1 Center for Craniofacial Molecular Biology School of Dentistry University of
Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033,
USA
2 Department of Cancer Biology, Vanderbilt University, 22 South Pierce Avenue,
PRB Room 649, Nashville, TN 37232, USA
* Author for correspondence (e-mail: ychai{at}usc.edu)
Accepted 7 July 2003
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SUMMARY |
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Key words: Cranial neural crest (CNC), Calvaria development, Palatogenesis, TGFß type II receptor signaling
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Introduction |
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The mammalian palate develops from two primordia: the primary and the
secondary palate. The primary palate represents only a small part of the adult
hard palate and is the part anterior to the incisive fossa. The secondary
palate is the primordium of the hard and soft palate in adults. Palate
development is a multi-step process that involves palatal shelf growth,
elevation, midline fusion of palatal shelves and the disappearance of the
midline epithelial seam. The palatal structures are composed of the
CNC-derived ectomesenchyme and pharyngeal ectoderm
(Ferguson, 1988;
Shuler, 1995
;
Wilkie and Morriss-Kay, 2001
;
Zhang et al., 2002
).
TGFß signaling plays a pivotal role in regulating palatogenesis.
During mouse palatal development, both TGFß1 and TGFß3 are expressed
in the medial edge epithelium (MEE) of the palatal shelves, whereas TGFß2
expression is restricted to the CNC-derived mesenchyme beneath the MEE
(Fitzpatrick et al., 1990;
Pelton et al., 1990
). Upon
fusion of the palatal shelves and disappearance of the midline epithelial
seam, the expression of TGFß1 and TGFß3 is lost, suggesting crucial
functions of TGFß signaling in regulating palatal fusion.
Loss-of-function mutation of Tgfb2 or Tgfb3 results in cleft
palate. Tgfb2-null mutant mice exhibit anteroposterior cleft of the
secondary palate with only 23% phenotype penetrance
(Sanford et al., 1997
).
Significantly, Tgfb3-null mutation results in 100% penetrance of
cleft secondary palate (Kaartinen et al.,
1995
; Proetzel et al.,
1995
). The etiology of cleft palate in Tgfb3-null mutant
mice is apparently due to a failure of fusion of palatal shelves, which has
been rescued by addition of exogenous TGFß3 in an in vitro organ culture
system (Brunet et al., 1995
;
Taya et al., 1999
). Subsequent
studies have shown that TGFß3 is specifically required for the fusion of
palatal shelves, probably by enhancing the transformation of MEE cells into
the palatal mesenchyme and inducing apoptosis in the MEE
(Sun et al., 1998
;
Martinez-Alvarez et al.,
2000
).
TGFß IIR is expressed in both the MEE and CNC-derived palatal
mesenchyme (Wang et al., 1995;
Cui et al., 1998
). The
physiological function of TGFß IIR in regulating palatogenesis is not
known because Tgfbr2-null mutation results in early embryonic
lethality, thus, making it impossible to investigate the functional
significance of this signaling molecule in regulating palatogenesis
(Oshima et al., 1996
). Up
until now, most of the palatogenesis studies, such as the ones involving
TGFß signaling, have mainly focused on the molecular regulation of the
fate of MEE cells. Although CNC cells are critical for palatogenesis, very
little is known about the molecular mechanism that regulates the fate of the
CNC-derived palatal mesenchyme during palatogenesis.
The vertebrate skull includes both the neurocranium (such as the calvaria
and base of skull) and viscerocranium (such as mandible, zygoma, maxilla,
etc.). Calvaria formation is a complex and lengthy developmental process that
is initiated during embryogenesis and is completed in adulthood. The size
flexibility of the calvaria is crucial for accommodating the rapid growth of
the brain. Both the mesoderm and CNC-derived ectomesenchyme contribute to the
cranial skeletogenic mesenchyme, which gives rise to bony elements (such as
frontal, parietal and occipital bones) collectively known as the calvaria
(Wilkie and Morriss-Kay,
2001). Studies have shown that the dura mater, a dense fibrous
membrane underneath the calvaria, and cranial sutures provides crucial
regulatory signals for calvaria development. To date, studies suggest that
cranial sutures function as signaling centers for bone growth and remain
patent postnatally to accommodate cranium expansion. Premature closure of
cranial sutures affects the growth of the calvaria and results in
craniosynostosis (Wilkie and Morriss-Kay,
2001
).
Multiple growth and transcription factors play pivotal roles in regulating
the osteogenic ability of cranial sutures. In particular, TGFß signaling
stimulates osteogenic progenitor cell proliferation and can induce premature
suture obliteration in cultured fetal rat calvaria, suggesting that TGFß
signaling plays an important regulatory role in postnatal calvaria development
(Opperman et al., 2000). In
addition, TGFß signaling within the immature dura mater (in newborn and
immature animals) possesses the ability to induce calvaria bone repair, while
diminished TGFß signaling within the mature dura mater fails to repair
calvarial defect, suggesting that TGFß signaling is a crucial regulator
for calvarial ossification (Greenwald et
al., 2000
). TGFß IIR is expressed in the dura mater and
cranial sutures, presumably playing an important role during skull development
(Pelton et al., 1990
;
Lawler et al., 1994
;
Wang et al., 1995
).
Collectively, these studies have demonstrated that TGFß signaling has an
important regulatory function for postnatal cranial suture patency and skull
repair. However, it remains unclear what the physiological function of
TGFß signaling is in regulating the initiation and development of the
calvaria during embryogenesis.
To investigate the role of TGFß signaling in regulating the fate of CNC cells during palate and calvaria development, we performed tissue-specific Tgfbr2 gene ablation using Cre/loxP recombination exclusively in the cranial neural crest lineage. Our study shows that loss of Tgfbr2 in the CNC cells results in cleft secondary palate and calvaria defects with 100% phenotype penetrance. Specifically, conditional Tgfbr2 mutation inhibits cyclin D1 expression and affects CNC cell proliferation in the palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Disruption of TGFß signaling in the CNC severely impairs cell proliferation in the dura mater, consequently resulting in calvaria agenesis. We provide the first in vivo evidence that TGFß signaling within the CNC-derived dura mater provides essential inductive instruction for both the CNC- and mesoderm-derived calvarial bone development.
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Materials and methods |
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Generation of Tgfbr2fl/fl;Wnt1-Cre mutant mice
and histological analysis
All mouse embryos used in this study were maintained on C57BL6/J
background. Mating Tgfbr2fl/+;Wnt1-Cre with
Tgfbr2fl/fl mice generated
Tgfbr2fl/fl;Wnt1-Cre null alleles that were genotyped
using PCR primers as previously described
(Chytil et al., 2002). All
samples were fixed in 10% buffered formalin and processed into serial paraffin
wax-embedded sections using routine procedures. For general morphology,
deparaffinized sections were stained with Hematoxylin and Eosin using standard
procedures.
Analysis of cell proliferation, death and density
DNA synthesis activity within the palate or skull was monitored by
intraperitoneal BrdU (5-bromo-2'-deoxy-uridine, Sigma) injection (100
µg/g body weight) at E12.5, E13.4 and E14.5. One hour after the injection,
mice were sacrificed and embryos were fixed in Carnoy's fixative solution and
processed. Serial sections of the specimen were cut at 5 µm intervals.
Detection of BrdU labeled cells was carried out by using a BrdU Labeling and
Detection kit and following manufacturer's protocol (Boehringer Mannheim).
BrdU-positive and total number of cells within the palatal mesenchyme or MEE
of palatal shelf were counted from five randomly selected sections per sample.
Five palate samples were evaluated from each experimental group. TUNEL assay
was performed using the In Situ Cell Death Detection (fluorescein) kit (Roche
Molecular Biochemicals) by following the manufacturer's protocol. Cell density
analysis was performed by counting the number of cells per unit area from 20
randomly selected sections per experimental group. Student's t-test
was applied for statistical analysis. A P value of less than 0.05 was
considered statistically significant.
Palatal shelf organ cultures
Timed-pregnant mice were sacrificed on postcoital day 13.5 (E13.5).
Genotyping was carried out as described above. The palatal shelves were
microdissected and cultured in serumless chemically defined medium as
previously described (Shuler et al.,
1991). After 3 days in culture, palates were harvested, fixed in
10% buffered formalin and processed.
Western analysis
The total protein concentration in the palates was determined by comparison
with BSA standards. Seventy-five micrograms total protein from each sample was
loaded in each well on a 12% polyacrylamide gel. Western analysis was carried
out as previously described (Chai et al.,
1999). Antibodies used: anti-cyclin D1 and anti-CDK4 (BD
Biosciences), anti-Msx1 (kindly provided by P. Denny, USC) and
anti-ß-actin (Santa Cruz Biotechnology).
Whole-mount skeletal staining
The three-dimensional architecture of the skeleton was examined using a
modified whole-mount Alcian blue-Alizarin Red S staining protocol (details
available upon request).
Immunohistochemistry
Sectioned immunohistochemistry was performed with an Immunostaining kit
(Zymed) according to manufacturer's directions. The following antibodies were
used for this experiment: anti-BrdU (Sigma), anti-cyclin D1 (BD Biosciences)
and anti-p21 (Santa Cruz Biotechnology). Positive staining was shown in
orange-red for immunohistochemistry. The slides were counterstained with
Hematoxylin.
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Results |
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The complete failure of mouse secondary palate fusion was first detected in Tgfbr2fl/fl;Wnt1-Cre mutant embryos at E14.5 when normal palatal fusion had just occurred (Fig. 2A,B). We compared cross-sections of E14.5 Tgfbr2fl/fl;Wnt1-Cre mutant embryonic heads with the ones of Tgfbr2+/fl;Wnt1-Cre or Tgfbr2fl/fl littermate embryos. There was decreased cellular density (P<0.05) in the elevated palatal shelf mesenchyme of Tgfbr2fl/fl;Wnt1-Cre mutant embryos (4298±275 cells/mm2) when compared with the normal developing palate (5174±168 cells/mm2), in which fusion occurred with the partial disappearance of the midline epithelium (Fig. 2C-F). At E16.5, both of the palatal shelves had elevated into horizontal position but failed to fuse at the midline in Tgfbr2fl/fl;Wnt1-Cre mutant embryos, while completed palatal fusion was observed in the control samples (Fig. 2G,H). The CNC-derived palatal mesenchyme began to form an aggregated cell mass as a prelude to palatal bone development in the control sample (Fig. 2I), while CNC cell condensation was not observed in the Tgfbr2fl/fl;Wnt1-Cre mutant embryo (Fig. 2J). At birth, complete cleft secondary palate was observed in Tgfbr2fl/fl;Wnt1-Cre mutant mice with 100% (36/36 newborn pups) phenotype penetrance (Fig. 2L).
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Next, we hypothesized that the failure of palatal fusion in the Tgfbr2fl/fl;Wnt1-Cre mutant mice was due to insufficient extension of the palatal shelves towards the midline. To test our hypothesis, we performed palatal fusion analysis by using a palatal shelf organ culture model. At E13.5, the developing palatal shelves were pointing downwards on both sides of the tongue. Each isolated pair of palatal shelves was placed in culture with the two segments just touching at the medial edge and kept in the original anteroposterior orientation, thus preventing any variability in growth rates from adversely affecting palatal development. During the 3 day culture period, both wild-type and Tgfbr2fl/fl mutant palatal specimens fused. All cultured wild-type palatal shelves (n=32 pairs) showed complete fusion with normal disappearance of the MEE and development of a confluent palatal mesenchyme (Fig. 3A,C). Furthermore, osteoid-like structure was present in the cultured palatal shelf, suggesting that palatal bone formation was initiated in vitro (Fig. 3C, insert). Although all cultured Tgfbr2fl/fl;Wnt1-Cre mutant palatal shelves also showed fusion (n=9 pairs), some fused palates (4/9, 44%) had residual epithelium (arrow) at the midline, indicating a possible delay in the fusion process (Fig. 3B,D). Nevertheless, the MEE cells were competent to facilitate palatal fusion in Tgfbr2fl/fl mutant samples once the palatal shelves were placed in close contact. In addition, osteoid-like structure was present (Fig. 3D, insert), suggesting that there was normal palatal bone formation in the cultured Tgfbr2fl/fl;Wnt1-Cre mutant palatal shelf.
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TGFß signaling is known to regulate the expression of transcription
factors which in turn may regulate the fate of CNC cells by controlling the
progression of cell cycle (Moses and
Serra, 1996; Han et al.,
2003
). Exogenous TGFß can repress the transcriptional
activity of the Msx1 gene in the palatal mesenchyme in vitro
(Nugent and Greene, 1998
). We
examined the expression level of Msx1 in the developing palate by western
analysis. Msx1 expression level was identical between the wild type and the
Tgfbr2fl/fl;Wnt1-Cre mutant samples at E13.5
(Fig. 5Q). Significantly, Msx1
expression level was significantly elevated (2.5 times) in the palate of the
Tgfbr2fl/fl;Wnt1-Cre mutants when compared with the Msx1
expression level in the controls (Fig.
5).
TGFß signaling in the CNC-derived dura mater is required for
calvaria development
During skull development, TGFß ligand and its type II receptor are
colocalized within the craniofacial mesenchyme and may regulate its
differentiation (Fitzpatrick et al.,
1990; Pelton et al.,
1990
; Lawler et al.,
1994
). A high level of TGFß IIR mRNA expression is apparent
in the meninges surrounding and covering the developing brain, suggesting an
important functional role of this receptor in regulating the dura mater
development (Wang et al.,
1995
). Recently, it was shown that CNC cells contribute to the
formation of the meninges, which underlies the entire calvaria
(Jiang et al., 2002
).
Remaining unclear is the functional significance of TGFß signaling in
regulating the development of the dura mater as well as the consequence of an
impaired dura formation in regulating the patterning of intramembranous bone
development.
By analyzing the Wnt1-Cre;R26R embryos, we found that the CNC-derived dura mater covered the entire surface of the developing brain in the wild-type sample at E14.5 (Fig. 7A, blue). In Tgfbr2fl/fl;R26R;Wnt1-Cre mutant embryos, dura development was severely impaired on the surface of the developing brain (Fig. 7B). Specifically, instead of having a well-defined dura that contained blood vessels as seen in the wild-type samples, the Tgfbr2fl/fl mutant embryos showed a single cell layer, poorly developed dura mater (Fig. 7C,E). As shown in Fig. 4, there was no CNC migration defect in the Tgfbr2fl/fl mutant embryos. This dura development defect resulted from severely impaired CNC cell proliferation activity in the Tgfbr2fl/fl mutant embryos, while active CNC cell proliferation was observed in the dura of wild-type controls at E14.5 (Fig. 7D,F). Although there was only a poorly defined dura in the Tgfbr2fl/fl mutants at E14.5, it suggested that CNC cells were able to contribute to early dura development. However, there was a specific requirement for TGFß signaling during the continued dura development. As craniofacial development continued, the impaired TGFß signaling in the CNC-derived dura mater failed to induce parietal bone formation (rostral region), while there was proper parietal bone development in the wild type samples at E16.5 (Fig. 7G,H). Eventually, the failure of inducing bone formation by the dura led to severely impaired calvaria development.
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Discussion |
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Until now, the function of TGFß signaling in regulating the
CNC-derived palatal mesenchyme is not well understood. TGFß subtype
expression is conspicuous in the cranial neural crest-derived mesenchyme
during early mouse craniofacial development
(Heine et al., 1987;
Massague, 1990
). The presence
of TGFß and its cognate receptors is obvious in the mesenchyme during
crucial epithelial-mesenchymal interactions related to the formation of the
palate, tooth, and Meckel's cartilage
(Nugent and Greene, 1998
;
Hall, 1992
;
Chai et al., 1994
;
Wang et al., 1995
;
Lumsden and Krumlauf, 1996
;
Ito et al., 2002
). Although
the TGFß type II receptor is strongly expressed in the CNC-derived
palatal mesenchyme, mice deficient in Tgfbr2 die before the formation
of the palate, making it impossible to investigate the functional significance
of TGFß signaling in regulating the fate of CNC cells during
palatogenesis (Wang et al.,
1995
; Oshima et al.,
1996
). Our animal model of Tgfbr2 conditional gene
ablation in the neural crest cells offers a unique opportunity to investigate
the functional mechanism of TGFß signaling in regulating the fate of the
CNC-derived palatal mesenchyme. Owing to the lack of a CNC migration defect in
Tgfbr2fl/fl;Wnt1-Cre mutant mice, we conclude that
TGFß IIR is not crucial for the proper migration of CNC cells into the
first branchial arch. The cell proliferation defect in the CNC-derived palatal
mesenchyme of Tgfbr2fl/fl;Wnt1-Cre mutant mice clearly
indicates that TGFß signaling is specifically required in the palatal
mesenchyme prior to palatal fusion. We propose that TGFß directly or
indirectly regulates the expression of cell cycle regulators (such as cyclin
D1) to control the progression of the cell cycle in CNC-derived palatal
mesenchyme, and this regulation is crucial for proper palatal mesenchymal cell
proliferation. Decreased palatal mesenchyme cell proliferation has resulted in
compromised palatal shelf extension and failure of palatal fusion in
Tgfbr2fl/fl mutant mice. It is important to note that our
animal model does not address whether TGFß IIR regulates the
non-CNC-derived palatal mesenchymal cell proliferation, because
Wnt1-Cre does not cause Tgfbr2 deletion in this particular
cell population. In addition, although cyclin D1 expression is significantly
downregulated in the palatal mesenchyme of Tgfbr2fl/fl
mutant mice, it is unlikely that a compromised cyclin D1 expression is
directly responsible for causing the cleft palate defect in
Tgfbr2fl/fl mutant mice because cyclin D1-null mutant mice
do not have cleft palate (Fantl et al.,
1995
). Other cycle regulators (such as CDK inhibitors p21 or
p18INK4c) appear to be unaffected when we compared their expression
patterns within the palatal mesenchyme between the wild-type and the
Tgfbr2fl/fl mutant mice. Clearly, the method by which
TGFß signaling controls the progression of the CNC-derived palatal
mesenchyme cell cycle during palatogenesis is complex; it will be the focus of
our future studies.
Cell-autonomous requirement for TGFß IIR in cranial neural crest
during palatogenesis
Contrary to the successful fusion of our cultured
Tgfbr2fl/fl;Wnt1-Cre mutant palatal shelves, cultured
Tgfb3-null mutant palatal shelves fail to fuse, even when they are
placed in close contact in vitro
(Kaartinen et al., 1997).
Despite clear adherence, the cultured Tgfb3-null mutant palatal
shelves show persistent MEE cells and intact basement membrane. Significantly,
supplementation of exogenous TGFß3 facilitates the successful fusion of
Tgfb3-null mutant palatal shelves in vitro with transformation of the
MEE and degradation of the underlying basement membrane. Clearly, TGFß3
is specifically required in regulating the fate of MEE cells during palatal
fusion. The successful signaling of TGFß3 requires an integral TGFß
receptor complex. Indeed, TGFß IIR is also expressed in the MEE prior to
palatal fusion (Cui et al.,
1998
). Our palatal organ culture experiment suggests that the
basic TGFß signaling cascade in MEE cells is intact despite the null
mutation of Tgfbr2 in the CNC-derived palatal mesenchyme. It also
demonstrates that there is a cell-autonomous requirement for TGFß
signaling in the CNC-derived palatal mesenchyme during palatogenesis. In human
clefting birth defects, failure of palatal fusion after proper palatal
adhesion (such as the one in Tgfb3-null mutant mice) only represents
a small percentage of the cleft palate cases, while failure of palatal shelf
extension (such as the one in Tgfbr2fl/fl;Wnt1-Cre mutant
mice) is associated with the majority of the cleft palate cases. Hence, the
Tgfbr2fl/fl;Wnt1-Cre mutant mice will serve as an
important animal model for the investigation of the molecular etiology of
human cleft palate.
Inductive signaling within the CNC-derived dura mater is critical for
both the CNC- and non-CNC-derived calvarial bone development
Defects in the development of the dura mater and calvaria bone have
significant implications. A recent study has shown that the mammalian frontal
bones are neural crest derived (still controversial for avian) and that the
parietal bones are of mesodermal origin. Furthermore, the dura mater that
underlies the parietal bones is neural crest-derived and is sensitive to
retinoic acid exposure during parietal bone ossification, suggesting that
intramembranous ossification of this mesodermal bone requires interaction with
the CNC-derived meninges (Jiang et al.,
2002). Here, the defects of both frontal and parietal bones
suggest that the CNC-derived dura mater is crucial for the induction of
CNC-derived frontal bone and mesoderm-derived parietal bone formation. We
hypothesize that the dura mater produces inductive signaling which interacts
with the overlaying mesenchyme, whether neural crest or mesodermally derived,
to control the initiation and patterning of frontal and parietal bones during
calvaria development. Furthermore, our study indicates that TGFß
signaling plays a pivotal role in regulating the proliferation of the
CNC-derived dura mater. Aberrant TGFß signaling results in compromised
dura mater development and consequently, in calvaria development defects.
TGFß is known to regulate the fate of multipotential progenitor cells
instructively by regulating the expression or function of tissue-specific
transcription factors (Moses and Serra,
1996). For example, TGFß downregulates the expression of
homeobox gene Msx1 and affects cell fate determination in limb
development (Ganan et al.,
1996
). The expression patterns of TGFß and Msx1 have
significant overlaps during palatal development and suggest an epistatic
relationship between these genes when CNC-derived cells become committed to
form the palatal mesenchyme (Pelton et
al., 1990
; Ferguson,
1994
). Overexpression of TGFß suppresses transcriptional
activity of the Msx1 gene in the palatal mesenchyme in vitro
(Nugent and Greene, 1998
).
Similarly, TGFß signaling may regulate the expression of the
Msx2 gene during calvaria development. TGFß IIR and Msx2 are
co-expressed in the CNC-derived meninges prior to calvaria formation. We have
shown here that Msx1 expression is significantly elevated while cyclin D1
expression is greatly reduced in the palatal mesenchyme of the
Tgfbr2fl/fl;Wnt1-Cre mutant embryos, suggesting that
TGFß may regulate the expression of the Msx1 gene, which in turn
controls the progression of the CNC cell cycle during palatogenesis. A recent
in vitro study has shown that Msx1 gene expression maintains cyclin
D1 gene expression and controls cell cycle progression, thereby regulating
terminal differentiation of progenitor cells during embryonic development
(Hu et al., 2001
). Our in vivo
data suggests that the outcome of Msx1-regulated cyclin D1 expression might be
tissue type-dependent. As suggested in the previous study, cyclin D1 is likely
to be an indirect target of Msx1 during embryonic development.
Furthermore, our study supports the previously proposed model that reconciles
the observed phenotype similarities between the Msx1 loss- and
gain-of-function mutations in the context of cell cycle regulation
(Hu et al., 2001
). In
addition, mutations of the TGFß IIR may also impinge on BMP signaling
within the developing CNC and the CNC-derived mesenchyme, because there is
significant overlap between the expression patterns of BMP and TGFß
during craniofacial development. TGFß IIR can bind to BMPs, and the
dominant-negative mutation of TGFß IIR attenuates both BMP and TGFß
signaling (Massague, 1990
;
ten Dijke et al., 1994
;
Dumont and Arteaga, 2003
).
Potentially useful regionally restricted branchial arch and/or palatal
mesenchyme markers (such as members of the homeobox-containing genes) need to
be analyzed to dissect the TGFß signaling cascade in regulating the fate
of CNC cells during craniofacial morphogenesis.
The broad spectrum of phenotypic abnormalities suggests that TGFß signaling is crucial for the transcriptional regulation of multiple regulatory signaling cascades during embryogenesis. We provide an animal model for investigating the molecular mechanism of cleft palate, calvaria agenesis and other CNC-related congenital malformations and demonstrate that TGFß IIR signaling is specifically required in regulating the fate of CNC cells during craniofacial development. Future studies using this animal model will provide useful information on the mechanism of TGFß IIR signaling in both normal and abnormal human development. In addition, genetic screening of the Tgfbr2 mutation among individuals with secondary palate cleft and skull malformations may provide crucial information in linkage analysis to investigate the etiology of congenital malformations.
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ACKNOWLEDGMENTS |
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