ventral neuroepithelium. For example, when beads soaked with
recombinant BMP4 or BMP5 are implanted into the neural tube of the chicken
forebrain, loss of Shh expression in the ventral forebrain is
observed to cause cyclopia and HPE (Golden
et al., 1999
). In mice genetically deficient for noggin,
a BMP4 antagonist, expression of dorsal cell fates is normal but
Shh-dependent ventral cell fate is lost progressively
(McMahon et al., 1998
).
Finally, in mice homozygous for the deletion of chordin and
heterozygous for the noggin gene defect, deficiencies of both BMP
antagonists also cause loss of Shh expression, specifically in the
AEP, and holoprosencephalic syndrome, a phenotype highly reminiscent of
defects in megalin/ embryos
(Anderson et al., 2002
).
As well as with alterations in Bmp4 and Shh pathways,
megalin-deficient embryos also present with changes in expression of
Fgf8 and Pax6. Fgf8 promotes rather than induces
rostroventral telencephalic fate (Wilson
and Houart, 2004), and its expression is considered to be
dependent on coordinated Shh and Bmp signaling
(Ohkubo et al., 2002
).
Therefore reduction in ventral Fgf8 gene expression and the extension
of the signal along the dorsal midline probably reflects secondary alterations
in dorsoventral patterning of the forebrain and abnormalities in formation of
midline tissue. Similarly, the expansion of Pax6 into ventral parts
of the telencephalon of receptor-deficient mice is probably a consequence of
absence of SHH in the AEP, as described for other models with specific loss of
Shh expression in the rostral forebrain
(Huh et al., 1999
;
Gofflot et al., 1999
).
The role of megalin in patterning of the rostral neural tube: a working model
What may be the mechanism whereby megalin affects dorsoventral
specification of the neural tube? Given the competing action of dorsal and
ventral signaling factors on neural tube patterning one may envision two
scenarios. In one model, the receptor may be essential to induce Shh
expression in cells of the AEP and its absence results in loss of SHH activity
and in impaired ventral cell fate. Thus, overexpression of the dorsal markers
such as BMP4 would be a consequence of the lack of ventral SHH signals. In an
alternative model, megalin may act as a negative regulator of Bmp4
activity in the dorsal neuroepithelium and receptor deficiency causes an
increase in dorsal BMP4 signals, an effect known to suppress Shh
(Golden et al., 1999;
McMahon et al., 1998
;
Liem et al., 2000
). Based on
the fact that increases in Bmp4 expression and signaling through
phosphorylated Smad proteins at E9.5 clearly precede defects observed in
Shh at E10.5, we favor the latter model.
A role of megalin in suppression of Bmp4 expression is supported
by findings obtained in this study and by work from others. In the early
forebrain, BMP4 is believed to induce its own expression via a positive
feedback loop; thus, increases in BMP4 activity are expected to induce
Bmp4 transcription (Blitz et al.,
2000). Furthermore, the importance of restricting BMP signals
during development is well established and several mechanisms have evolved to
negatively regulate morphogen action, including soluble BMP antagonists and
dominant negative pseudo-receptors
(Balemans and Van Hul, 2002
;
Onichtchouck et al., 1999
).
Taken together with the ability of megalin to catabolize BMP4
(Fig. 9), these findings
provide an explanatory model in which megalin acts as an endocytic receptor
for BMP4 in the neural tube, antagonizing morphogen signaling. As a
consequence of receptor deficiency, BMP4 activity may be locally increased,
causing activation of Bmp4 transcription and stimulation of rostral
and dorsal patterning and suppression of ventral patterning. A similar
function for megalin as a clearance receptor for parathyroid hormone (PTH)
suppressing signaling via the PTH receptor in the kidney has been documented
before (Hilpert et al., 1999
).
Few established cell lines express the receptor megalin, and BN16 cells are
the system of choice for testing megalin-dependent endocytosis. The lack of
expression of BMP receptors in BN16 cells (R.S., unpublished) currently
precludes directly testing the consequence of megalin activity on BMP
signaling in these cells. Therefore, future efforts should be directed toward
establishing suitable cellular systems (e.g. neural tube explants) to address
this question.
In conclusion, our studies have uncovered an important novel activity of
LRPs as modifiers of BMP4- and SHH-dependent patterning of the forebrain, and
the role that is played by megalin in this process. These findings have
identified megalin as an important factor in axial embryonic pattern formation
and characterized a novel molecular pathway that contributes to abnormal
specification of the ventral forebrain and to HPE, the most common
developmental brain anomaly in humans
(Wallis and Muenke, 1999).
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ACKNOWLEDGMENTS |
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Footnotes |
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