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First published online March 20, 2009
doi: 10.1242/10.1242/dev.028167
Center for Oral Biology and Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
* Author for correspondence (e-mail: Rulang_Jiang{at}urmc.rochester.edu)
Accepted 11 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cleft palate, Fgf10, Osr2, Osr2-IresCre, Palate development, Shh, Smo, Tissue-specific gene inactivation
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Disturbances of the growth, elevation or fusion of the
palatal shelves result in cleft palate, one of the most common birth defects
in humans.
Classic organ culture assays and recent palatal explant studies with
exogenous recombinant signaling molecules have demonstrated that growth and
patterning of the developing palatal shelves depend on epithelial-mesenchymal
interactions (Tyler and Koch,
1977; Ferguson and Honig,
1984; Zhang et al.,
2002; Rice et al.,
2004; Yu et al.,
2005). During early palate development, expression of several
signaling molecules and transcription factors, including Bmp4, Fgf10, Msx1 and
Shox2, was found specifically restricted in the anterior palate
(Zhang et al., 2002;
Alappat et al., 2005;
Yu et al., 2005) (reviewed by
Hilliard et al., 2005). Bmp4
and Msx1 appeared to function in a positive feedback loop to regulate
mesenchyme proliferation in the anterior palate
(Zhang et al., 2002). In
addition, mesenchymal Bmp4 was required to maintain Shh expression in the
anterior palatal epithelium (Zhang et al.,
2002) and exogenous Shh protein was capable of stimulating palatal
mesenchyme proliferation in palate explant cultures
(Zhang et al., 2002;
Rice et al., 2004). The
mesenchymally expressed Fgf10 regulates palatal epithelial cell survival and
proliferation (Rice et al.,
2004; Alappat et al.,
2005). Both Fgf10 and its epithelial receptor Fgfr2b are also
required for maintenance of Shh expression in the palatal epithelium
(Rice et al., 2004). These
data suggest that Shh plays important roles in palatal growth and
patterning.
Shh is a member of the hedgehog family of secreted proteins and plays
crucial roles in diverse developmental processes, including left-right axis
establishment, dorsoventral patterning of the neural tube, endoderm
development, anteroposterior patterning of the developing limb, and brain
development and patterning (reviewed by
Ingham and McMahon, 2001;
McMahon et al., 2003;
Roessler and Muenke, 2003).
Shh signals to recipient cells through binding to the patched family of
receptors, Ptch1 or Ptch2. In the absence of ligands, the patched receptors
inhibit the function of another transmembrane protein, smoothened (Smo), an
obligatory component of the hedgehog signaling pathway. Activation of Smo upon
Shh signaling leads to activation of downstream genes by the Gli family
transcription factors. Interestingly, Ptch1 and Gli1 are
also among the downstream target genes of the Shh pathway, thus establishing a
regulatory feedback loop (reviewed by
Ingham and McMahon, 2001;
McMahon et al., 2003).
Shh signaling plays essential roles in craniofacial development. Mutations
in SHH in humans cause holoprosencephaly (reviewed by
Cohen, 2004), whereas a
targeted null mutation in Shh in mice resulted in severe cranial
deficiencies (Chiang et al.,
1996). Inhibition of Shh signaling in the chick facial primordia
with a function-blocking antibody inhibited facial outgrowth, whereas ectopic
application of active Shh protein caused facial overgrowth
(Hu and Helms, 1999). Ahlgren
and Bronner-Fraser showed that inhibition of Shh in the cranial mesenchyme
caused extensive cell death (Ahlgren and
Bronner-Fraser, 1999). Thus, Shh signaling appears to regulate
both proliferation and survival of the neural-crest-derived cranial
mesenchyme. However, the severe cranial deficiency in Shh null
mutants and early embryonic lethality of mutant mouse embryos lacking either
Ptch1 or Smo prevented a direct genetic analysis of the
molecular mechanisms involving Shh signaling in palate development
(Chiang et al., 1996;
Goodrich et al., 1997;
Zhang et al., 2001).
The adaptation of the Cre/loxP system for temporally and/or
spatially controlled gene inactivation in mice made it possible to
systematically dissect the genetic pathways in specific developmental
processes (Jiang and Gridley,
1997; Sauer,
1998). Whereas mice homozygous for null mutations in Shh
failed to develop most facial structures
(Chiang et al., 1996), the
K14-Cre;Shhc/n mice, in which Shh was inactivated
specifically in epithelial cells after embryonic day 11.5 (E11.5), developed
most facial structures but exhibited tooth developmental arrest
(Dassule et al., 2000). The
K14-Cre transgenic mice also expressed Cre in the oral epithelium,
including in the palatal epithelium, during palatal outgrowth
(Dassule et al., 2000;
Vaziri Sani et al., 2005).
About 85% of the K14-Cre;Shhc/n mice had cleft palate
(Rice et al., 2004;
Gritli-Linde, 2007).
Unfortunately, there has been no report regarding palate development in the
K14-Cre;Shhc/n mutant mice other than the brief
descriptions of the late-term cleft palate phenotype. Similarly, while mice
lacking Gli2 and mice with neural-crest-specific inactivation of
Smo have been reported to have palatal defects
(Mo et al., 1997;
Jeong et al., 2004), how the
Shh signaling pathway regulates palate development remains to be elucidated.
Here we show, through analysis of mice with specific inactivation of
Smo in the palatal mesenchyme, that Shh signaling regulates
expression of a number of signaling molecules and transcription factors in the
palatal mesenchyme to coordinate the epithelial-mesenchymal interactions that
control palatal outgrowth.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Shh+/n (Shhtm1Amc/J),
Shhc/c (Shhtm2Amc/J) and
Smoc/c (Smotm2Amc/J) mice have been
described previously (St-Jacques et al.,
1998; Dassule et al.,
2000; Lewis et al.,
2001; Long et al.,
2001) and were purchased from the Jackson Laboratory (Bar Harbor,
ME). The R26R reporter mice
(Soriano, 1999) were also
purchased from the Jackson Laboratory. K14-Cre transgenic mice (line
43) (Andl et al., 2004) were
acquired through a Material Transfer Agreement with Dr Sarah Millar at the
University of Pennsylvania and provided by Dr Yang Chai (University of
Southern California). K14-Cre transgenic mice were maintained by
crossing to C57BL/6J mice. Shh+/n mice were also
maintained by crossing to the C57BL/6J mice, whereas Shhc/c,
Smoc/c and Osr2-IresCre mice were maintained as
homozygotes in their own strain background. Male K14-Cre transgenic
mice were crossed to Shh+/n female mice to generate
K14-Cre;Shh+/n males, which were subsequently crossed to
Shhc/c mice to generate K14-Cre;Shhc/n
embryos for analysis. Independently, Osr2IresCre/IresCre
homozygous male mice were crossed to Smoc/c female mice to
generate Osr2-IresCre;Smo+/c male mice, which were
subsequently crossed to Smoc/c female mice to generate
Osr2-IresCre;Smoc/c embryos for analysis. Although the
Ires-Cre insertion in the Osr2IresCre allele did
not disrupt Osr2 gene function
(Lan et al., 2007), all
experimental analysis of Osr2-IresCre;Smoc/c embryos used
Osr2-IresCre;Smo+/c littermates as controls. Genotyping of
mice and embryos were carried out by allele-specific PCR as previously
described (Dassule et al.,
2000; Long et al.,
2001; Lan et al.,
2007).
Histology and in situ hybridization analyses
For histology, embryos were fixed in Bouin's fixative, dehydrated through
graded alcohols, embedded in paraffin, sectioned at 7 µm thickness, and
stained with Hematoxylin and Eosin. For in situ hybridization, embryos were
fixed overnight at 4°C in 4% paraformaldehyde in PBS, dehydrated through
graded alcohols, embedded in paraffin and sectioned at 5 µm thickness. In
situ hybridization of tissue sections were performed as described previously
(Zhang et al., 1999). At least
three pairs of control and mutant embryos were analyzed for each developmental
stage.
X-gal staining and skeletal analysis
Embryos were dissected in ice-cold PBS and stained with X-gal for
β-galactosidase detection as described previously
(Hogan et al., 1994).
Cryosections were counterstained with Eosin following X-gal staining. Skeletal
preparations were made from newborn mice as described previously
(Martin et al., 1995).
Detection of cell proliferation and immunohistochemical staining
For detection of cell proliferation in the palatal shelves, timed pregnant
female mice were injected intraperitoneally on gestational day 12.5 or 13.5
with BrdU (Roche) labeling reagent (45 µg/g body weight). One hour after
injection, embryos were dissected, fixed, embedded in paraffin and sectioned
in the coronal plane for immunodetection of BrdU using the BrdU labeling and
detection kit (Roche) as described previously
(Lan et al., 2004). Following
BrdU immunostaining, the embryonic sections were counterstained with Nuclear
Fast Red to visualize all cellular nuclei. Sections were selected from the
middle of the anterior halves (corresponding to the anterior margin of the
maxillary first molar tooth buds) and posterior regions (posterior to the
maxillary first molar tooth buds) of the palatal shelves in comparable
positions in the control and mutant embryos. Cell counts were recorded
separately for the palatal epithelium and mesenchyme in each of the bilateral
palatal shelves from five continuous sections of each region of the palate.
The cell proliferation index was calculated as a percentage of the cell nuclei
with BrdU labeling. Data were collected from at least three pairs of mutant
and control littermates. Students' t-test was used to analyze the
significance of difference and a P-value less than 0.05 was
considered statistically significant.
Immunohistochemical detection of cyclin D1 was carried out with paraffin
sections of paraformaldehyde-fixed embryos using a rabbit polyclonal antibody
against cyclin D1 (Thermo Scientific, Fremont, CA) at 1:200 dilution and
detected using the Zymed Histostain Plus Kit (Zymed Laboratories) as
previously described (Casey et al.,
2006).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Gritli-Linde, 2007). To
investigate the roles of Shh signaling in palate development, we generated
K14-Cre;Shhc/n mutant mice using a different
K14-Cre transgenic mouse line (line 43)
(Andl et al., 2004) from that
used in the previous studies, hoping to achieve higher penetrance of the cleft
palate phenotype. However, we observed that about 70% (12 of 17) of our
K14-Cre;Shhc/n mutant mice exhibited cleft palate
(Fig. 1B; and data not shown).
To examine whether Shh signaling plays a crucial role in palatal outgrowth, we
first compared cell proliferation during early palate development in four
pairs of K14-Cre;Shhc/n mutant and control littermates by
examining BrdU incorporation at E13.5. Of the four
K14-Cre;Shhc/n mutant embryos, one displayed significant
reduction in the numbers of BrdU-labeled cells in both the palatal epithelium
and mesenchyme (Fig. 1C,D), one
displayed significant reduction in BrdU-labeled cells in the epithelium only,
one displayed reduction in BrdU-labeled cells in the palatal mesenchyme only
and one had similar cell proliferation in both the palatal epithelium and
mesenchyme to that in the control embryos (see Table S1 in the supplementary
material). These data suggest that the inactivation of Shh in the
palatal epithelium may be variable in the different
K14-Cre;Shhc/n mutant mice.
To examine the efficiency of Shh inactivation in the palatal
epithelium in K14-Cre;Shhc/n mutant mice, we compared
expression of Ptch1 and Gli1, two known target genes of Shh
signaling, in the developing palatal shelves in the mutant and control
littermates. At E13.5, both Ptch1 and Gli1 are expressed in
a lateral-to-medial gradient in the mesenchyme of the developing palatal
shelves in the control embryos (Fig.
1E,G). In the E13.5 K14-Cre;Shhc/n mutant
embryos, expression of both Ptch1 and Gli1 in the developing
palatal shelves is downregulated but substantial amounts of Ptch1 and
Gli1 mRNAs remained in the palatal mesenchyme in all three mutant
embryos examined (Fig. 1F,H).
By contrast, expression of both mRNAs is nearly completely downregulated in
the developing tooth epithelium in the K14-Cre;Shhc/n
mutant embryos in comparison with control littermates
(Fig. 1E-H). Both
Ptch1 and Gli1 are also highly expressed in the mesenchymal
cells in the mandibular ossification areas, which are regulated by Ihh instead
of Shh and are not altered in the K14-Cre;Shhc/n mutant
embryos (Fig. 1E-H). As Shh
expression has been detected only in the palatal epithelium but not in the
palatal mesenchyme (Zhang et al.,
2002; Rice et al.,
2006), and as Ihh expression is restricted to the areas
undergoing ossification whereas Dhh is not expressed in the
developing palate (Rice et al.,
2006), the sustained expression of Ptch1 and
Gli1 in the palatal mesenchyme indicates that the inactivation of
Shh in the palatal epithelium in the
K14-Cre;Shhc/n mutant embryos was incomplete, which is the
most likely reason for the great variability in palatal mesenchyme
proliferation at E13.5 and the incomplete penetrance of the cleft palate
phenotype at birth.
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). The
Osr2-IresCre mice express Cre recombinase in the palatal mesenchyme
from the onset of palatal outgrowth at E11.5
(Lan et al., 2007)
(Fig. 2A). Importantly,
although Osr2-IresCre mice express Cre in several other tissues in
the developing craniofacial complex (Lan
et al., 2007) (see Fig. S1 in the supplementary material), Cre
activity was restricted in the mesenchyme and was not detected in the
epithelium of the developing secondary palate
(Fig. 2B). In the
Osr2-IresCre;Smoc/c embryos, whereas Ptch1 and
Gli1 were expressed in the palatal epithelial cells and several other
craniofacial tissues at similar levels to those in control littermates (see
Fig. S2A-D in the supplementary material), expression of both genes was
dramatically downregulated in the palatal mesenchyme by E13
(Fig. 2C-F). These data
demonstrate that signaling downstream of Shh remained intact in the palatal
epithelium and was efficiently blocked in the palatal mesenchyme in
Osr2-IresCre;Smoc/c mutant embryos. Examination of
Osr2-IresCre;Smoc/c mutant pups at birth revealed that
100% (n=22) of them had cleft palate
(Fig. 3), indicating that Shh
signaling in the palatal mesenchyme is required for palate development. In
addition to the cleft palate defect, the
Osr2-IresCre;Smoc/c mutant mice exhibited open eyelids and
smaller but thickened tympanic rings (Fig.
3B,F). As neither the Osr2-IresCre heterozygous mice nor
the Osr2-IresCre;Smo+/c mice had these defects
(Fig. 3A,C,E; and data not
shown), they probably resulted from loss of Smo-mediated hedgehog signaling in
the developing eyelids and other craniofacial tissues where Cre was also
expressed (see Fig. S1 in the supplementary material). Consistent with this
hypothesis, expression of both Ptch1 and Gli1 was also
downregulated in the developing eyelid tissues in the
Osr2-IresCre;Smoc/c mutant mice, in comparison with the
control littermates (see Fig. S2A-D in the supplementary material).
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To investigate whether palatal shelf retardation in the Osr2-IresCre;Smoc/c mutant embryos was due to impaired cell proliferation, we analyzed BrdU incorporation in E13 embryos. Indeed, the Osr2-IresCre;Smoc/c mutants consistently exhibited significant reductions in cell proliferation throughout the anteroposterior axis in the palatal mesenchyme (Fig. 5). Interestingly, the Osr2-IresCre;Smoc/c mutant embryos also exhibited significant reduction in cell proliferation in the anterior, but not in the posterior, palatal epithelia (Fig. 5C,F). As these embryos had Smo inactivation in the palatal mesenchyme but not in the palatal epithelium, the significant reduction in cell proliferation in the anterior palatal epithelium suggests that Shh-Smo signaling controls expression of mesenchymal factors involved in the regulation of palatal epithelial proliferation.
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; Ishibashi and
McMahon, 2002; Lobjois et al.,
2004; Mill et al.,
2005; Hu et al.,
2006). Immunohistochemical staining using a Ccnd1-specific
antibody showed that it is strongly expressed in both the palatal epithelium
and mesenchyme at E13.5 in control embryos
(Fig. 6A). Expression of Ccnd1
was substantially diminished in the palatal mesenchyme in
Osr2-IresCre;Smoc/c mutant littermates
(Fig. 6B). Ccnd2 is
expressed strongly in the palatal epithelium and relatively weakly throughout
the palatal mesenchyme in control embryos at E13.5
(Fig. 6C). In the
Osr2-IresCre;Smoc/c mutant embryos, whereas Ccnd2
mRNA expression in the palatal epithelium was similar to that in the control
embryos, its expression in the palatal mesenchyme was clearly reduced
(Fig. 6D). Thus, Shh signaling
regulates palatal mesenchyme cell proliferation at least in part through the
maintenance of Ccnd1 and Ccnd2 expression.
Alterations in Bmp2 and Bmp4 expression in the developing palatal mesenchyme of Osr2-IresCre;Smoc/c mutant embryos
In palatal explant culture assays, exogenous Shh protein induced
Bmp2 but not Bmp4 mRNA expression in the anterior palate
(Zhang et al., 2002). In E13.5
wild-type embryos, both Bmp2 and Bmp4 mRNAs are expressed in
the distal mesenchyme underlying the medial edge epithelium in the anterior
palate, and neither is expressed in the posterior palatal mesenchyme
(Zhang et al., 2002;
Hilliard et al., 2005).
Bmp2 mRNA expression in the palatal mesenchyme was downregulated in
Osr2-IresCre;Smoc/c mutant embryos in comparison with that
in control littermates (Fig.
7A,B), confirming the hypothesis that Shh-Smo signaling is
required for maintenance of Bmp2 mRNA expression in the anterior
palatal mesenchyme in vivo. However, Bmp4 expression in the anterior
palatal mesenchyme was upregulated, particularly in the lateral region of the
anterior palatal mesenchyme, in Osr2-IresCre;Smoc/c mutant
embryos compared with control littermates
(Fig. 7C,D). Expression of
Msx1, a known downstream target of Bmp4 signaling, was also
upregulated in the anterior palatal mesenchyme in the
Osr2-IresCre;Smoc/c mutant embryos
(Fig. 7E,F). By contrast,
expression of Shox2, encoding a transcription factor specifically
expressed in and required for development of the anterior palate
(Yu et al., 2005), was not
significantly altered in the Osr2-IresCre;Smoc/c mutant
embryos compared with control littermates
(Fig. 7G,H), indicating that
the effects of Smo inactivation on the expression of Bmp2,
Bmp4 and Msx1 in the palatal mesenchyme are specific and not due
to tissue retardation.
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). To better understand the Shh signaling pathway in palate
development, we examined expression of these Fox genes in the
developing palatal shelves in the Osr2-IresCre;Smoc/c
mutant embryos and control littermates. Expression of Foxc1, Foxc2
and Foxd2 was absent or very weak in the control palatal mesenchyme
at E13.5 and was not altered in the Osr2-IresCre;Smoc/c
mutant littermates (data not shown). Foxf1a mRNA was highly
specifically expressed in the lateral palatal mesenchyme as well as in the
lingual side of the molar tooth mesenchyme in the E13.5 control embryos
(Fig. 8A,C). In the
Osr2-IresCre;Smoc/c littermates, Foxf1a mRNA
expression was specifically downregulated in the palatal mesenchyme in both
the anterior and posterior palate, whereas its expression in the tooth
mesenchyme persisted (Fig.
8B,D). Foxf2 mRNA was highly expressed in the palatal
mesenchyme and in the developing tongue musculature at E13.5 in control
embryos (Fig. 8E,G). Expression
of Foxf2 mRNA was specifically downregulated in the palatal
mesenchyme, whereas strong expression persisted in the developing tongue, in
the Osr2-IresCre;Smoc/c littermates
(Fig. 8F,H). Thus, similar to
that in the early developing facial primordia, expression of Foxf1a
and Foxf2 in the developing palatal mesenchyme is also positively
regulated by Shh-Smo signaling.
To further investigate the molecular mechanisms of Shh-Smo signaling in
palate development, we compared expression of several other mesenchymal
transcription factors known to regulate palate development. Osr2
encodes a zinc finger protein specifically required for palatal mesenchyme
cell proliferation (Lan et al.,
2001; Lan et al.,
2004). Whereas Osr2 mRNA expression was similar in the
Osr2-IresCre;Smoc/c mutant and control embryos at E12.5
(data not shown), there was a significant reduction in Osr2 mRNA
expression in the palatal mesenchyme in
Osr2-IresCre;Smoc/c mutant embryos by E13.5
(Fig. 9A,B). By contrast,
Osr2 mRNA expression in the mesenchyme lingual to the first molar
tooth buds remained at similar levels in the control and mutant littermates
(Fig. 9A,B). Moreover,
expression of three other transcription factor genes, Osr1, Pax9 and
Tbx22, were not significantly altered in the
Osr2-IresCre;Smoc/c mutant palate from that in the control
littermates (Fig. 9C,D; and
data not shown). Thus, Smo activity appears to be specifically required for
the maintenance of Osr2 mRNA expression in the palatal
mesenchyme.
Shh-Smo signaling is required for maintenance of Fgf10 expression in the palatal mesenchyme
Fgf10 is expressed in the palatal mesenchyme and regulates palatal
epithelial cell proliferation (Rice et
al., 2004). Interestingly, Fgf10 mRNA is strongly
expressed in the anterior half, with little expression in the posterior half,
of the developing palatal shelves (Alappat
et al., 2005; Welsh et al.,
2007). We found that Fgf10 mRNA expression in the
developing anterior palatal mesenchyme was dramatically reduced in
Osr2-IresCre;Smoc/c mutant embryos by E13.5, in comparison
with control littermates (Fig.
9E,F). Interestingly, Fgf10 expression was also
downregulated in the developing eyelid mesenchyme in
Osr2-IresCre;Smoc/c mutant embryos, in comparison with the
control littermates (see Fig. S2E,F in the supplementary material). Expression
of Fgf10 in both the developing palate and eyelid overlapped with
that of Ptch1, and the downregulation of Fgf10 expression
also correlated with downregulation of Ptch1 in these tissues, in the
Osr2-IresCre;Smoc/c mutant embryos (Fig. S2A,B,E,F in the
supplementary material). Fgf10 also plays a crucial role in regulating cell
proliferation during eyelid development
(Tao et al., 2005). Similar to
Osr2-IresCre;Smoc/c mutant mice,
K14-Cre;Shhc/n mutant mice exhibited open eyelids at birth
(Dassule et al., 2000). Taken
together, these data suggest that Fgf10 acts downstream of Shh signaling to
regulate development of the eyelid and secondary palate.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), it is not
known what signals induce initial palatal outgrowth and how various molecular
pathways are integrated to regulate the epithelial-mesenchymal interactions
controlling palatal growth. Shh is expressed in the oral epithelium
before palatal outgrowth and becomes restricted to the medial and lateral
epithelium of the downward-growing palatal shelves
(Rice et al., 2006). In
palatal explant culture assays, exogenous Shh protein increased palatal
mesenchyme cell proliferation, and a function-blocking Shh antibody inhibited
palatal mesenchyme proliferation (Zhang et
al., 2002). Whereas targeted null mutations in the Shh
gene resulted in severe craniofacial defects in homozygous mutant mice,
K14-Cre-mediated inactivation of Shh in epithelial tissues, including
oral ectoderm, caused incomplete penetrance of cleft palate
(Rice et al., 2004;
Gritli-Linde, 2007). We found
that the incomplete penetrance of cleft palate in the
K14-Cre;Shhc/n mutant mice was due to incomplete
inactivation of Shh activity in the palatal epithelium. The different
K14-Cre transgenic mouse strains resulted in different penetrance of
the cleft palate phenotype in the K14-Cre;Shhc/n mutant
mice, probably due to transgene positional effects and different mouse strain
background effects on Cre expression.
In this study, we used the recently generated Osr2-IresCre knockin
mice to genetically dissect the molecular mechanism involving Shh signaling in
palate development. Although the Osr2-IresCre mice also express Cre
in several other craniofacial tissues (see Fig. S1 in the supplementary
material), they express Cre throughout the palatal mesenchyme but not in the
palatal epithelium (Fig. 2B).
Indeed, we found that the Osr2-IresCre;Smoc/c mutant
embryos exhibited dramatic reduction in expression of Ptch1 and
Gli1 mRNAs in the palatal mesenchyme by E13. Interestingly, although
Osr2-IresCre;Smoc/c mutant mice exhibited complete
penetrance of cleft palate, their palatal shelves appeared morphologically
normal before E13 and were elevated by E14.5, in contrast to the previously
described severely retarded palatal shelves that failed to elevate in the
K14-Cre;Shhc/n mutant mice
(Rice et al., 2004;
Gritli-Linde, 2007). These
differences in palatal phenotype may be due to loss of Shh signaling in both
the epithelium and mesenchyme in the K14-Cre;Shhc/n mutant
mice compared with loss of Shh signaling only in the palatal mesenchyme in the
Osr2-IresCre;Smoc/c mutant mice. It is also possible that
the high penetrance of cleft palate phenotype in the
Osr2-IresCre;Smoc/c mutant mice resulted from a
combination of palatal specific defects and secondary effects of loss of Smo
in other Cre-expressing craniofacial tissues. Nevertheless, the
Osr2-IresCre;Smoc/c mutant mice provided an opportunity to
clarify the role of Shh signaling in the reciprocal epithelial-mesenchymal
interactions during palate development.
The epithelially expressed Shh signals directly to the palatal mesenchyme to regulate palatal outgrowth
In palatal explant culture assays, Zhang et al. showed that exogenous Shh
protein induced Bmp2, but not Bmp4, mRNA expression in the
anterior palatal mesenchyme (Zhang et al.,
2002). Zhang et al. further showed that both exogenous Shh and
Bmp2 proteins induced palatal mesenchyme proliferation and that exogenous
noggin protein blocked the effect of Shh on palatal mesenchyme proliferation
(Zhang et al., 2002), which
led to the hypothesis that Bmp2 mediated the mitogenic effect of Shh signaling
on the palatal mesenchyme. Whereas our data confirm a role for Shh-Smo
signaling in maintenance of Bmp2 expression in the anterior palatal
mesenchyme, it should be noted that Bmp2 expression in the anterior
palatal mesenchyme is highly restricted to just subjacent to the medial edge
epithelium and at very low abundance compared with its abundant expression in
many other regions of the developing craniofacial complex (see Fig. S3 in the
supplementary material). Moreover, whereas Shh was shown to induce
Bmp2 expression in the anterior but not the posterior palatal
explants (Zhang et al., 2002),
we found that mesenchymal cell proliferation was significantly reduced in both
anterior and posterior regions of the developing palate in the
Osr2-IresCre;Smoc/c mutant embryos, indicating that other
factors also mediate the mitogenic effects of Shh signaling in the palatal
mesenchyme. Furthermore, we found that the expression levels of Ccnd1 protein
and of Ccnd2 mRNA were both reduced in the palatal mesenchyme of
Osr2-IresCre;Smoc/c mutant embryos, compared with the
control littermates. Shh signaling has been shown to regulate Ccnd1
and/or Ccnd2 expression in multiple tissues and Gli proteins have
been shown to bind to specific sites in the Ccnd1 and Ccnd2
gene promoters (Kenney and Rowitch,
2000; Long et al.,
2001; Ishibashi and McMahon,
2002; Yoon et al.,
2002; Mill et al.,
2005; Hu et al.,
2006). Gli1, Gli2 and Gli3 are all expressed in
the palatal mesenchyme during palatal outgrowth
(Rice et al., 2006). Thus, it
is likely that Shh-Smo signaling directly activates Gli-mediated
transcriptional activation of Ccnd1 and Ccnd2 in the palatal
mesenchyme to promote palatal outgrowth.
Foxf1, Foxf2 and Osr2 may be downstream effectors of Shh signaling during palatal outgrowth
During early craniofacial development, expression of several Fox family
genes, including Foxf1a and Foxf2, depended on Shh-Smo
signaling (Jeong et al.,
2004). Foxf1a expression in the developing lung
mesenchyme, a non-neural-crest-derived tissue, was also positively regulated
by Shh signaling (Mahlapuu et al., 2001a). We found that Foxf1a and
Foxf2 were expressed in the developing palatal mesenchyme in the
control embryos and both were downregulated in the palatal mesenchyme of the
Osr2-IresCre;Smoc/c mutant embryos. Whereas
Foxf1a-/- null mouse embryos died before craniofacial
morphogenesis, Foxf2-/- mutant mice exhibited complete
cleft palate (Mahlapuu et al., 2001b; Wang
et al., 2003). Jeong et al. proposed a Fox code for mediating the
roles of Shh-Smo signaling during early facial development
(Jeong et al., 2004). It is
likely that Foxf1a and Foxf2 also mediate some of the effects of Shh-Smo
signaling during palate development.
We found that Osr2 mRNA expression was downregulated in the
palatal mesenchyme of Osr2-IresCre;Smoc/c mutant mouse
embryos. Although Osr2 was not expressed in many tissues that receive
active Shh signaling, such as in the ventral neural tube, Osr2 mRNA
expression overlapped with Ptch1 mRNA expression during palate
development, with each exhibiting a lateral-to-medial gradient as the palatal
shelves grew vertically from E12 to E13.5
(Lan et al., 2001;
Lan et al., 2004;
Rice et al., 2006). To confirm
that Shh signaling plays a role in the maintenance of Osr2 mRNA
expression in the developing palatal mesenchyme, we examined Osr2
mRNA expression in K14-Cre;Shhc/n mutant embryos and found
that it was also downregulated in the palatal mesenchyme in those embryos by
E13.5 (data not shown). Osr2 is one of a few transcription factors with a
proven role in regulating palatal mesenchyme cell proliferation
(Lan et al., 2004). It is
possible that Shh regulates palatal mesenchyme cell proliferation in part
through maintenance of Osr2 expression.
Shh signaling coordinates the reciprocal epithelial-mesenchymal interactions during palatal outgrowth
We found that Osr2-IresCre;Smoc/c mutant mice exhibited
significant reduction in cell proliferation in the anterior palatal
epithelium. As Shh-Smo signaling remained intact in the palatal epithelium in
these mutants, we hypothesized that Shh signaling controls the expression of a
mesenchymal factor required for palatal epithelial cell proliferation. Rice et
al. showed that Fgf10, expressed in the developing palatal
mesenchyme, was required for palatal epithelial cell proliferation
(Rice et al., 2004).
Interestingly, Fgf10 is normally strongly expressed in the anterior
half, with very little expression in the posterior half, of the developing
palate (Alappat et al., 2005;
Hilliard et al., 2005;
Welsh et al., 2007). We found
that expression of Fgf10 mRNA in the anterior palatal mesenchyme was
dramatically downregulated in Osr2-IresCre;Smoc/c mutant
embryos, suggesting that Shh-Smo signaling in the palatal mesenchyme
secondarily affected palatal epithelial cell proliferation through regulation
of Fgf10 expression.
Shh has been shown to regulate Fgf10 expression during development
of several other vertebrate organs. Shh induced Fgf10 mRNA expression
in the chick limb bud (Ohuchi et al.,
1997). Exogenous Shh also induced, and a function blocking Shh
antibody inhibited, Fgf10 mRNA expression in the developing genital
tubercle mesenchyme (Haraguchi et al.,
2001). We found, in addition to downregulation of Fgf10
in the palatal mesenchyme, Osr2-IresCre;Smoc/c mutant mice
exhibited downregulation of Fgf10 expression in the developing eyelid
mesenchyme. By contrast, Shh inhibited Fgf10 expression in the
developing lung mesenchyme and Shh-/- mutant mice
exhibited more widespread expression of Fgf10 mRNA in the developing
lung mesenchyme (Bellusci et al.,
1997; Litingtung et al.,
1998). Thus, Fgf10 expression may be either positively or
negatively regulated by Shh signaling depending on the specific developmental
context. It is possible that similar effectors downstream of Shh-Smo signaling
exist in the developing eyelid, palate, limb bud and genital tubercle
mesenchyme for the maintenance of Fgf10 mRNA expression.
Interestingly, Rice et al. showed that exogenous Fgf10 protein induced
Shh mRNA expression in the palatal epithelium of wild-type embryos
and that Shh mRNA expression was reduced in the palatal epithelium in
Fgf10-/- and Fgfr2b-/- mutant embryos
(Rice et al., 2004). Whereas
our data indicate secondary effects of Smo inactivation in the
palatal mesenchyme on palatal epithelial cell proliferation, Rice et al.
showed that cell proliferation was significantly reduced not only in the
palatal epithelium but also in the palatal mesenchyme in
Fgf10-/- and Fgfr2b-/- mutant mice
(Rice et al., 2004). Taken
together, these data suggest that Shh and Fgf10 function in a positive
feedback loop to regulate palatal outgrowth.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1387/DC1
|
Footnotes |
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REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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