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First published online 23 October 2008
doi: 10.1242/dev.025767
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1 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA
70118, USA.
2 Section of Oral Biology, The Ohio State University College of Dentistry,
Columbus, OH 43210, USA.
3 Department of Physiology and Cell Biology, Faculty of Medical Sciences, Kobe
University, Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe
650-0017, Japan.
4 Center for Animal Resources and Development, Kumamoto University, Honjo 2-2-1,
Kumamoto 860-0811, Japan.
* Author for correspondence (e-mail: ychen{at}tulane.edu)
Accepted 7 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Wnt5a, Ror2, Palatogenesis, Cleft palate, Non-canonical Wnt pathway, Cell migration
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Okano et al., 2006;
Gritli-Linde, 2007). Many
genes involved in palate development exhibit regional differential expression
along the anteroposterior (AP) axis. The anterior and posterior palatal
mesenchyme also shows different molecular and cellular responses to growth
factors. This tissue and molecular heterogeneity could lead to differential
cell behaviors, such as cell proliferation, production of different
extracellular matrix and cell migration, in the palate along the AP axis.
However, despite rapid progress in the field, the exact cellular and molecular
mechanisms that are involved in morphological changes during palate
development remain unknown.
The Wnt family genes encode a large and diverse group of signaling
molecules in the patterning, proliferation and differentiation of a variety of
organs and cell types (Cadigan and Nusse,
1997). Wnt ligand binds to its receptor Frizzled (Fz) and Lrp5/6
to activate the canonical Wnt/β-catenin signaling pathway, or functions
through β-catenin-independent pathways, the noncanonical pathways,
including the planar cell polarity (PCP) and Wnt/Ca2+ pathways
(Veeman et al., 2003). Wnt
ligands are typically classified into canonical and noncanonical Wnts by the
pathways they work through. Wnt5a is a representative of the noncanonical Wnt
class. One function of the noncanonical pathway is its inhibition of the
canonical Wnt signaling. In Xenopus, overexpression of Wnt5a
blocks secondary axis induction by Wnt8
(Torres et al., 1996). In the
developing limbs, Wnt5a blocks canonical Wnt pathway by promoting degradation
of β-catenin in a GSK3-independent way
(Topol et al., 2003).
Noncanonical Wnt signaling also regulates, via the PCP pathway, convergent
extension movement, an essential cell migration process during vertebrate
gastrulation (Mlodzik, 2002).
Altered Wnt5a function leads to dysregulated convergent extension movement in
vertebrates (Moon et al.,
1993; Kilian et al.,
2003). It was recently reported that an alternative Wnt receptor,
Ror2, an orphan tyrosine kinase, mediates Wnt5a-initiated noncanonical
signaling and is required for the Wnt5a-mediated inhibition of the canonical
signaling (Oishi et al., 2003;
Mikels and Nusse, 2006).
Interestingly, Wnt5a is also capable of activating Wnt/β-catenin
signaling in the presence of Fz4 and Lrp5
(Mikels and Nusse, 2006). In
addition, the orphan receptor Ryk could also bind to Wnt5a besides to other
Wnt ligands (Lu et al., 2004;
Keeble et al., 2006).
Wnt signaling was initially implicated in palatogenesis based on the
findings that WNT3 mutations in humans and Wnt9b targeted
inactivation in mice cause a cleft palate phenotype with an incomplete
penetrance (Niemann et al.,
2004; Carroll et al.,
2005). Mapping of the chromosomal translocation in individuals
with non-syndromic cleft lip and palate identified a region containing
WNT5A (Blanton et al.,
2004). Although β-catenin expression was reported in the
developing palate (Martinez-Alvarez et
al., 2000; Tudela et al.,
2002; Nawshad and Hay,
2003), specific ablation of β-catenin in the
epithelial-derived tissues by a K14-Cre transgenic allele appeared
not to affect palate formation (Huelsken
et al., 2001).
We report here that Wnt5a and its receptors exhibit a gradient expression along the AP axis in the developing palate, consistent with a complete cleft palate phenotype in Wnt5a-/- mice. We analyzed the developing palate of Wnt5a mutants at the histological, cellular and molecular levels. We show for the first time the existence of active directional migration of mesenchymal cells in the palate, which is disrupted in the Wnt5a-/- palate. We further reveal a chemotactic role for Wnt5a in the palate. Genetic studies and in vitro assays provide evidence that Ror2 mediates Wnt5a signaling through a noncanonical pathway to regulate cell proliferation and cell migration during palatogenesis.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), TOPGAL
(DasGupta and Fuchs, 1999) and
Actinβ-Egfp transgenic mice were purchased from the
Jackson laboratories. Generation of Ror2 mutant mice has been
described previously (Takeuchi et al.,
2000). The genotype of these mice were determined by PCR-based
methods as described previously (DasGupta
and Fuchs, 1999; Yamaguchi et
al., 1999; Takeuchi et al.,
2000). Actinβ-Egfp mice were identified by
Egfp expression under UV light.
Histology, in situ hybridization, BrdU labeling and TUNEL assays
Mouse embryos were dissected in ice-cold PBS and fixed in 4%
paraformaldehyde (PFA)/PBS overnight at 4°C before being dehydrated
through graded ethanol and embedded in paraffin. Samples were sectioned at 10
µm for histological analysis and section in situ hybridization. Detection
of cell proliferation was performed with the bromodeoxyuridine (BrdU) labeling
and Detection Kit (Roche Diagnostics Corporation, Indianapolis), and the TUNEL
assay was performed to detect cell apoptosis, as described previously
(Zhang et al., 2002;
Alappat et al., 2005). For in
situ hybridization experiments, at least three samples were used for each
gene. For BrdU labeling, nine sections from three samples were counted and
applied for statistical calculation. BrdU-labeled cells were counted and
presented as percentage of total nuclei within arbitrarily defined areas, and
Student's t-test was used to determine significance of
differences.
In vitro organ culture, tissue grafting, DiI cell labeling, and bead implantation
Secondary palatal shelves were microdissected from E12.5 or E13.5 embryos,
orientated with dorsum upwards, and placed on filter in Trowell type organ
cultures, as described previously (Zhang
et al., 2002). For in vitro palate fusion assay, paired palatal
shelves from E13.5 embryos were placed in organ culture as described
previously (Taya et al.,
1999). Samples were harvested after 3 days in culture and
processed for histological analysis. For tissue grafting, donor palatal
mesenchyme from E12.5 Actinβ-Egfp embryos were
carefully dissected with fine forceps, minced into small tissue pieces and
implanted into the same position in explanted host palatal shelves from
aged-matched embryos. Explants were cultured in 
-MEM medium plus 10%
FBS, monitored for cell migration under a fluorescence microscope at 24-hour
intervals for 72 hours. For DiI labeling experiment, the lipophilic dye DiI
(1,1-dioctadecyl-3,3,39,39-tetramethyl-indocarbocyanine perchlorate; Molecular
Probes) was initially prepared at a concentration of 0.5% in 100% ethanol and
then diluted 1:9 with 0.3 M sucrose containing 0.1% Nile Blue sulfate. The DiI
solution was injected into explanted palatal shelves using a micropipette
(Li and Muneoka, 1999).
Protein-soaked beads were implanted to explanted palatal shelves, about
250-300 µm away from the DiI injection site. Migration of DiI-labeled cells
was monitored in a similar way for monitoring Egfp-expressing cell
migration. Bead preparation and implantation were performed as reported before
(Chen et al., 1996;
Zhang et al., 2002). Affi-Gel
blue agarose beads (100-200 µm in diameter, from Bio-RAD, Hercules, CA)
were used as carriers for Shh, Wnt5a and Bmp4, and heparin beads (Sigma, St
Louis, MO) were used as carriers for Fgf10. All the proteins were purchased
from R&D Systems (Minneapolis, MN).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In an
effort to identify genes that exhibit such an expression pattern in the
palate, we compared the gene expression profiles from the anterior (anterior
to the first molar) versus posterior (posterior to the first molar) region of
the E13.5 palatal shelves by Affimetrix chip microarray assays. Several genes
that encode components of Wnt signaling pathway, including Wnt5a,
frizzled 4 (Fzd4), Ror2 and β-catenin (Catnb),
showed differential expression levels between the anterior and posterior
palate, which was subsequently confirmed by in situ hybridization assays.
Wnt5a expression was detected in the palatal mesenchyme, in a graded
fashion along the AP axis (higher levels in the anterior region)
(Fig. 1A,E). A gradient
expression for Wnt5a along the nasal-oral axis was also observed
(Fig. 1A,E). Fzd4 and
Ror2 were also expressed, in a similar AP gradient fashion, in the
epithelium and mesenchyme of the palate, with a restricted mesenchymal domain
adjacent to the medial edge epithelium (MEE)
(Fig. 1B,C,F,G). A graded
expression of β-catenin, mainly in the MEE, was also detected along the
AP axis of the palate (Fig.
1D,H).
Wnt5a mutant mice exhibit complete cleft secondary palate
The expression of Wnt5a and its receptors Fzd4 and
Ror2 suggests a role for these genes in palatogenesis. The fact that
Ror2 knockout mice exhibit a cleft palate phenotype
(Schwabe et al., 2004) and
that individuals with non-syndromic cleft lip and palate have been linked to
chromosomal translocation containing WNT5A locus
(Blanton et al., 2004) promoted
us to examine a cleft palate phenotype in Wnt5a-/- mice.
Gross morphological and histological examinations revealed a complete cleft
palate phenotype in Wnt5a-/- mice
(Fig. 2A,B). In the mutants,
the deformed palatal shelves were distinguishable as early as E12.5 but became
pronounced by E13.5. At E13.5, when the palatal shelves in the wild type take
the vertical position on both sides of the tongue, the anterior region of the
mutant palatal shelves remained in the horizontal position
(Fig. 2C,D). By contrast, the
posterior portion of the mutant palatal shelves grew in a vertical growth
position (Fig. 2E,F).
Morphologically, the mutant anterior palatal shelf appeared round, shortened
and blunt-ended, lacking the ventrolateral indention of the shelf
(Fig. 2D), whereas the
posterior shelf was shortened, broadened and wedge-shaped
(Fig. 2F). At E14.5, the
wild-type palatal shelves have elevated to above the tongue and fused at the
midline (Fig. 2G,I). In
Wnt5a mutant, the anterior region of the palatal shelves remained in
horizontal level but was too short to make a contact
(Fig. 2H). However, the
posterior palatal shelves failed to elevate
(Fig. 2J). The lack of contact
between the palatal shelf and tongue/mandible suggests an intrinsic defect in
elevation. The gradient expression of Wnt5a along the AP axis of the
developing palate appears to account for the distinct morphological defects
found in the anterior and posterior palate of the mutant. In addition,
Wnt5a mutants also displayed a short and small tongue, and a deformed
oral cavity, which may contribute, to some extent, to the cleft palate
defect.
Wnt5a mutant exhibits an impaired palatal growth
To reveal the cellular mechanisms responsible for the palate deformation in
Wnt5a-/- mice, we carried out cell proliferation and
apoptosis assays as well as in vitro palate fusion assay. We determined cell
proliferation rate in the wild type and Wnt5a-/- palates
at E13.5 by BrdU labeling. As controls, BrdU-positive cells were also counted
in the maxillary mesenchyme lateral to the palate where Wnt5a is not
expressed. An altered cell proliferation rate was found in the mutant palatal
mesenchyme (Fig. 3).
Intriguingly, a significantly elevated cell proliferation level was seen in
the anterior palate, whereas a decreased rate occurred in the posterior
palate. This differential alteration of cell proliferation rate in the
Wnt5a-/- palate along the AP axis implicates dual roles
for Wnt5a in the regulation of cell proliferation. It is interesting to note
that, in the anterior palate of the mutant, the mesenchymal cells on the
future nasal side were much more mitotic than cells on the future oral side.
This dysregulated cell proliferation level in the future nasal aspect
apparently accounts for, at least partially, the deformed shape of the mutant
palatal shelves and a failure in change of palate growth direction from the
horizontal to vertical position. However, TUNEL assays revealed a comparable
level of cell apoptosis in the wild-type and Wnt5a-/-
palatal shelves at E13.5 (data not shown). To determine whether the formation
of a cleft palate in Wnt5a mutants could also be attributed to a
failure in palate fusion, we employed an in vitro organ culture system to
examine palate fusion. The results demonstrated that pairs of palatal shelves
from E13.5 Wnt5a-/- embryos, when placed in contact with
the MEE and facing one another, were able to fuse, in a way identically to the
wild-type controls (data not shown). The dysregulated cell proliferation in
the palatal mesenchyme therefore represents a major cellular process that
causes a cleft palate formation in Wnt5a mutants.
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), Shh in the MEE of the anterior palate was
downregulated (Fig. 4A,B),
whereas an ectopic Shh expression was observed in the nasal side
epithelium of the posterior palate, immediately adjacent to the ectopically
expressed Bmp4 domain in the palatal mesenchyme
(Fig. 4F,H). Ptch1, a
downstream target and receptor of Shh, displayed altered expression pattern
accordingly along the AP axis (Fig.
4I,J,M,N). However, as the mutant palatal shelves are so
malformed, the exact position where Shh is ectopically activated in
the palate remains to be determined. Nevertheless, these observations place
Wnt5a upstream of Shh and Bmp4, consistent with the
previous report in the developing lung (Li et al.,
2002;
2005). Msx1, a
downstream target of Bmp4, was partially downregulated in the
anterior palatal mesenchyme of the Wnt5a-/- palate
(Fig. 4K,
Fig. 5L). Although the
expression of Bmp2, a downstream target of Shh that mediates
the mitotic effect of Shh in the anterior palate
(Zhang et al., 2002), remained
unchanged in the anterior palate of the mutant (data not shown), a significant
downregulation of Bmp2 was observed in the posterior palatal
mesenchyme (Fig. 4O,P). It
appears that other upstream factors besides Shh are also involved in
regulating Bmp2 expression. By contrast, Fgf10 and its
receptor Fgfr2 exhibited comparable expression level and pattern in
both wild-type and mutant palate (data not shown). These results demonstrate
that Wnt5a acts differentially, in term of gene expression
regulation, in the anterior and posterior palate.
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). We failed to detect TOPGAL expression in the developing
palate of both wild type and Wnt5a mutants (data not shown),
suggesting that the canonical Wnt signaling pathway may not be involved in
palate development. The fact that mice lacking Fz4, the receptor that is
expressed in the developing palate and mediates the canonical signaling of
Wnt5a, survive to adulthood without a defective palate
(Hsieh et al., 2005), implies
an involvement of noncanonical pathway. The fact that both
Ror2-/- and Wnt5a-/- mice exhibit a
cleft palate phenotype strongly implicates an overlapping role for these two
molecules. We therefore examined the cleft palate defect in
Ror2-/- embryos. Similar to Wnt5a mutant, we
found a downregulated Shh in the MEE and an enhanced cell
proliferation rate in the anterior palatal mesenchyme of
Ror2-/- embryos (Fig.
5). We subsequently tested a genetic interaction between
Wnt5a and Ror2 by compounding Wnt5a+/-
and Ror2+/- to generate double heterozygous mice.
Wnt5a+/-;Ror2+/- mice indeed exhibited
cleft palate defects that phenocopy that of Ror2-/-
mutants, including downregulated Shh expression in the MEE and
upregulated cell proliferation rate in the mesenchyme of the anterior palate,
particularly in the nasal aspect (Fig.
5). These results demonstrate an epistatic effect of these two
genes on palate development. However, the posterior palatal shelves of
Ror2-/- and
Wnt5a+/-;Ror2+/- mice did not show an
altered cell proliferation rate, consistent with an extremely low level of
Ror2 expression in this region. As the palatal phenotypes in
Ror2-/- and
Ror2+/-;Wnt5a+/- mice appear milder
when compared with that of Wnt5a mutants, Ror2 is not the
only receptor mediating the function of Wnt5a during palatogenesis.
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Wnt5a-deficiency leads to altered cell migration in the palatal mesenchyme
In the assays for gene expression, we made an interesting observation about
Sox9 expression, a HMG box gene whose mutation also leads to cleft
palate formation (Bi et al.,
1999; Mori-Akiyama et al.,
2003). Sox9 is expressed and regulated by Shox2
in the anterior palatal mesenchyme (Fig.
4Q) (Yu et al.,
2005; Gu et al.,
2008). Consistent with an unaltered Shox2 expression
(Fig. 4S,T), Sox9
expression was maintained in the anterior palatal mesenchyme of Wnt5a
mutant (Fig. 4R). However, a
close examination identified a shifted expression domain of Sox9. In
the controls, Sox9 expression was restricted to the distal region
(the MEE side) of the anterior palate (Fig.
4Q). In the mutant, however, Sox9 expression domain was
shifted to the future nasal side (Fig.
4R), which could reflect a positional change of the
Sox9-expressing cells in the palate. Based on this assumption, we
reasoned that a constant cell migration exists in the developing palatal
mesenchyme. Aberrant cell migration could lead to a change in position of the
mesenchymal cells within the Wnt5a-/- palate.
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Ror2 mediates the chemotactic activity of Wnt5a in the developing palate
We tried to determine whether Wnt5a acts as a chemoattractant for
mesenchymal cells of the palate. We coupled DiI-cell labeling/tissue grafting
with bead implantation in explanted palatal shelves. As shown in
Fig. 8, BSA-soaked beads (1
mg/ml) had no influence on migration direction of DiI-labeled cells or grafted
Egfp-expressing cells (26/28). By contrast, DiI-labeled cells/grafted
Egfp-expressing cells exhibited a directional migration towards
Wnt5a-soaked beads (0.5 mg/ml) in 24 hours
(Fig. 8B). This chemotactic
effect of Wnt5a was seen in 69% of cases (25/36). We also found that cells
responded to the chemotactic action of Wnt5a identically, regardless where the
beads were implanted, implying that exogenous Wnt5a has overcome the
endogenous gradient. To further test whether Wnt5a is essential and sufficient
to drive AP cell movement in the palate, we applied Wnt5a beads onto the
anterior side of E12.5 Wnt5a-/- palatal shelves in culture
and monitored the movement of DiI-labeled cells in the posterior palate. A
constant cell migration towards the anterior aspect (exogenous Wnt5a source)
was observed (6/10) (data not shown).
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; Yamamoto et al.,
2007). To determine whether Ror2 also mediates the chemotactic
activity of Wnt5a in the developing palate, we performed DiI labeling and bead
implantation on the explanted palatal shelves from E12.5
Ror2-/- embryos. As expected, Wnt5a beads did not exhibit
a chemotactic effect on the DiI-labeled cells (9/10). Occasionally, very weak
chemotactic response was observed. A few labeled cells could be found around
the Wnt5a bead after an extended culture period
(Fig. 8C).
Several other growth factors are expressed in the anterior palate,
including Bmp4, Fgf10 and Shh (Zhang et
al., 2002; Rice et al.,
2004; Alappat et al.,
2005). We wondered whether any of these factors are also
implicated in regulating cell migration. We found that Shh (0.5 mg/ml) and
Bmp4 (0.4 mg/ml) did not act either as a chemoattractant or a chemorepellent
in the palate (data not shown). By contrast, Fgf10, a known chemoattractant
for several types of cells (Park et al.,
1998; Ceccarelli et al.,
2007; Natanson-Yaron et al.,
2007), exhibited a chemotactic potency for the palatal mesenchymal
cells (Fig. 8D), which was
observed in 63% of total cases (10/16).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). During palate development, palatal shelves undergo rapid
outgrowth and significant changes in palate shape and position. Cell
proliferation and extracellular matrix production and deposition within the
palatal mesenchyme contribute to these processes significantly
(Ferguson, 1988;
Gritli-Linde, 2007). The
morphological movement of the palatal shelf has been considered an active
process of shelf elevation. However, an active directional cell migration by
the mesenchymal cells during palate development has not been reported. In this
paper, we demonstrate the existence of two streams of active directional cell
migration: in the posterior palate, cells migrate towards to anterior aspect,
whereas in the anterior palate, cells move towards the lateral aspect. Though
its biological significance in palate formation is yet to be elucidated, the
directional cell migration should have a crucial role in palate patterning
(shaping) and elevation. The Wnt5a-/- deformed palate
could be partially attributed to the aberrant cell migration. During palate
shelf elevation, different means (rotation versus remodeling) are thought to
be used in different regions of the palate along the AP axis
(Okano et al., 2006). The
distinct directions of cell migration in the anterior and posterior palate may
represent a cellular process underlying these two different elevation
mechanisms. Computer modeling studies will be required to model cell migration
directionality associated with palate elevation.
Wnt5a has been implicated in cell migration in developing vertebrate
embryos and in invasive cancer cells
(Weeraratna et al., 2002;
Jopling and den Hertog, 2005;
Kim et al., 2005;
Witze et al., 2008). Here, we
show a defective cell migration in the Wnt5a-/- palate.
Our results prove that Wnt5a is a potent and specific chemoattractant for the
palatal mesenchymal cells. The gradient of Wnt5a expression along the
AP axis therefore sets a chemotactic base for the posterior-to-anterior
migration of the mesenchymal cells in the palate. Such graded expression was
also observed for Wnt5a in those organs and tissues that undergo outgrowth,
including the limb and tailbud (Yamaguchi
et al., 1999). Consistent with the graded fashion of
Wnt5a expression in the distal limb mesenchyme, active
proximal-distal cell migration has been reported in developing chick limb buds
(Li and Muneoka, 1999).
Although defective cell migration has not yet been reported in the limbs of
mice lacking Wnt5a, a reduced Wnt5a dose indeed impairs cell
migration in fish fins, the homolog of the tetrapod limbs (Sakahuchi et al.,
2006). In addition, impaired cell migration has been reported in the
developing pancreas of Wnt5a-/- embryo
(Kim et al., 2005). In the
developing palate, Ror2 appears to be essential for the chemotactic action of
Wnt5a, as cells lacking Ror2 failed to respond to Wnt5a. It has been
shown that Ror2 is required for Wnt5a-induced migration of cultured cells by
mediating filopodia formation (Nishita et
al., 2006). By contrast, it has recently been shown that Wnt5a can
also act as a chemorepellent for neuronal cells
(Keeble et al., 2006). This
chemorepulsive action of Wnt5a is mediated by Ryk. Wnt5a thus has dual roles
in the regulation of cell migration, depending on the presence of specific
receptor. As Ryk is also expressed in the developing palate in a
punctuate pattern (Halford et al.,
2000), the Ryk-expressing cells could move in a direction
away from the source of Wnt5a. This potential chemorepulsive action of Wnt5a
may account partially for the lateral directionality of mesenchymal cells
migration in the anterior palate, which warrants future investigation.
Many growth factors and receptors are expressed in a specific temporal and
spatial manner in the developing palate
(Gritli-Linde, 2007). Of them,
some are proven chemoattractants. We demonstrate in this study that, besides
Wnt5a, Fgf10 is also a potent chemoattractant for mesenchymal cells of the
palate. Fgf10 is expressed in the lateral region of the anterior
palate at E12.5 and E13.5 (Alappat et al.,
2005), which could at least partially account for the lateral
migration of mesenchymal cells. As the primary Fgf10 receptor Fgfr2b is only
expressed in the palatal epithelium (Rice
et al., 2004), this chemotactic action of Fgf10 is probably
mediated by other Fgf receptor(s) in the mesenchyme. This idea is supported by
the fact that exogenous Fgf10 inhibits cell proliferation in the anterior
palatal mesenchyme (Yu et al.,
2005). As multiple chemoattractants and chemorepellents are likely
to be present in the developing palate, the directionality of cell migration
is probably controlled by a combinatorial effect of multiple factors.
The gradient expression of Wnt5a and its receptors Fz4
and Ror2 correlates with the tissue heterogeneity along the AP axis
of the developing palate. We observed in the Wnt5a-/-
palate altered expression of several genes crucial for palate development.
However, a closer examination of gene expression reveals distinct or even
opposite responses of the anterior and posterior palatal tissues to the
absence of Wnt5a. For example, Shh and Bmp4 were
downregulated in the anterior palate, but were ectopically activated in the
posterior region. In addition, opposite cellular responses (cell
proliferation) were found in the anterior and posterior palatal mesenchyme
lacking Wnt5a, with an increased level of cell proliferation in the
anterior but a decreased level in the posterior palate. Shh has been shown to
positively regulate cell proliferation in the palatal mesenchyme
(Zhang et al., 2002). However,
the altered Shh expression patterns do not correlate with the changes
in cell proliferation rate in the Wnt5a-/- palate. One
would expect a decreased cell proliferation rate in the anterior region of the
Wnt5a-/- palate where Shh is downregulated.
However, a hyperproliferation of mesenchymal cells was instead observed,
suggesting an involvement of multiple signaling pathways in cell proliferation
regulation in the anterior palate where Wnt5a-mediated signaling acts as a
negative regulator. By contrast, the proliferation rate in the posterior
palate of Wnt5a mutant is significantly decreased when compared with
the controls, revealing a positive role for Wnt5a in cell proliferation
regulating in the posterior palate. As the number of migrating mesenchymal
cells in the palate appears small, the altered expression of these crucial
morphogenetic genes and the aberrant cell proliferation appear to have major
impact on cleft palate formation in Wnt5a mutants.
Wnt5a has been previously shown to exert distinct effects on cell
proliferation in different developing organs. Inactivation of Wnt5a
results in reduced proliferation rate of the progenitor cells in the
developing limbs, but leads to an increased level of cell proliferation during
distal lung morphogenesis and mammary tissues
(Yamaguchi et al., 1999;
Li et al., 2002;
Li et al., 2005;
Roarty and Serra, 2007). Wnt5a
has also been shown to inhibit B cell proliferation and functions as a tumor
suppressor in hematopoietic tissue (Liang
et al., 2003). However, in the developing palate, Wnt5a appears to
exert opposite roles in the regulation of gene expression and cell
proliferation in different regions. The differential expression of Wnt5a
receptors (such as Ror2 and Fz4) in the palatal shelf along the AP axis could
account for the distinct functions of Wnt5a. It is possible that other
unidentified Wnt5a receptors are expressed and involved in mediating Wnt5a
signaling in the palate. However, several signaling pathways initiated by
other growth factors, including BMP, FGF and TGFβ, have been proven to
differentially operate and exert their distinct effects on cell proliferation
regulation in different domains of the developing palate along the AP axis. A
combinatorial effect of, and a finely tuned balance between, these signaling
pathways appear essential for the precise control of normal palate growth. The
absence of Wnt5a could disrupt the closely linked signaling network,
leading to dysregulated cell proliferation rate.
Wnt5a has dual roles in signaling: activating either the Wnt canonical or
noncanonical signaling pathways. At least two known Wnt5a receptors, Fz4,
which mediates the activation of the canonical pathway by Wnt5a, and Ror2,
which mediates the noncanonical signaling, are expressed in an overlapping
pattern with Wnt5a in the developing palate. However, activities of the
canonical Wnt signaling were not detected in the developing palate in
wild-type and Wnt5a-/- mice, excluding an involvement of
the canonical Wnt signaling in palate development. However, we cannot rule out
the possibility that the TOPGAL transgenic allele is not sensitive enough to
detect a low signaling level. Wnt5a appears to regulate palatogenesis via a
noncanonical Wnt pathway that does not antagonize the canonical signaling. The
fact that Fz4-null mice do not exhibit a cleft palate defect and that
the inactivation of both Lef1 and Tcf4, the essential
nuclear mediators of the canonical Wnt signaling, does not cause an abnormal
palate formation, further supports this conclusion
(Hsieh et al., 2005;
Brugmann et al., 2007). Several
lines of evidence support the conclusion that Ror2 mediates the function of
Wnt5a in regulating palatogenesis: (1) Wnt5a and Ror2 are
co-expressed in the anterior palatal mesenchyme; (2) Wnt5a and
Ror2 exert an epistatic effect in palate development; (3) the similar
cleft palate phenotype, including elevated cell proliferation rate and altered
expression pattern of downstream genes, are seen in both Wnt5a and
Ror2 mutants; (4) exogenous Wnt5a protein fails to rescue (reduce)
cell proliferation rate in Ror2-/- palatal mesenchyme; (5)
exogenous Wnt5a protein fails to exert chemotactic effect on the palatal cells
lacking Ror2. In fact, Ror2-mediated Wnt5a signaling can active the
Wnt-JNK pathway and/or inhibit Wnt canonical signaling cascade
(Oishi et al., 2003;
Mikels and Nusse, 2006;
Schambony and Wedlich, 2007).
Although the activity of Wnt/β-catenin canonical signaling is not
detected or elevated in Wnt5a-/- palate, it still remains
unknown whether Wnt5a/Ror2 acts through the JNK pathway to regulate
palatogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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