1 Alkek Institute of Biosciences and Technology, Texas A&M System Health
Science Center, 2121 Holcombe Boulevard, Houston, TX 77030, USA
2 Department of Molecular Genetics, University of Texas M.D. Anderson Cancer
Center, 1515 Holcombe Boulevard, Houston, TX 77030,USA
3 Laboratory of Reproductive and Developmental Toxicology, National Institutes
of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
4 Department of Pediatric Dentistry, School of Dental Medicine, University of
Connecticut Health Science Center, 263 Farmington Avenue, Farmington, CT
06030, USA
* Author for correspondence (e-mail: jmartin{at}ibt.tamhsc.edu)
Accepted 5 January 2005
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SUMMARY |
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Key words: Bmp, Palate, Morphogenesis
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Introduction |
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Recent findings have shown that in some families CL/P and CP may result
from the same genetic lesion. For example, a nonsense mutation (ser104stop) in
the Msx1 homeobox gene, a transcriptional regulator that is a
downstream component of bone morphogenetic protein (Bmp) signaling pathways,
resulted in both cleft palate (CP) and cleft lip/palate (CL/P) in a Dutch
family (van den Boogaard et al.,
2000). Moreover, common mutations in p63 have also been
shown to cause CL/P and CP within a family
(Barrow et al., 2002
). Recent
evidence suggests that p63 is a direct downstream target of Bmp
signaling in zebrafish (Bakkers et al.,
2002
). Taken together, these data suggest that Bmp-signaling has a
role in two forms of orofacial clefting that were previously considered to be
distinct.
Bmp ligands are expressed in the facial primordia
(Ashique et al., 2002;
Barlow and Francis-West, 1997
;
Francis-West et al., 1994
),
and are known to signal through broadly expressed type I and type II
serine/threonine kinase receptors (von
Bubnoff and Cho, 2001
). Bead-implantation experiments in chick
embryos led to the conclusion that both reduction and enhancement of Bmp
signaling within facial primordia caused defective lip fusion
(Ashique et al., 2002
).
Although underscoring the importance of tightly regulated Bmp signaling in lip
and palate fusion, the transient nature of bead implantation experiments, as
evidenced by the requirement for multiple rounds of bead insertion, may fail
to uncover the complete requirements for Bmp function in orofacial
development.
Direct investigation of the type 1A Bmp receptor gene Bmpr1a (also
referred to as Alk3), and Bmp4 in craniofacial development
has been hampered by the early embryonic lethality of the germline null mutant
mice (Mishina et al., 2002;
Mishina et al., 1995
;
Winnier et al., 1995
). To
circumvent this early lethality, we used conditional null alleles of
Bmpr1a and Bmp4 to directly investigate Bmp signaling in lip
and palate fusion in mouse embryos. We report here that the
Bmpr1a-dependent pathway is a major regulator of lip and palate
fusion and tooth morphogenesis. Inactivation of Bmpr1a in the
craniofacial primordia resulted in CL/P with tooth agenesis. However, we
provide evidence that the mechanisms underlying lip clefting and cleft
secondary palate are distinct. We also report that deficiency of Bmp4
resulted in isolated cleft lip. Taken together, our results reveal that Bmp
signaling has distinct functions in lip fusion versus secondary palate
development.
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Materials and methods |
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Palate in vitro organ culture
Palatal shelves were harvested at 13.0 dpc and 14.0 dpc from wild-type and
mutant embryos. The dissected palatal shelves were cultured on 6.5-mm
transwell (Costar), keeping the paired shelves with their medial edge
epithelia (MEE) in close apposition without apparent distortion of the tissue
shape. Paired palatal shelves were cultured overnight using DMEM supplemented
with 1% penicillin/streptomycin (Chai et
al., 1998).
Separation of epithelium from mesenchyme
Branchial arches were dissected and placed in a Petri dish containing HBSS
(Sigma) with 10% FCS. After dissection, HBSS was replaced with dispase II (0.8
U/ml from Roche). Explants were incubated for 1 hour at room temperature.
Forceps were used to gently separate the epithelium from mesenchyme.
Histologic analysis
Mouse tissues were fixed in buffered formalin overnight, dehydrated through
graded alcohols, and embedded in paraffin wax. Paraffin blocks were sectioned
at 7-10 µm and stained with Hematoxylin and Eosin.
Skeletal preparations
After scalding, mice were eviscerated, fixed in 95% ethanol and stained
with Alcian Blue (0.015% Alcian Blue dissolved in 20% acetic acid and 76%
ethanol). Following two washes with 95% ethanol, the sample was cleared by 2%
KOH at room temperature. Bone was stained with Alizarin Red (50 mg Alizarin
Red in one liter of 2% KOH).
Analysis of BrdU incorporation
Pregnant mice were intraperitoneally injected with BrdU (100 mg/kg body
weight) 1 hour prior to sacrifice. Embryos were fixed overnight in 10%
formalin at 4°C, dehydrated through an ethanol series, cleared in xylene,
embedded in paraffin wax and sectioned at 5 µm. BrdU was detected
immunohistochemically with a cell proliferation kit (Invitrogen), according to
the manufacturer's instructions.
Whole-mount ß-gal staining
After dissection, the embryos were fixed in fresh fix buffer [(0.2
glutaraldehyde, 2% formalin, 5 mM EGTA, 2 mM MgCl2, in 0.1 M
phosphate buffer (pH 7.3)] for 30 minutes. Following three washes with rinse
buffer [0.1% sodium dexycholate, 0.2% NP40, 2 mM MgCl2, in 0.1 M
phosphate buffer pH 7.3)], the samples were stained with staining buffer (1
mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, in
rinse buffer) overnight at room temperature.
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Results |
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Signaling through Bmpr1a is required for lip and secondary palate fusion
At 18.5 dpc, we found that all Nestin cre;Bmpr1a n/f mutant
embryos showed a strong phenotype with bilateral cleft lip and palate
(Fig. 2A-D). Histological
sections of the forming secondary palate at multiple time points revealed that
at 13.5 dpc, both the wild-type and the Nestin cre;Bmpr1a n/f palatal
shelves grew vertically down the side of the tongue, although the mutant
shelves were slightly smaller than the control
(Fig. 2E,F). Moreover, at 14.5
dpc, the palatal shelves elevated correctly in both wild-type and mutant
embryos (Fig. 2G,H). However,
after palatal shelf elevation, the mutant palatal shelves failed to grow
together and fuse (Fig. 2I,J).
These results suggest that the cleft secondary palate in Nestin cre;Bmpr1a
n/f embryos was secondary to failure of palatal shelf growth.
|
Bmpr1a deficient palatal shelves fuse in vitro
If the cleft secondary palate of Nestin cre;Bmpr1a n/f mutants was
caused by a proliferation defect in the maxillary mesenchyme, then mutant
palatal shelves may still retain the ability to fuse in vitro. To investigate
this notion, organ culture experiments were performed. Palatal shelves were
harvested at 13.0 and 14.0 dpc and cultured overnight with the medial edge
epithelium (MEE) in contact. The Nestin cre;Bmpr1a n/f palatal
shelves harvested at both 13.0 and 14.0 dpc fused in organ culture
(Fig. 2K-N). Moreover, in the
palatal shelves harvested at 13.0 dpc, MEE degeneration was clearly visible
(Fig. 2M,N). These data
indicate that the Nestin cre;Bmpr1a n/f palatal shelves retain the
ability to fuse and support the hypothesis that the mechanism underlying cleft
secondary palate in the Nestin cre;Bmpr1a n/f embryos was a failure
of palatal shelf outgrowth. However, further experiments are required to prove
that the MP proliferation defect caused cleft secondary palate in Nestin
cre;Bmpr1a n/f embryos.
Defective patterning of the secondary palate in Nestin cre;Bmpr1a deficient embryos
Recent work has suggested that Bmp signaling plays a role in anterior
posterior (AP) patterning of the palatal shelves
(Zhang et al., 2002). To
investigate whether AP patterning was defective in the Nestin
cre;Bmpr1a mutant palatal shelves, we examined the expression of
Msx1 in the wild-type and Nestin cre;Bmpr1a mutant secondary
palates. Msx1 continued to be expressed in the anterior secondary
palate of Nestin cre;Bmpr1a mutant embryos
(Fig. 3A-B). The expression of
Msx1 in the MP of mutant embryos suggests that Bmp signals are
transduced to the MP mesenchyme in the absence of Bmpr1a. This is
likely to reflect the contribution of other type I receptors, such as
Bmpr1b (also known as Alk6), to Bmp signaling in the MP.
|
Elevated apoptosis in the fusing lip region of Nestin cre mutant embryos
To gain insight into the molecular mechanism underlying cleft lip in
Nestin cre;Bmpr1a mutants, we examined markers of the edge epithelium
of the MP and medial nasal process (MNP). Fgf8 is expressed in the
epithelium of the nasal processes and the MP in chick embryos
(Helms et al., 1997).
Moreover, Fgf signaling is known to closely interact with Bmp
signaling pathways to regulate craniofacial and tooth development
(Neubuser et al., 1997
;
Tucker et al., 1998
). In
addition to Fgf8, we examined expression of p63 and
Pitx1, which are expressed in the edge ectoderm of the fusing lip.
P63 has also been implicated in cleft lip and palate in human
patients (Celli et al., 1999
),
and Pitx1 is a known target gene for Fgf8 in mandibular development
(St Amand et al., 2000
).
To precisely define the onset of the lip fusion defect, we examined Fgf8 expression in embryos that had been carefully staged by counting somite number (Fig. 4A-D). In 29-somite embryos (10.0 dpc), Fgf8 expression was similar in the wild-type and Nestin cre;Bmpr1a mutant embryos (Fig. 4A,B). Fgf8 was expressed in the proximal part of the nasal process ectoderm but was not yet expressed in the fusing region. By contrast, 31-somite embryos (10.25 dpc) expressed Fgf8 in the ectoderm at the lip fusion point, whereas Nestin cre;Bmpr1a mutants failed to upregulate Fgf8 in this region (Fig. 4C,D). This difference in Fgf8 expression was more pronounced in 10.5 dpc (34-35 somites) embryos (Fig. 4E,F). In addition to diminished Fgf8 expression, we found that Nestin cre;Bmpr1a mutants also had downregulated expression of both p63 and Pitx1 in the fusing ectoderm of the nasal processes in 10.5 dpc embryos (Fig. 4G-L).
|
Bmpr1a deficiency in oral ectoderm results in defective tooth morphogenesis
Consistent with the role of Msx1 in tooth agenesis in human
patients, we detected tooth defects in Nestin cre;Bmpr1a mutants. At
16.5 dpc, mandibular and maxillary first and second molars are normally in the
late and early cap stage of development, respectively
(Fig. 5A,B). The inner and
outer enamel epithelium in the developing molars are separated by the stellate
reticulum. The maxillary molars in Nestin cre;Bmpr1a n/f 16.5 dpc
embryos were arrested at the bud stage, with an invaginated dental lamina
encircled by condensed mesenchyme (Fig.
5A,C). In contrast to the defective maxillary molars, Nestin
cre;Bmpr1a n/f 16.5 dpc embryos had well-developed cap-staged mandibular
molars (Fig. 5B,D). In 16.5 dpc
wild-type embryos, there are two well-developed incisor tooth germs in each
arch (Fig. 5E,F). In the
Nestin cre;Bmpr1a mutant embryos, development of the mandibular
incisors was normal but mutant embryos lacked maxillary incisor teeth
(Fig. 5G,H).
|
Bmp4 deficiency resulted in isolated cleft lip
In order to gain insight into the ligands that would be involved in cleft
lip and palate, we examined expression of Bmp4 and Bmp2. At
10.5 dpc, we found that Bmp2 and Bmp4 are co-expressed in
the edge epithelium at the point of fusion between the MNP and the MP
(Fig. 6A,B). We next used a
conditional null allele of Bmp4, the Bmp4floxneo
allele, to address the functional role of Bmp4 in CL/P. The
Bmp4floxneo allele has LoxP sites flanking exon 4 that
encodes the mature Bmp4 ligand. Removal of the LoxP flanked region has been
shown to result in a null allele (Liu et
al., 2004).
|
We examined Nestin cre;Bmp4 n/f mutant embryos at two developmental timepoints. At 12.0 dpc, we found that all Nestin cre;Bmp4 n/f mutant embryos had a bilateral delay in fusion of the MNP and MP (Fig. 6E,F). At 14.5 dpc, two out of nine Nestin cre;Bmp4 n/f mutants had unilateral isolated cleft lip (Fig. 6G,H). Taken together, these data reveal that Bmp4 functions in the ectoderm of the nasal processes to regulate lip fusion. Moreover, these findings suggest that most Nestin cre;Bmp4 n/f mutants spontaneously repair the cleft lip that we observed at 12.0 dpc.
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Discussion |
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Bmp signaling in secondary palate growth and AP patterning
Development of the secondary palate is a complex process that requires
outgrowth, elevation and fusion of the palatal shelves
(Ferguson, 1988). Our analysis
revealed that Bmpr1a is required for proliferation of MP mesenchyme.
The in vitro explants showed that the Bmpr1a mutant palatal shelves
are competent to fuse when placed together in culture. Previous work in
secondary palate development uncovered a genetic pathway in which Msx1 and
Bmp4 function in an autoregulatory loop to regulate proliferation in anterior
palate mesenchyme (Zhang et al.,
2002
). Our data reveal that Bmp signaling is also required for
cell proliferation at earlier stages, in the MP mesenchyme.
Two previous studies looking at Bmp signaling in chick embryos reached
conflicting conclusions regarding the regulation of cell proliferation in the
facial prominences. One study concluded that implantation of Bmp-soaked beads
in MP mesenchyme resulted in elevated proliferation
(Barlow and Francis-West,
1997). The second study, while concluding that Noggin
reduced cell proliferation, found that implanted Bmp2 beads failed to
influence cell proliferation (Ashique et
al., 2002
). Our data support the notion that Bmp signaling
promotes cell proliferation of MP mesenchyme.
We found that Bmp signaling is required to restrict expression of
Barx1 and Pax9 in the forming secondary palate. It is
interesting to note that in the developing mandible, Bmp signaling has been
shown to repress expression of Pax9 and Barx1, suggesting a
conservation of pathways in the palate and mandible
(Neubuser et al., 1997;
Tucker et al., 1998
;
Zhang et al., 2002
). Moreover,
the changes in gene expression in the Nestin cre;Bmpr1a mutant
secondary palates support the idea that cleft secondary palate is not a
consequence of failed lip fusion.
We have found evidence for genetic redundancy among Bmp receptors in the
MP. The Nestin cre;Bmpr1a mutant embryos still express Msx1
in the palatal mesenchyme, suggesting that Bmp signals are transduced in the
absence of Bmpr1a. Neural crest specific ablation of another type 1
Bmp receptor (Alk2) also resulted in cleft palate, although this may
be secondary to mandibular defects (Dudas
et al., 2004). Another possible candidate for redundancy is
Bmpr1b, which is expressed in craniofacial processes (W.L. and
J.F.M., unpublished). It will be important to explore the question of genetic
redundancy between the type 1 Bmp receptors in future experiments.
Bmp signaling in lip fusion
Our data suggest that a Bmp4-Bmpr1a autoregulatory loop in the
edge epithelium is necessary for lip fusion. These findings implicate the
Bmp4-BmpR1A pathway in human clefting syndromes, and are consistent with
previous observations showing the association between the Bmp-target
Msx1 and CL/P (van den Boogaard
et al., 2000). It was previously shown that an Arg239Pro
Msx1 mutation resulted in tooth agenesis but no clefting in human
patients (Vastardis et al.,
1996
). The second family with a Ser104stop mutation in
Msx1 had both CL/P and selective tooth agenesis
(van den Boogaard et al.,
2000
). The defects in both families are likely to result from
haploinsufficiency and suggest that the Arg239Pro Msx1 mutation
retained more residual function than the Ser104 stop mutation
(Hu et al., 1998
).
Our observation that most Nestin cre;Bmp4 n/f mutant embryos
repair their cleft lip also implicates Bmp4 in clinical cases of
microform cleft lip that have been observed in human patients. It is notable
that congenital heart disease has been associated with microform clefting
(Castilla and Martinez-Frias,
1995; Grech et al.,
2000
). We, and others, have recently reported that Bmp4
has a direct role in cardiac morphogenesis, suggesting an important role for
Bmp4 in human congenital heart disease
(Jiao et al., 2003
;
Liu et al., 2004
).
Our findings reveal that Bmpr1a is required for survival of the
edge epithelium and mesenchyme of the MNP. Work in chick embryos has shown
that during lip fusion, the outermost periderm epithelium of the frontonasal
mass and MP undergoes apoptosis. This exposes a fusion-competent basal
epithelium, allowing basal cells to fuse through a process that may involve
desmosomes. Cells within the fusing region, or the seam, undergo an epithelial
to mesenchymal transition during remodeling of the lip
(Sun et al., 2000). Our data
suggest that, in Bmpr1a mutants, premature apoptosis in the edge
epithelium of the MNP is responsible for clefting of the lip.
The role of programmed cell death in facial process fusion is
controversial. The epithelial-mesenchymal transformation of the MEE to palate
mesenchyme has been considered to be the critical determinant for secondary
palate fusion (Fitchett and Hay,
1989). However, recent studies have challenged this model by
suggesting that apoptosis is required for normal fusion to occur
(Cuervo and Covarrubias, 2004
;
Cuervo et al., 2002
). With
regard to the data presented here, it is known that premature induction of
apoptosis in palatal shelves inhibited fusion, perhaps as a result of reduced
cell adhesion (Cuervo et al.,
2002
). Further experiments will be required to determine whether
failed fusion of the edge epithelium in Nestin cre;Bmpr1a
n/f mutants is secondary to reduced cellular adhesion.
It is also notable that, in addition to elevated apoptosis in Nestin cre;Bmpr1a n/f mutant nasal ectoderm, there was elevated apoptosis in the underlying mesenchyme (Fig. 4M-O). This suggests that the defect in lip fusion may result from loss of the MNP mesenchyme in addition to the mechanisms discussed above. Further experiments will be required to investigate this.
The in situ analysis presented here revealed defects in the expression of
ectodermal markers of the fusing lip, Fgf8, Pitx1 and p63,
in Nestin cre;Bmpr1a n/f mutants. Fgf8 expression in the
fusing lip region of the Nestin cre;Bmpr1a n/f mutants revealed a
failure to upregulate Fgf8 in 31-somite embryos prior to induction of
apoptosis. In the mandibular process, Fgf8 has an important role in
promoting the survival of underlying mesenchyme
(Trumpp et al., 1999).
Therefore, in Nestin cre;Bmpr1a n/f mutants, the failure of
Fgf8 upregulation may directly result in elevated apoptosis and lip
clefting. This notion is also supported by recent observations in chick
embryos treated with retinoic acid inhibitors. In these retinoid deficient
embryos, defective closure of the nasal pit was associated with elevated
apoptosis and downregulation of Fgf8 in the lateral nasal process
(Song et al., 2004
). However,
further experiments will be required to definitively show that apoptosis is
the cause of lip clefting in the Nestin cre;Bmpr1a n/f mutants.
An alternative hypothesis is suggested by the observation that p63
has previously been shown to play an important role in differentiation of
embryonic epidermis (Mills et al.,
1999; Yang et al.,
1999
). Furthermore, recent work uncovered multiple, functional
Smad regulatory elements in the zebrafish p63 gene
(Bakkers et al., 2002
). Taken
together with our observation that p63 is reduced in the edge
epithelium of the MNP and MP of Nestin cre;Bmpr1a n/f mutants, these
data suggest the existence of a linear genetic pathway important for
maturation and subsequent fusion of the edge epithelium. The connection of
p63 to clefting in humans makes this idea worthy of further
investigation (Celli et al.,
1999
).
Bead implantation experiments performed in chick embryos concluded that, in
the maxillary epithelium, Bmp signaling promoted cell death and loss of
epithelium. Beads soaked in Noggin resulted in reduced cell death in the
epithelium of the frontonasal mass, and thus epithelial integrity was
maintained in the pre-fusion phases. Moreover, Fgf8 expression in the
frontonasal process was increased (Ashique
et al., 2002). By contrast, our data suggest that Bmp signaling in
the epithelium is required for cell survival and upregulation of Fgf8
expression. The different results may stem from the large doses of Noggin that
were necessary to obtain phenotypes in the chick experiments. It is also
notable that we inactivated Bmp signaling in the nasal process epithelium,
whereas Noggin was placed in the mesenchyme and so may have indirectly
disrupted an unidentified signal from mesenchyme to epithelium.
Bmp signaling in tooth morphogenesis
It has been proposed that Bmp signaling has multiple roles in tooth
development, including tooth type morphogenesis, tooth organ placement, and
signaling within and from the enamel knot
(Jernvall et al., 1998;
Neubuser et al., 1997
;
Tucker et al., 1998
). The
Nestin cre;Bmpr1a n/f mutants display different tooth phenotypes in
the mandible and maxilla. In the maxilla, molars arrest at the bud stage,
while incisors arrest at an earlier stage. In the mandible, molars arrest at
the cap stage, while incisor development is normal. Recently published
observations showing that inactivation of Bmpr1a in dental epithelium
resulted in arrest of tooth development at the bud stage
(Andl et al., 2004
) suggest
that the distinct tooth phenotypes we observed are likely to result from
variable cre activity. Nonetheless, our data also raise the possibility that
different teeth may have different requirements for levels of Bmp signaling to
complete organogenesis.
Our RT-PCR analysis showed that the Nestin cre;Bmpr1a n/f mutants
mandibular ectoderm had mosaic cre activity, whereas maxilla had a more
complete deletion of Bmpr1a at 11.5 dpc. The cap stage arrest is
consistent with previous data showing that Bmp4 signaling to the forming
enamel knot is crucial for the progression of tooth development
(Bei et al., 2000;
Chen et al., 1996
;
Jernvall et al., 1998
).
Bmp4 in mesenchyme also has been proposed to induce expression of p21, and cell cycle arrest in cells of the prospective enamel knot. Once induced in enamel knot, Bmp4 functions to regulate tooth organ shape by controlling cell cycle progression and apoptosis. It is plausible that mosaic disruption of Bmpr1a in the dental epithelium would allow tooth germ development to progress beyond the bud stage and arrest at stages when enamel knot function would become important. Future experiments will be needed to investigate these ideas in more detail.
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ACKNOWLEDGMENTS |
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