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First published online 21 March 2007
doi: 10.1242/dev.02837
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The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA.
* Author for correspondence (e-mail: tom.gridley{at}jax.org)
Accepted 15 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cleft palate, Snail, Slug, Epithelial-mesenchymal transition, Periderm cells, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Murray, 2002). Formation of
the mammalian secondary palate begins with the appearance of bilateral
outgrowths from the maxillary processes, which grow and extend vertically
along the sides of the tongue (Ferguson,
1988). At a specific point in development, mechanical forces
resulting from the forward extension of the mandible depress the tongue,
allowing the palatal shelves to elevate and come into close approximation with
each other. Through the specific expression of cell surface molecules and
alterations in the morphology of cells along the medial edge epithelia (MEE),
the shelves adhere and form a single structure bisected by a layer of
epithelium, known as the medial epithelial seam (MES). The MES subsequently
disappears, leading to a continuous palatal shelf consisting of a mesenchymal
core bounded by the nasal and oral epithelium.
Disruption of any of these processes can result in cleft secondary palate.
For example, Tgfb3-null mice exhibit defects in palatal shelf fusion
resulting from a failure of the MEE to acquire the necessary adhesive
phenotype (Gato et al., 2002;
Taya et al., 1999;
Tudela et al., 2002). Mutation
of the Msx1 gene, however, results in cleft palate due to reduced
proliferation in the palatal shelves during the vertical growth phase (Zhang
et al., 2002). Cleft palate is also seen in mice exhibiting defects in palatal
shelf elevation, such as Jag2-null mice, in which fusions between the
tongue and palatal shelves are observed
(Casey et al., 2006;
Jiang et al., 1998a).
The Pierre Robin Sequence (also termed Robin Sequence) is a human
developmental malformation characterized by mandibular retrognathia (normal
size receded mandible) or micrognathia (abnormally small mandible),
glossoptosis (rearward and downward displacement of the tongue) and cleft
palate. Cases of Pierre Robin Sequence are both phenotypically and genetically
heterogeneous (Cohen, 1999;
Houdayer et al., 2001;
Jakobsen et al., 2006;
Jamshidi et al., 2004;
Melkoniemi et al., 2003;
Ounap et al., 2005;
Ricks et al., 2002). The
association of mandibular defects with palatal clefting has been observed in
several mouse models of cleft palate, including Hoxa2
(Gendron-Maguire et al.,
1993), Egfr
(Miettinen et al., 1999) and
Dmm (disproportionate micromelia, a dominant mutant allele of the
Col2a1 gene) (Ricks et al.,
2002) mutant mice. In both Egfr-null and Dmm
mice, growth and development of Meckel's cartilage is affected, delaying the
necessary forward movement of the mandible and tongue.
The mechanism by which the MES degrades to form a continuous mesenchymal
palatal shelf has been a matter of intense debate. One proposal is that the
loss of epithelial cells is caused by an epithelialmesenchymal transition
(EMT) (Fitchett and Hay,
1989). Although there is substantial data to suggest this process
occurs in a portion of the cells in the MES, recent lineage tracing studies
appear to rule out the possibility that these cells persist in the resulting
mesenchymal core of the palate (Vaziri Sani et al., 2005). Alternate models
propose that MES cells undergo apoptosis or migrate to either the oral or
nasal epithelium. It is likely that a combination of these events leads to the
eventual dissolution of the MES. During embryogenesis, the oral and nasal
epithelium is bilayered, consisting of a cuboidal basal epithelial cell layer
and a flattened, transient periderm layer on the surface
(Fitchett and Hay, 1989).
Current data suggest that, upon palatal shelf contact, periderm cells migrate
toward accumulations of epithelial cells at the oral and nasal aspects of the
shelf, where they undergo apoptosis
(Carette and Ferguson, 1992;
Cuervo and Covarrubias, 2004;
Martinez-Alvarez et al.,
2000b). Loss of the periderm layer has been proposed to be
important for reinforcing the adhesion between the shelves mediated by the
underlying basal cell layer (Fitchett and
Hay, 1989; Nawshad et al.,
2004).
Members of the Snail gene family play a central role in the patterning of
vertebrate embryos. These genes drive epithelialmesenchymal transitions
throughout development by directly repressing the transcription of genes
encoding components of cellcell adhesive complexes in epithelia
(Nieto, 2002). However,
members of the Snail gene family also have roles in other processes, such as
the protection of cells from programmed cell death, the establishment of
left-right asymmetry and the regulation of cell motility (reviewed by
Barrallo-Gimeno and Nieto,
2005). Although at least some Snai2-/- mice
are viable and fertile (Jiang et al.,
1998b), they exhibit an interesting array of phenotypes, including
an increased sensitivity in the hematopoietic lineages to induction of
apoptosis by ionizing radiation (Inoue et
al., 2002; Inukai et al.,
1999). We have recently shown that conditional deletion of the
Snai1 gene in the epiblast results in randomization of left-right
axis specification in mice (Murray and
Gridley, 2006). Mutations of the Snai1 and Snai2
genes have also been shown to interact genetically with a Twist1
mutation during the formation of cranial sutures
(Oram and Gridley, 2005).
Given these observations, we have extended our analysis of the functions of
Snai1 and Snai2 genes during development of other
craniofacial structures in mice, including the secondary palate. Here we
report that approximately 50% of Snai2-/- mice present
with cleft palate at birth, and the penetrance of this phenotype increases to
100% on a Snai1 heterozygous background. Moreover, embryos with
conditional deletion of the Snai1 gene in neural crest cells on a
Snai2 null genetic background exhibit multiple craniofacial
abnormalities, including defects such as micrognathia and cleft palate that
are similar to those seen in Pierre Robin Sequence patients. These results
demonstrate crucial roles for Snail gene family members in palatal shelf
fusion and craniofacial morphogenesis in mice.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) null
allele, Snai2del1 and Snai2lacZ null
alleles (Jiang et al., 1998b),
and Snai1flox conditional allele
(Murray et al., 2006) have
been described previously.
Snai1flox/flox mice were
maintained as homozygotes and bred to Snai2+/- mice for
the experiments described in this report. Wnt1-Cre mice
(Danielian et al., 1998) were
obtained from the Jackson Laboratory. Typically, compound heterozygous mice
carrying Snai1 and Snai2 null alleles were bred to single
heterozygous Snai2 female mice to generate Snai1+/-
Snai2-/- and Snai1+/+
Snai2-/- progeny. Similarly, male mice harboring
Wnt1-Cre, Snai1 and Snai2 alleles were crossed to
Snai1flox/flox
Snai2+/- females, and embryos were isolated at varying
gestational stages. Embryos of the genotype Wnt1-Cre/+
Snai1flox/- are designated
Snai1 conditional knockout (Snai1-cko), while those also
null for Snai2 are designated Snai1/2 double
knockout (Snai1/2-dko). ROSA26-EGFP mice
(Mao et al., 2001) containing
a Cre-inducible GFP reporter in the ROSA26 locus were obtained from the
Jackson Laboratory. The ROSA26-EGFP mice were bred to Wnt1-Cre/+
Snai1flox/flox
Snai2+/- mice and subsequently bred to homozygosity for the
ROSA26-EGFP reporter and Snai1flox alleles. Embryos or
neonatal mice were genotyped by allelespecific PCR of DNA isolated from the
yolk sac or tail.
In situ hybridization
Radioactive in situ hybridization was performed essentially as described
(Krebs et al., 2001), except
33P-UTP was used instead of 35S as the labeled
nucleotide. Exposure times for each probe were determined empirically.
Radioactive probes were made using the Promega (Madison, WI) Riboprobe kit,
using 33P-labeled UTP (Perkin-Elmer, Waltham, MA). Whole-mount in
situ hybridization was performed as described
(Krebs et al., 2001), and
digoxigenin-labeled riboprobes were generated using the Roche (Indianapolis,
IN) labeling kit according to the manufacturer's instructions.
Histology and immunostaining
Histology, immunohistochemistry and immunofluorescence were performed on 7
µm sections of paraformaldehyde-fixed, paraffin-embedded embryos.
Antibodies for keratin 6 (Clone LHK6B, NeoMarkers, Fremont, CA) and
phospho-histone H3 (Upstate Biotechnology, Charlottesville, VA) were diluted
1:100 in 4% goat serum/PBS. Quantitation of phospho-histone H3 staining was
performed by counting equivalent areas of eight separate sections from two
embryos for each age/genotype. Data is presented as the mean±s.e.m.,
and P-values were computed using Student's t-test. An
antigen retrieval step comprising boiling for 10 minutes in 10 mM citric acid
was performed for both antibodies. For keratin 6, an Alexa Fluor 488-labeled
secondary antibody (Invitrogen, Carlsbad, CA) was used at a 1:400 dilution and
samples were counterstained with Hoescht 33342 (Invitrogen, Carlsbad, CA) and
mounted using Vectashield hard set mounting media (Vector Labs, Burlingame,
CA). Phospho-histone H3 staining was visualized using diaminobenzidine (DAB)
following incubation with a peroxidase conjugated secondary antibody (Jackson
ImmunoResearch, West Grove, PA) diluted to 1:2000. EGFP was visualized by
immunostaining with a chicken anti-GFP antibody (Aves Labs, Tigard, OR) and
Alexa Fluor 488-conjugated anti-chicken secondary antibodies. For TUNEL
analysis, samples were analyzed using the In Situ Cell Detection Kit,
Fluorescein (Roche, Indianapolis, IN). Coronal cryostat sections of embryonic
day 14.5 (E14.5) Snai2lacZ embryos were stained for
ß-galactosidase activity, as described
(Oram et al., 2003).
Skeletal staining
Newborn mutant mice and littermate controls were stained with Alcian Blue
and Alizarin Red to visualize skeletal and cartilaginous elements. Embryos
were skinned, eviscerated and fixed overnight in 100% ethanol (EtOH). They
were then stained overnight in 0.015% Alcian Blue, 0.005% Alizaran Red in 5%
acetic acid/70% EtOH. Clearing was performed in 1% potassium hydroxide/20%
glycerol, after which they were brought into 100% glycerol for photography and
storage. A modification of this method omitting the Alizarin Red was used to
visualize the cartilaginous skeleton of E14.5 embryos. Embryos were then
washed in 70% EtOH and cleared in 1:2 benzyl alcohol:benzyl benzoate.
Electron microscopy
Mandibles of E14.5 and E17.5 embryos were removed to visualize the palate,
and the embryos were fixed at 4°C with 2.5% glutaraldehyde in 0.1 M sodium
cacodylate, pH 7.4. Following washes, embryos were post-fixed in 1% osmium
tetroxide in 0.1 M sodium cacodylate for 2 hours at 4°C, dehydrated and
dried under CO2. Samples were then mounted, sputter coated with
gold to 15 nm, and examined at 20 kV with a Hitachi 3000N scanning electron
microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Jiang et al., 1998b).
Observation of the progeny of Snai2+/- intercrosses after
birth revealed a striking reduction in the number of
Snai2-/- mice at weaning. When observed shortly after
birth, some Snai2-/- progeny presented with severe
abdominal distention from air accumulation and absence of milk in the stomach,
both due to cleft secondary palate. Further analysis revealed that
approximately 50% of Snai2-/- neonates presented with
cleft palate. Penetrance of cleft palate increased to 100% in
Snai1+/- Snai2-/- neonatal mice, revealing
functional redundancy between these two gene family members
(Fig. 1A,B). Histological
analysis revealed that the palatal shelves of
Snai1+/-Snai2-/- embryos at E15.0 elevated
normally and came into contact at the midline
(Fig. 1C,D). However, the
shelves failed to form an MES and fuse. It is likely that the subsequent
growth of the head caused separation of the unfused palatal shelves, resulting
in the cleft secondary palate observed at birth.
To better understand the potential roles of the Snai1 and
Snai2 genes in palate development, we analyzed and compared their
expression patterns during palatogenesis. Snai1 gene expression was
assessed by in situ hybridization, while Snai2 expression was
determined by ß-galactosidase expression of the
Snai2lacZ allele
(Jiang et al., 1998b). At
E13.5, the Snai1 gene was expressed throughout the palatal shelf
mesenchyme, with particularly high levels in the cells underlying the MEE at
the anterior and along the medial aspect of the shelf mesenchyme at the
posterior (Fig. 1E,F). At
E14.5, strong expression was observed in the palatal mesenchyme just adjacent
to the newly formed MES (Fig.
1G,H), consistent with a previous report
(Martinez-Alvarez et al.,
2004). Snai2 expression at E14.5 was more diffuse, but
was present throughout the palate mesenchyme and epithelium
(Fig. 1I,J). Interestingly, we
noted an enhancement of ß-galactosidase expression from the
Snai2lacZ allele on a Snai1+/-
heterozygous genetic background (Fig.
1J versus 1I), suggesting that a compensatory mechanism regulating
gene expression levels may exist between these two genes.
|
), and
mutations in the Tgfb3 gene have been associated with nonsyndromic
cleft palate in humans (Lidral et al.,
1998). The palatal shelves of Tgfb3-null mice grow,
elevate and meet at the midline normally but fail to adhere and fuse
(Kaartinen et al., 1995;
Taya et al., 1999). This is
consistent with the expression pattern of the Tgfb3 gene along the
MEE, and is very similar to the phenotype of Snai2-/- and
Snai1+/- Snai2-/- mice at E14.5. Therefore, we
examined whether alterations in Tgfb3 expression could explain the
phenotypes observed in Snai2-/- and
Snai1+/- Snai2-/- mice. However, expression of
the Tgfb3 gene was unaltered in either Snai2-/-
or Snai1+/- Snai2-/- embryos
(Fig. 2A-C). Similarly, the
Irf6 gene, which is mutated in some human cleft palate patients and
is expressed throughout the palatal shelf epithelium
(Knight et al., 2006;
Kondo et al., 2002), was
expressed normally in Snai2-/- and Snai1+/-
Snai2-/- mutant embryos
(Fig. 2D-F).
Palatal shelf adhesion and fusion requires a series of modifications in the
morphology and expression of cell surface proteins of the MEE. As the shelves
approach each other, periderm cells along the apical surface of the MEE bulge
and extend filopodia and lamellipodia from their surface
(Martinez-Alvarez et al.,
2000a; Taya et al.,
1999). In Tgfb3 mutant mice, these cellular movements are
defective and the shelves subsequently fail to adhere. Although Tgfb3
expression appeared normal in Snai1+/-Snai2-/-
mutants, it is possible that Snai1 and Snai2 function lies
downstream of TGFß3 function. Scanning electron microscopy revealed that
both control and Snai1+/- Snai2-/- palatal
shelves exhibited the normal presence of both bulging cells and lamellipodia
on the MEE of E14.5 embryos (Fig.
3A,B). Numerous studies have highlighted the importance of
apoptosis in palatal shelf fusion (Carette
and Ferguson, 1992; Cuervo and
Covarrubias, 2004;
Martinez-Alvarez et al.,
2000b). Along with their described roles in regulating EMT, the
Snai1 and Snai2 genes also have been demonstrated to
regulate apoptosis (Inoue et al.,
2002; Inukai et al.,
1999; Vega et al., 2004). Therefore, we examined whether there
were any alterations in apoptosis in the developing palate of
Snai1+/- Snai2-/- mutant embryos at E14.5.
Control embryos displayed substantial apoptosis along the MES
(Fig. 3E, arrowheads),
concentrated at the oral and nasal aspects of the seam in accumulations of
epithelial cells termed epithelial triangles
(Fig. 3C,E, arrows). In mutant
embryos, however, we observed a dramatic reduction in the number of apoptotic
cells at the intersection of the two palatal shelves, suggesting that failure
to undergo normal programmed cell death might account for the cleft palate
phenotype in Snai1+/- Snai2-/- mutants
(Fig. 3D,F). Although an
occasional TUNEL-positive cell along the MEE of mutant shelves was observed,
comprehensive analysis of these embryos (n=4, at least 20 sections
per embryo along the anteroposterior axis) revealed that this was a rare
event. Indeed, apoptosis of periderm cells has been observed before MES
formation, prompting the suggestion that removal of the periderm is necessary
for adhesion (Fitchett and Hay,
1989). Countering this model is the observation using cell surface
labeling techniques that periderm cells migrate toward the epithelial
triangles after adhesion, where they undergo extensive apoptosis
(Cuervo and Covarrubias, 2004).
Thus, it is likely that a combination of migration to epithelial triangles and
apoptosis are required for the proper formation of the MES per se, a structure
that is not formed in Snai1+/- Snai2-/- mutant
embryos. Periderm cells marked by expression of keratin 6
(Mazzalupo and Coulombe, 2001)
were observed migrating into the epithelial triangles of control embryos
(Fig. 3G), while in mutant
embryos (Fig. 3H) keratin
6-expressing cells were confined to a discrete domain along the medial aspect
of the adjacent palatal shelves. Just before fusion, K6-positive cells were
present on the medial aspects of the palatal shelves of both control and
mutant embryos (data not shown). Migration of periderm cells is thought to
play an important role in epithelial fusions
(Mazzalupo and Coulombe,
2001), and a defect in periderm cell migration thus represents a
logical explanation, along with a concomitant reduction in apoptosis, for the
fusion defect observed in Snai1+/- Snai2-/-
mutant palates.
|
). We used
the Wnt1-Cre line (Danielian et
al., 1998) to delete the Snai1 gene in palatal shelf
mesenchyme, because an earlier study had shown Wnt1-Cre to be an
effective deleter in the vast majority of palatal mesenchyme cells
(Ito et al., 2003). Although
the Snai1 gene has been proposed to play an important role in the
formation of the neural crest, we have shown that, surprisingly,
Snai1 function is not required for neural crest cell delamination and
migration through E9.5 in mice (Murray and
Gridley, 2006). Thus, this approach permitted us to test both the
role of the Snai1 gene in palatal shelf growth and fusion and its
role in the development of neural-crest-derived structures after E9.5. In
order to determine the effectiveness of Snai1 deletion in the
appropriate cell types, we analyzed Snai1 expression by whole-mount
in situ hybridization. Snai1 expression in the branchial arches was
completely lost in Wnt1-Cre
Snai1flox/- (designated
Snai1-cko) embryos by E9.5
(Fig. 4A,B). Surprisingly, the
Snai1-cko mice were viable, fertile and exhibited no obvious
phenotypic abnormalities (data not shown).
Redundant function of the Snai1 and Snai2 genes in cranial neural crest cells
As we did not observe any obvious phenotypic abnormalities in
Snai1-cko mice, we bred the Snai2+/-
allele into this cross and examined the effects of compound mutant alleles on
palate development. We observed the expected frequencies of cleft palate in
Snai2-/- and Snai1+/-
Snai2-/- mice. However, Wnt1-Cre
Snai1flox/- Snai2-/-
mice (hereafter designated Snai1/2-dko) exhibited an
extensive array of craniofacial defects, including cleft palate. We observed a
striking difference in the phenotype of the developing palate in
Snai1/2-dko embryos compared with
Snai1+/-Snai2-/- embryos. Rather than elevating
normally and failing to fuse, as the palatal shelves of
Snai1+/- Snai2-/- embryos did, the palatal
shelves of Snai1/2-dko embryos remained in their vertical
growth orientation and failed to elevate
(Fig. 4C,D). Other craniofacial
defects observed in Snai1/2-dko neonates included presence
of an abnormal mandible that was significantly shorter than that of control
mice (Fig. 4E,F). The mandible
of the Snai1/2-dko neonates appeared to be missing the
rostral portion of the Meckel's cartilage and was fused at the midline
(Fig. 4G,H). In addition,
Snai1/2-dko mice had a dome-shaped skull, shortened parietal
bones and an enlarged frontal foramen (Fig.
4I,J). This spectrum of defects is also seen in mice with
mutations in neural crest regulatory genes such as Alk2
(Acvr1 - Mouse Genome Informatics)
(Dudas et al., 2004),
AP-2a (Tcfap2a - Mouse Genome Informatics)
(Brewer et al., 2004) and
Tgfbr2 (Ito et al.,
2003). Our results demonstrate that although the Snai1
and Snai2 genes play no detectable role in the early generation and
emigration of the mouse neural crest
(Murray and Gridley, 2006),
these genes are important for proper differentiation and patterning of cranial
neural-crest-derived structures.
|
|
). At E13.5, the Pax9 gene is expressed in the
palatal shelf mesenchyme and in the condensing mesenchyme surrounding the
developing molars (Fig. 5A),
and a similar pattern was observed in Snai1/2-dko
embryos at both E13.5 and 14.5 (Fig.
5B,C). Similarly, no difference between control and mutant embryos
was noted in the expression of the Msx1 gene
(Fig. 5D-F), which is crucial
for normal growth of the anterior palatal shelves (Zhang et al., 2002).
Expression of the Shh gene is dynamic in the developing palate, and
is restricted to a small domain of thickened oral epithelium at E13.5
(Fig. 5G), where it plays a
role in providing a mitogenic signal to the adjacent mesenchyme. In
Fgf10-/- and Fgfr2-/- mice,
Shh expression is disrupted, concomitant with a decrease in
proliferation in the nascent palatal shelves
(Rice et al., 2004). In
Snai1/2-dko mice, the Shh gene was
expressed in the proper domain at both stages, but at a somewhat reduced
intensity (Fig. 5H,I). The
Osr1 gene is expressed in a dynamic mediolateral pattern in the
developing palatal shelf and has been suggested to play an important role in
palatal shelf patterning (Lan et al.,
2004). Control embryos displayed Osr1 expression in the
lateral aspects of the vertical palatal shelf
(Fig. 5J), while staining in
Snai1/2-dko mutant shelves at E13.5 and 14.5 was
reduced somewhat, although some expression was noted in the proper domains
(Fig. 5K,L). Given the role of
the Shh gene in regulating cell division
(Ingham and McMahon, 2001), we
analyzed cell proliferation in control and mutant embryos using a marker of
mitosis, phospho-histone H3. When compared to an equivalent area of E13.5
control palatal shelves (78.4±10), we could not detect any significant
changes in the numbers of mitotic cells in
Snai1/2-dko palatal shelves at either E13.5
(80.2±10, P=0.28) or E14.5 (85.5±15, P=0.15)
(Fig. 6). Thus, the subtle
change in Shh expression observed in
Snai1/2-dko mice does not appear to result in any
major perturbation of the proliferative potential of the developing palatal
shelves. These results demonstrate that there are no major disruptions in
Snai1/2-dko mutants in the expression of several
genes central to the control of palatal shelf patterning.
|
), we performed lineage-tracing analyses on
E11.5, 12.5 and 14.5 embryos utilizing ROSA26-EGFP reporter mice
(Mao et al., 2001). We
observed no obvious differences in the distribution of EGFP-labeled cells in
Snai1/2-dko embryos at any of these stages (see Fig. S1 in
the supplementary material), indicating that there are no major disruptions of
neural crest cell migration in Snai1/2-dko embryos. It is
notable that subtle differences in the size and shape of the anterior palatal
shelves were observed in some Snai1/2-dko mutants
(Fig. 5E,
Fig. 6D; see Fig. S2 in the
supplementary material). These differences were apparent in only a subset of
Snai1/2-dko mutant embryos (see Fig. S2 in the supplementary
material), as there appears to be considerable variability in the extent of
outgrowth in control anterior palatal shelves (see Fig. S2A versus S2C in the
supplementary material). Although we did not observe any differences in cell
proliferation between control and mutant embryos, we cannot exclude the
possibility that delayed outgrowth of the anterior palatal shelves plays a
role in the cleft palate phenotype of Snai1/2-dko
embryos.
Cleft palate in Snai1/2-dko mice is probably secondary to defects in mandible development
As patterning and growth of the palatal shelves in
Snai1/2-dko mice appeared to be essentially normal, we
examined whether the palatal shelf defects might be secondary to the
craniofacial defects noted at birth. Mandibular malformations, such as
micrognathia, are proposed to be the underlying cause of cleft palate in
several mouse mutants, such as Egfr-/-
(Miettinen et al., 1999),
Hoxa2-/-
(Gendron-Maguire et al., 1993)
and Dmm (Ricks et al.,
2002) mutant mice. Because the forward growth of the mandible
driven by the extension of the neural-crest-derived Meckel's cartilage
provides the mechanism to lower the tongue and permit palatal shelf elevation,
any defects in this process could result in a cleft palate phenotype similar
to that observed in Snai1/2-dko mice. In light of the
mandibular abnormalities observed at birth
(Fig. 4F,H), we performed
Alcian Blue staining of the embryonic cartilage at E14.5 to determine if
defects in Meckel's cartilage could be observed at this crucial time point.
Meckel's cartilage was dramatically shorter in Snai1/2-dko
embryos compared with control embryos (Fig.
7). Therefore, we propose that that the underlying cause of cleft
palate in Snai1/2-dko embryos is abnormal development of
Meckel's cartilage, which in turn prevents the normal extension of the tongue
and elevation of the palatal shelves.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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|
) previously described high levels of Snai1 gene
expression in palatal shelf mesenchyme, and noted that a few MES cells express
the Snai1 gene during the fusion process, thus supporting a small
role for EMT in MES dissolution. Interestingly, they also observed a dramatic
increase in Snai1 expression in the MES of
Tgfb3-/- mutant palates, and proposed a model in which
increased compensatory expression of the Tgfb1 gene induces
Snai1 expression, thereby protecting these cells from apoptosis
(Barrallo-Gimeno and Nieto,
2005; Vega et al., 2004). This suggests a model in which the
Snai1 gene plays an important role in regulating the survival of MEE
and MES cells. We did not observe an increase in apoptosis in the mutant
palatal shelves, but instead observed a complete absence of cell death. This
might be secondary, however, to a more fundamental defect in palatal shelf
fusion. In support of this notion, we find that periderm cells remained at the
intersection of the two MEE layers, and did not migrate toward the oral and
nasal epithelia.
Defects in MES formation in Snai1+/- Snai2-/- mice
A crucial component of palate development is the adhesion of the palatal
shelves and subsequent transformation of the separate MEE into a single MES
(Ferguson, 1988). Normally, as
the palatal shelves approach each other, periderm cells of the bilayered MEE
bulge, form lamellipodia and express extracellular matrix proteins important
for adhesion on their surface. After contact is initiated, periderm cells
migrate to the oral and nasal aspects of the forming MES and undergo apoptosis
(Cuervo and Covarrubias, 2004).
The basal epithelial cells intercalate to form a single layer MES, which then
disappears as the basal cells also commit to an apoptotic program. Several
mouse models of cleft palate exhibit defects in this process, the classic
example being the Tgfb3-null mouse
(Kaartinen et al., 1995;
Taya et al., 1999). In these
embryos, palatal shelves elevate normally, but periderm cells fail to bulge
and do not form lamellipodia. Our results show that Snai1+/-
Snai2-/- mutant embryos display normal expression of the
Tgfb3 gene and the presence of bulging cells and lamellipodia on the
surface of the MEE. However, our data demonstrate that Snai1+/-
Snai2-/- embryos lack both the apoptosis normally observed at
the MES and the migration of periderm cells to form epithelial triangles. It
had been suggested that cell death of the periderm layer is crucial for the
proper intercalation and adhesion of the underlying basal cell layer
(Fitchett and Hay, 1989).
However, in vitro labeling experiments show that a substantial portion of the
periderm migrates to epithelial triangles at the nasal and oral aspects of the
MES (Carette and Ferguson,
1992), where they then undergo apoptosis
(Cuervo and Covarrubias, 2004;
Cuervo et al., 2002). Thus, a
model emerges wherein adhesion stimulates migration of the periderm, which in
turn allows for intercalation of the basal epithelial cells and formation of
the mature MES. One prediction from this model is that defects in periderm
cell migration would short-circuit the program and result in a lack of
periderm cell death and basal cell intercalation. Our data are consistent with
a failure of periderm cell migration in Snai1+/-
Snai2-/- mutants. The absence of apoptosis could then be
attributed to the persistence of the periderm on the MEE.
Could a failure to undergo EMT following MEE adhesion contribute to the cleft palate phenotype of Snai1+/- Snai2-/- embryos? Using fate-mapping analysis, Vaziri Sani et al. claimed that the absence of persistently labeled cells in the palatal shelf mesenchyme precluded EMT as a possible mechanism for palatal shelf fusion (Vaziri Sani et al., 2005). However, the data presented did not address whether MES cells could be observed undergoing EMT at the early stages of fusion, only that they did not ultimately contribute to the mesenchymal core. It is possible that EMT might occur in cells fated to undergo apoptosis, thus escaping identification in fate-mapping studies. Indeed, the migration of periderm cells presumably requires modification of their epithelial phenotype in order to migrate toward the epithelial triangles. Thus, it remains plausible that EMT is occurring at some point during the process of MES dissolution, although these cells ultimately do not contribute in a long-term manner to the palate mesenchyme.
Functions of Snail family genes in mouse neural crest development
We have reported previously the surprising result that neither the
Snai1 nor the Snai2 gene, alone or in combination, is
required for neural crest cell generation and delamination in mice, at least
through 9.5 days of gestation (Murray and
Gridley, 2006). It had been widely proposed that the
Snai1 gene would play an essential role in neural crest delamination
in mice, based on the observations that: (1) Snai1 is expressed in
the dorsal neural folds concomitant with neural crest cell delamination
(Locascio et al., 2002;
Sefton et al., 1998); (2)
ectopic Snai1 gene expression induces EMT in cultured epithelial
cells (Batlle et al., 2000;
Cano et al., 2000); (3) the
Snai2 gene plays a crucial role in neural crest cell delamination in
the chick (Nieto et al.,
1994); (4) Snai2-/- mice lack an obvious
neural crest phenotype (Jiang et al.,
1998b); and (5) the Snai1 and Snai2 genes are
expressed in a modular fashion across vertebrate species
(Locascio et al., 2002). The
results presented here reveal an entirely different role for the
Snai1 and Snai2 genes in craniofacial morphogenesis. While
loss of a single Snai1 allele in the context of a Snai2-null
background (Snai1+/- Snai2-/- mice) results in
palatal clefting due to defects in periderm cell migration and apoptosis,
removing the remaining Snai1 allele in the neural crest
(Snai1/2-dko mice), which populates a majority of the
palatal shelf mesenchyme, results in a very different cleft palate phenotype.
Marker analysis revealed only very subtle alterations in palatal shelf
patterning, including a small but clear decrease in the expression of
Shh in the oral epithelium and Osr1 in the mesenchyme.
Indeed, these changes may be related to the apparent small differences in
anterior palatal shelf outgrowth observed in Snai1/2-dko
embryos compared to some controls. It is unlikely, however, that these changes
are ultimately responsible for the observed cleft palate phenotype. In
addition to cleft palate, Snai1/2-dko embryos also exhibit
other craniofacial abnormalities, including micrognathia, fused mandible and
an enlarged parietal foramen in the skull vault. This combination of
phenotypes has been observed in other conditional neural crest mutants, such
as Alk2 (Dudas et al.,
2004), AP-2a (Brewer
et al., 2004) and Tgfbr2
(Ito et al., 2003), and
demonstrates that although the Snai1 and Snai2 genes are not
essential in the initial delamination and migration of neural crest cells from
the neural tube, they play a crucial role in the later development of
neural-crest-derived structures.
Snai1/2-dko embryos as a model for Pierre Robin Sequence
The Pierre Robin Sequence (also termed Robin Sequence) is a human
developmental malformation characterized by mandibular retrognathia (normal
size receded mandible) or micrognathia (abnormally small mandible),
glossoptosis (rearward and downward displacement of the tongue) and cleft
palate. Most newborns with Pierre Robin Sequence also exhibit respiratory and
feeding difficulties. Cases of Pierre Robin Sequence are both phenotypically
and genetically heterogeneous (Cohen,
1999; Houdayer et al.,
2001; Jakobsen et al.,
2006; Jamshidi et al.,
2004; Melkoniemi et al.,
2003; Ounap et al.,
2005; Ricks et al.,
2002). Pierre Robin Sequence can occur as an isolated nonsyndromic
form, or can occur in combination with other syndromes. Mutations in several
collagen genes, including COL2A1, COL11A1 and COL11A2, have
been found in subjects with nonsyndromic Pierre Robin Sequence
(Melkoniemi et al., 2003),
although it has not been established definitively that these mutations are
causal for the observed malformations. In addition, chromosomal translocations
(Jamshidi et al., 2004),
duplications (Ounap et al.,
2005) and deletions (Houdayer
et al., 2001) have been described that are associated with
nonsyndromic Pierre Robin Sequence.
A model for the etiology of the cleft palate defect in individuals with
Pierre Robin Sequence is that the small and/or abnormally positioned mandible
prevents the normal positioning of the tongue, thereby inhibiting palatal
shelf elevation and fusion. This model is supported by analyses of two mouse
models of Pierre Robin Sequence, the A/WySn inbred mouse strain
(Schubert et al., 2005) and
mice heterozygous for the disproportionate micromelia (Dmm) mutation
(Ricks et al., 2002), a
semidominant mutant allele of the Col2a1 gene
(Pace et al., 1997). Based on
a mechanical requirement for mandibular extension to drive the proper
depression of the tongue, we propose that a similar mechanism is responsible
for the cleft palate phenotype of Snai1/2-dko mice. This
interpretation is supported by the dramatic growth retardation of Meckel's
cartilage at E14.5, the crucial time for palatal shelf elevation.
Snai1/2-dko mice represent a novel model of Pierre Robin
Sequence, and should provide a useful tool for the study of this heterogeneous
developmental disorder in humans.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1789/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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