Cleft Palate and Aglossia Result from Perturbations in Wnt and Hedgehog Signaling

Abstract

Objective

The objective of this study was to explore the molecular basis for cleft secondary palate and arrested tongue development caused by the loss of the intraflagellar transport protein, Kif3a.

Design

Kif3a mutant embryos and their littermate controls were analyzed for defects in facial development at multiple stages of embryonic development. Histology was employed to understand the effects of Kif3a deletion on palate and tongue development. Various transgenic reporter strains were used to understand how deletion of Kif3a affected Hedgehog and Wnt signaling. Immunostaining for structural elements of the tongue and for components of the Wnt pathway were performed. BrdU activity analyses were carried out to examine how the loss of Kif3a affected cell proliferation and led to palate and tongue malformations.

Results

Kif3a deletion causes cranial neural crest cells to become unresponsive to Hedgehog signals and hyper-responsive to Wnt signals. This aberrant molecular signaling causes abnormally high cell proliferation, but paradoxically outgrowths of the tongue and the palatal processes are reduced. The basis for this enigmatic effect can be traced back to a disruption in epithelial/mesenchymal signaling that governs facial development.

Conclusion

The primary cilium is a cell surface organelle that integrates Hh and Wnt signaling, and disruptions in the function of the primary cilium cause one of the most common—of the rarest—craniofacial birth defects observed in humans. The shared molecular basis for these dysmorphologies is an abnormally high Wnt signal simultaneous with an abnormally low Hedgehog signal. These pathways are integrated in the primary cilium.

Keywords

craniofacial development embryonic Kif3a morphogenesis primary cilia

Cleft palate is one of the most common birth defects in humans. This congenital deformity is caused when the growth, elevation, and/or fusion of the palatal processes is disrupted. In contrast, incomplete development of the tongue (aglossia) is a very rare birth defect. Underdevelopment of the tongue (hypoglossia) has been reported in association with limb anomalies (Hall, 1971), but in more than 300 years, only 10 cases of isolated complete aglossia have been reported (Salles et al., 2008). We present evidence that development of the palate and the tongue both depend on the Hedgehog and Wnt pathways and that the two pathways are coordinated in a cellular organelle called the primary cilium.

Anatomically, the palate and tongue develop in concert. The secondary palate and the anterior two thirds of the tongue are both derived from the first branchial (pharyngeal) arch. Around embryonic day 9 (e9.0) in mice—and week 4 in humans—the first arch separates into the maxillary and the mandibular prominences, which ultimately give rise to the upper jaw (maxilla) and the lower jaw (mandible). Between e9.0 and e11.5, the small bud-like maxillary and mandibular prominences expand into large primordia, primarily through the proliferation of cranial neural crest cells. At e11.5, the maxillary prominences develop bilateral outgrowths along their medial edges; these outgrowths are the palatal processes. Initially the palatal processes grow toward the midline, but by e13.5 they reorient and grow downward, parallel to the lateral surfaces of the tongue. By el4.5, facilitated by growth of the mandible, the palatal processes elevate above the tongue, make contact, and fuse (for a complete description of palatogenesis, see Bush and Jiang [2012]).

The tongue begins to form at approximately the same time as the palate. At e11.5, the mandibular prominence develops outgrowths along its caudal edge; these outgrowths are the tuberculum impar and the lateral lingual prominences and together these will give rise to the anterior portion the tongue. By e13.5, musculoskeletal development begins in the jaws: Myogenic precursors are established within the tongue primordium (Han et al., 2012) and osteoid tissue forms within the mandibular prominences, thus giving rise to the tongue musculature and the jawbones. Cell proliferation, coupled with the expansion of the bony tissue, results in outgrowth of the lower jaw (Ramaesh and Bard, 2003). It is generally agreed that the growth of the mandible physically displaces the tongue anteriorly, out of the way of the elevating palatal processes. Disruptions in any of these embryonic movements can lead to palatal clefting.

Palatal and tongue development depend on molecular signals that are coordinated by the primary cilium. The primary cilium protrudes from the cell surface into the intercellular space where it has been likened to an antenna, capable of detecting and binding growth factors in the cell's local environment (Singla and Reiter, 2006). Accumulating data indicates that once bound, these microtubule-based organelles transport transcription factors from the tip of the cilium to the nucleus via a bidirectional system called intraflagellar transport (ift).

The essential nature of the primary cilium for human health was first demonstrated in 1976 when disruptions in components of the primary cilium were linked to human disease (Afzelius, 1976). For example, Ift proteins are essential for transport in and out of the primary cilium, and mutations in many of these components lead to cilia defects and human disease (reviewed recently in Yuan and Yang [2015]). Kif3a is an essential component of the Ift system, and embryos carrying null mutations die early during development (Marszalek et al., 1999). Ferrante et al. (2006) and Liu et al. (2014) have demonstrated that primary cilia mutations cause midline facial malformations and cleft palate. In this study, we analyzed how the removal of Kif3a from cells that populate the pharyngeal arches causes a concomitant arrest in both palatal and tongue development. These studies provide new clues into how the growth and differentiation of components of the mammalian face are coordinated through the action of the primary cilium.

Methods and Materials

Collection of Embryos

The Stanford Committee on Animal Research approved all experimental procedures. Transgenic mice were generated by intercrossing (Table 1). In all cases, timed matings were performed, and embryos were collected at indicated time points.

TABLE 1

Genotyping of Mouse Strains^*

Intercrossing	Mutant	Control	Cell Type That Was Labeled by LacZ or GFP Reporter
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl	Wnt1^Cre/+;Kif3a^fl/fl	Wnt1^Cre/-;Kif30^fl/fl
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl;R26R^LacZ/LacZ	Wnt1^Cre/+;Kif3^fl/fl;R26R^LacZ/+	Wnt1^Cre/+;Kif3a^fl/+;R26R^LacZ/+	Neural crest cells and its derivatives
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl;R26R^GFP/GFP	Wnt1^Cre/+;Kif3a^fl/fl;R26R^GFP/+	Wnt1^Cre/+;Kif3a^fl/+;R26R^GFP/+	Neural crest cells and their derivatives
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl;Gli^LacZ/+	Wnt1^Cre/+;Kif3a^fl/fl;Gli1^LacZ/+	Wnt1^Cre/+;Kif3a^fl/+;Gli1^LacZ/+	Hedgehog signaling responsive cells
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl;PTC^LacZ/+	Wnt1^Cre/+;Kif3a^fl/fl;PTC^LacZ/+	Wnt1^Cre/+;Kif3a^fl/+;PTC^LacZ/+	Hedgehog signaling responsive cells
Wnt1^Cre/+;Kif3a^fl/+ X Kif3a^fl/fl;Axin2^LacZ/LacZ	Wnt1^Cre/+;Kif3a^fl/fl;Axin2^LacZ/+	Wnt1^Cre/+;Kif3a^fl/+;Axin2^LacZ/+	Wnt signaling responsive cells
Lef1^+/-; TCF4^+/-X Lef1^+/-; TCF4^+/-	Lef1^+/-;TCF4^+/-	Lef1^-/-;TCF4^-/-

Kif3a = kinesin family member 3A; Lef1 = lymphoid enhancer-binding factor 1; TCF4 = T-cell factor 4; PTC = hedgehog-patched; R26R = Gt(ROSA)26; Cre = Cre recombinase; fl = LoxP flanked (floxed); LacZ = bacterial beta-galactosidase gene; GFP = green fluorescent protein.

Sample Preparation, Tissue Processing, Histology, and Bone and Cartilage Staining

Embryos were collected, fixed in 4% paraformaldehyde at 4°C overnight, dehydrated gradually into ethanol, and embedded in paraffin and cut at a thickness of 8 μm. Histological stains and Alizarin red/Alcian blue bone/cartilage staining were performed as described (Liu et al., 2014).

Xgal Staining

To visualize beta galactosidase activity, paraformaldehyde-fixed embryos were incubated with 30% sucrose overnight at 4°C, embedded in OCT media (TissueTek, Vogel, Germany), placed in dry ice, and cryo-sectioned into 10-μm thick coronal sections. Tissue sections were fixed with 0.2% gluteraldehyde and stained overnight at 37°C with 1 mg/ml Xgal solution.

Immunostaining and BrdU Staining

Lef1 and Nestin immunostaining were performed as described (Liu et al., 2014). Dilutions of Lef1 (Cell Signaling, Danvers, MA) and Nestin (Millipore, Billerica, MA) were both 1:200. BrdU staining was carried out by using a BrdU staining kit (Life Technologies, Carlsbad, CA) following manufacturer's instructions.

Results

The Axin2^LacZ/+ Strain of Wnt Reporter Mice Represents the Status of Endogenous Wnt Signaling in the Facial Prominences

In a previous study, we reported that animals lacking Kif3a in cranial neural crest cells exhibited a severe type of facial dysmorphology that included wide-spaced eyes (hypertelorism) and clefting (Brugmann et al., 2010). We attributed this dysmorphology to a perturbation in Wnt signaling. This conclusion was based on analyses of the Kif3a mutant crossed into the T cell factor (TCF) optimal-promoter beta-galactosi-dase (TOPgal) strain of transgenic Wnt reporter mice. This genetic strategy allowed us to compare domains of Wnt signaling in the faces of control (genetically normal) embryos with Kif3a mutant embryos. Analyses of these embryos demonstrated that there were regions of the developing Kif3a face in which Wnt signaling was elevated but other domains in which Wnt signaling was reduced (Brugmann et al., 2010). This result was confusing; how could the loss of Kif3a cause some cranial neural crest cells to act as if they were experiencing very high levels of Wnt signaling and nearby cranial neural crest cells to act as if they were missing a Wnt signal? We wondered if the Wnt reporter strain we had used was somehow at fault.

The TOPgal reporter strain was generated by random insertion (DasGupta and Fuchs, 1999). As a consequence, this strain shares risks common to most transgenic lines, including inconsistent reporter activity from one generation to the next (Al Alam et al., 2011; Sadelain et al., 2012). Here, we revisited the question of what molecular perturbations cause cleft palate and aglossia in Kif3a mutant embryos. We started by comparing this TOPgal reporter strain with a more reliable Wnt reporter strain, Axin2^LacZ/+, in which the reporter gene is under control of the endogenous Axin2 promoter (Lustig et al., 2002).

In age-matched embryos stained at the same time for Xgal, the Wnt responsive domains were much smaller in TOPgal embryos when compared with Axin2^LacZ/+ embryos (Fig. 1A and 1B). Whole-mount Xgal staining requires that the reagents penetrate the tissues of the embryo, and this can sometimes cause variations in Xgal staining intensity (Al Alam et al., 2011). To ensure that the differences in Xgal staining were not caused by penetration differences, we analyzed representative tissue sections from both strains of reporter mice. Each tissue section was cut to the same thickness and then stained using identical protocols for the same duration. Differences in Xgal staining were still obvious: In TOPgal embryos at e11.5, Xgal-positive cells were restricted to the mesenchyme of the maxillary prominences (Fig. 1C and 1E). At the same embryonic age in Axin2^LacZ/+ embryos, Xgal-positive cells were found throughout the mesenchyme of the maxillary and the frontonasal prominences. In addition, the ectoderm was Xgal positive (Fig. 1D and 1F).

FIGURE 1

Endogenous Wnt signaling is reflected by the pattern of Xgal staining in the facial prominences of Axin2^LacZ/+ mice. (A) Lateral view of e9.5 TOPgal embryo stained with Xgal. (B) Lateral view of e9.5 Axin2^LacZ/+ embryo stained with Xgal. (C) Coronal section of facial prominences stained with Xgal in e11.5 TOPgal embryo. (D) Coronal section of facial prominences stained with Xgal in e11.5 Axin2^LacZ/+ embryo. (E) Coronal section of maxillary prominence stained with Xgal in e11.5 TOPgal embryo. (F) Coronal section of maxillary prominence stained with Xgal in e11.5 Axin2^LacZ/+ embryo. (G) Coronal section of frontonasal prominence stained with Xgal in e11.5 TOPgal embryo. (H) Coronal section of frontonasal prominence stained with Xgal in e11.5 Axin2^LacZ/+ embryo. f, frontonasal; mx, maxillary. Scale bars = 50 μm.

These differences are significant; our previous conclusions regarding the role of Wnt signaling in the Kif3a facial phenotype were based on analyses of TOPgal embryos (e.g., Brugmann et al. [2010] and also see Ocbina et al. [2009]). Therefore, we decided to reevaluate how endogenous Wnt signaling was affected by Kif3a deletion, this time using the Axin2^LacZ/+ reporter strain of mice. To do this we conditionally eliminated Kif3a in the Axin2^LacZ/+ reporter strain, yielding Wnt1^Cre/+;Ki-f3a^fl/fl;Axin2^LacZ/+ offspring (hereafter referred to as Kif3a mutants) and their Wnt1^Cre/-;Kif3a^fl/fl;Axin2^LacZ/+ littermates (hereafter referred to as controls).

Kif3a Mutant Embryos Exhibit Palate and Tongue Defects

Neurocristopathies, including Waardenburg syndrome and Hirshsprung's disease, are a class of dysmorphologies caused by a disruption in the migration of neural crest cells (Bolande, 1997). We assessed Kif3a mutant embryos to determine if they fell within this class of dysmorphologies. By e12.5, cranial neural crest cells complete their migration into the facial prominences, and at this time point we found that both control and Kif3a mutant embryos exhibit relatively normal facial prominences (Fig. 2A and 2B). This result indicated that the Kif3a deletion did not produce a neurocristopathy-like defect.

FIGURE 2

A functional primary cilium is required for palatogenesis and glossogenesis. (A) Maxillary and mandibular prominences in embryonic day (e)12.5 (n = 3) control and (B) age-matched mutants (genotypes as indicated). (C) Maxillary and mandibular prominences in e16.5 (n = 3) control and (D) age-matched mutants. (E) When viewed from the ventral surface, the primary and secondary palates fuse to form the roof of the mouth. (F) In mutants the absence of the palatal processes exposes the nasal antrum; nasal turbinates are visible (asterisks). (G) Coronal tissue sections through the head region of an e18.5 (n = 3) control embryo, showing the basioccipital, the bilateral trigeminal ganglia (V), the soft palate, and the tongue. (H) Age-matched mutants lack midline structures including the basioccipital and the tongue. (I) Pentachrome staining of coronal tissue sections identifies in controls the osteoid matrix of the mandible and chondroid matrix of Meckel's cartilage (mc). (J) In mutants the bone and cartilage form, albeit with an abnormal morphology. bs, basisphenoid; bo, basioccipital; gg, genioglossus; mc, Meckel's cartilage; mn, mandible; mx, maxilla; ppmx, palatal process of the maxilla; t, tongue. Scale bars in A to D, G, H = 100 μm; I, J, K, L = 500 μm.

By e16.5, Kif3a embryos were distinguishable from control embryos in that growth of the mutant lower jaw was stunted (cf. Fig. 2C and 2D). The upper jaw was also affected; in control embryos, the primary and secondary palates fused to create the roof of the mouth (Fig. 2E), whereas 33% of mutant embryos lacked both primary and secondary palates, which left the nasal turbinates exposed (Fig. 2F).

Coronal tissue sections helped clarify the extent of the palatal defect and also revealed that 100% of the mutant embryos lacked a tongue. Tissue sections from control embryos showed the secondary palate flanked by the trigeminal ganglia, the basioccipital bone, and the tongue (Fig. 2G). In a subset of mutant littermates, the maxillary prominences failed to develop a palatal shelf, and only a thin fibrous band of tissue connected the left and right maxillary prominences (Fig. 2H). Despite these extreme dysmorphologies in the Kif3a mutants, neither osteogenic differentiation in the mandible nor chondrogenic differentiation in Meckel's cartilage were impeded (Fig. 2I and 2J).

Neural Crest Proliferation Requires a Functional Primary Cilium

Truncated upper and lower jaws strongly suggested that Kif3a mutants had a defect in cell proliferation. The facial prominences were examined at e12.5, with a focus on those regions of the developing face from which the palate and tongue arise (green and red boxes, Fig. 3). Using BrdU staining, mutant embryos exhibited a generalized increase in immunopositive cells in the palate when compared with controls (Fig. 3A through 3D). The same relationship held true in the tongue primordium, where the mutant embryos showed more BrdU-immunopositive cells (Fig. 3E and 3F). Quantification of the BrdU signal (Fig. 3G and 3H) clearly demonstrated that Kif3a deletion caused cranial neural crest cells to increase their rate of proliferation. How could an increase in proliferation result in a truncation in growth? Examination of control and mutant heads revealed a lateral expansion of the Kif3a mutants relative to their littermate controls (Fig. 3I and 3J). Therefore, the increased cell proliferation observed in the Kif3a mutants produced a much wider, but dramatically shorter, upper and lower jaw.

FIGURE 3

Primary cilia control neural crest proliferation. (A) Coronal tissue sections through the head region stained to detect BrdU (n = 3) of e12.5 control embryo and (B) mutant embryos (genotypes as indicated). (C) Sections of palatal process of e12.5 control (n = 3) and (D) mutant embryos (n = 3), stained to detect BrdU. (E) Sections of tongue of e12.5 control (n = 3) and (F) similar region of mutant embryos (n = 3), stained for BrdU. Quantification of BrdU^+ve cells in the (G) palatal process and (H) tongue. (I) Bone and cartilage staining of control and (J) Kif3a mutant embryos at e18.5. nc, nasal capsule; mn, mandible; pp, palatal process; t, tongue. Scale bars in A to D = 50 μm; I to J = 1 mm.

Hedgehog Responsiveness Is Obliterated by Kif3a Deletion

The complete absence of the palatal processes and the tongue gave us the opportunity to study the events that initiate the outgrowth of these structures from the maxillary and mandibular prominences. Other groups have eliminated essential components of the primary cilia using the same Wnt1^Cre deleter strain of mice, and alternatively attributed the resulting tooth defects to either ectopic (Ohazama et al., 2009) or reduced (Nakatomi et al., 2013) Hedgehog (Hh) signaling. To gain some clarity on how Kif3a deletion affected Hh signaling, we crossed the mutant strain of mice with a strain of Hh reporter mice (Goodrich et al., 1996). This genetic strategy allows for a comparison of Hh signaling in the developing faces of control and mutant embryos using Xgal staining.

When compared with control embryos, a generalized loss in Xgal staining was observed in the palatal and mandibular mesenchyme of mutant embryos (Fig. 4A and 4B). In control embryos, Xgal staining was obvious in the palatal mesenchyme, whereas in mutants, Xgal staining was reduced (Fig. 4C and 4D). In the control tongue, Xgal staining was obvious in both the mesenchyme and ectoderm, whereas in the mutant tongue Xgal staining was absent (Fig. 4E and 4F). These data clearly showed that the deletion of Kif3a rendered tissues unable to respond to a Hh signal.

FIGURE 4

Kif3a deletion renders neural crest mesenchyme incapable of responding to Hh signals. (A) Coronal tissue sections stained with Xgal from e12.5 (n = 3) control and (B) mutant embryos (n = 3); genotypes as indicated. (C) Xgal stained palatal tissues from control and (D) mutant embryos. (E) Xgal stained tongue tissue from a control embryo; (F) Xgal stained midline tissue from a mutant embryo. (G) Coronal tissue sections stained for Xgal from control and (H) mutant embryos; genotypes as indicated. (I) Xgal stained palatal tissues from control and (J) mutant embryos. (K) Xgal stained tongue tissue from a control embryo; (L) Xgal stained midline tissue from a mutant embryo. Abbreviations as in previous figures. Scale bars in A, B, I to J = 50 μm; C to H = 100 μm.

We verified these results using a second Hh reporter strain of mice (Bai et al., 2002) and as predicted by the results noted previously, the deletion of Kif3a rendered neural crest cells incapable of responding to a Hh signal (Fig. 4G and 4H). This was obvious, for example, in the palatal processes: Normally the palatal mesenchyme is sensitive to a Hh signal (Fig. 4I), but in mutants the palatal processes were devoid of Hh responsiveness (Fig. 4J). In the developing tongue, control embryos showed Hh responsiveness in the mesenchyme and ectoderm (Fig. 4K); in mutants, Hh responsiveness was lost in the mesenchyme (Fig. 4L). We noted one other interesting feature: In the region where the tongue should have formed, a few isolated Hh responsive cells were observed within the deciliated mesenchyme (Fig. 4L).

Deciliated Cells Show Potentiated Wnt Responsiveness

At this point, our data demonstrated that a Hh signal from the endoderm acts on mesenchymal cells to initiate growth of the tongue primordium, and in Kif3a mutants, although the Hh initiating signal in the endoderm remains intact, the mesenchyme is unresponsive to the Hh signal. Consequently, cell proliferation and outgrowth of the tongue primordium is blocked.

Primary cilia have also been implicated in the modulation of Wnt signaling (Corbit et al., 2008; Lancaster et al., 2011; Liu et al., 2014). Lancaster and colleagues (2011) suggested that cells lacking primary cilia, such as in the Kif3a mutant, have potentiated Wnt responsiveness, whereas cells with primary cilia have inhibited Wnt responses. If these in vitro data were correct, they would predict that Wnt signaling would be elevated in the Kif3a mutant mesenchyme and reduced in the surrounding ectoderm.

We examined e12.5 control and mutant embryos and initially found no obvious difference in the level or pattern of Wnt responsiveness for each condition (Fig. 5A through 5D), but when we examined older (e16.5) embryos we found increased Wnt signaling in both the ectoderm and mesenchyme of the mutants (n = 3 for each condition; Fig. 5E and 5F; see also Supplemental Fig. 1). Thus, these in vivo data were in agreement with the predictions of Lancaster and colleagues (2011): In Kif3a mutant cells that had lost their primary cilia, Wnt responsiveness was significantly elevated.

FIGURE 5

Deciliated neural crest mesenchyme becomes hyper-responsive to Wnt signals. (A) Whole-mount Xgal staining of e12.5 (n = 3) control and (B) mutant embryos; genotypes as indicated. (C) Coronal tissue sections stained with Xgal from e12.5 (n = 3) control and (D) mutant embryos; genotypes as indicated. (E) Tissue sections stained with Xgal from e16.5 (n = 3) control tongue and (D) mutant midline tissues. E16.5 (n = 3) tissue sections immunostained with Lef1 from control (G) and (H) mutant embryos. E16.5 (n = 3) tissue sections immunostained with Nestin from control (I, K) and (J, L) mutant embryos. Whole mount images of (M) e16.0 (n = 3) control and (N) mutant embryos; genotypes as indicated. Pentachrome-stained tissue sections from (Q) control and mutant (P) embryos. (S) Schematic illustrating step 1, where Hh (purple stars) and Wnt (blue stars) signals from the ectoderm and act on the underlying mesenchyme (arrows). This step is equivalent in control and Kif3a conditional mutants. Step 2: in response to the Hh signal, mesenchymal cells express Gli1 and Ptc (green octagons); in response to the Wnt signal, mesenchymal cells express Axin2 (yellow octagons). Mutant embryos fail to elaborate a Hh response in the form of Gli1 or Ptc expression (red X); in response to the Wnt signal, mutants exhibit a hyper-response in the form of aberrantly up regulated Axin2. Step 3: mesenchymal cells signal back to the ectoderm, inducing epithelial specializations, such as taste buds, to form. In mutants, the lack of a mesenchymal Hh response results in aberrantly elevated Hh signaling within the ectoderm (purple stars); the hyper-response to Wnt causes the mutant ectoderm to repress further Wnt signals. Abbreviations as in previous figures. ep, epithelium; pc, pharyngeal cleft. Scale bars in A to D, I, J, O, P = 500 μm; E to H, K, L = 100 μm; M, N, Q, R = 50 μm.

Integrated Wnt Signaling Is Required for Taste Bud Development

Around e16.5, the tongue epidermis begins to develop taste buds, and these taste buds are indicated by the expression of Sonic hedgehog (Shh; reviewed in Kapsimali and Barlow [2013]). Shh expression in the tongue epithelia is regulated by Wnt signaling (Iwatsuki et al., 2007). Because both Hh and Wnt signaling were disrupted in other oral tissues, we closely examined the mutant oral epithelia for clues as to how these pathways were disrupted during taste bud development.

In controls, Hh signaling was strongly expressed in tongue epithelia; in mutants, Hh signaling was also strongly expressed in the epithelia (Fig. 4). This was expected because the initiating Hh signal originates from the ectoderm, where primary cilia are intact. Wnt signaling, on the other hand, was lost in the mutant epithelia. When compared with its periodic expression in control tongue epidermis, mutant epidermis showed only one or two sites of Wnt responsiveness (Fig. 5E and 5F). The Wnt responsive status of the taste buds was confirmed by columns of Lef1 expressing cells in the epithelia of control embryos (Fig. 5G); mutant epithelia showed only a few clusters of Lef expressing cells, predominantly in the midline (Fig. 5H). Thus, Wnt signaling is elevated in deciliated mesenchyme and depressed in the cilia-containing epithelium, which collectively prevents tongue and taste bud development.

The body of the tongue is composed of cranial neural crest cells and mesodermal cells, the latter of which give rise to the Nestin positive musculature of the tongue (Fig. 5I and 5K). Despite the absence of a tongue proper, mesodermal differentiation into the genioglos-sus muscle proceeded without disruption in mutant embryos (Fig. 5J and 5L). Thus, neither Hh nor Wnt signaling from cranial neural crest cells is required for tongue muscle differentiation.

We previously reported that reduced Wnt signaling caused by loss of the transcriptional targets Lef1 and Tcf4 results in cleft palate and maxillary hypoplasia (Brugmann et al., 2007). Predictions from our current study suggest that Lef1^-/-;Tcf4^-/- mutants should also exhibit tongue anomalies. Indeed, we found that they exhibited underdeveloped maxillary prominences and a notably foreshortened mandible that, relative to controls, caused the tongue to protrude (Fig. 5M and 5N). Histologic sections confirmed hypoglossia in the mutants (Fig. 5O and 5P), and gross examination of the tongue revealed a lack of taste buds (Fig. 5Q and 5R). Collectively, these analyses demonstrate that Wnt signaling is essential for not only the development of the tongue but also the epithelial specializations that provide this structure its unique chemosensory function.

Discussion

The development of the tongue and palate are coordinated during embryogenesis, and disruptions in the growth and/or differentiation of one structure can directly impact the growth and differentiation of the other. For example, abnormal adhesions between the palate and tongue epithelia because of mutations in the interferon regulatory factor 6 gene prevent palatal shelfelevation, and the result is cleft palate in both humans and mice (Casey et al., 2006; Ingraham et al., 2006; Fakhouri et al., 2014).

An abnormally shaped tongue can also lead to cleft palate. For example, genetic inactivation of the Tak1 gene in neural crest cells results in the formation of an abnormally tall tongue that physically hinders palatal shelf elevation (Song et al., 2013). An underdeveloped mandible can contribute to palatal clefting: Clinicians have long recognized that patients with very small mandibles (micrognathia/retrognathia) frequently have tethered tongues (ankyloglossia) and cleft palate. Examples of this combination of facial features can be seen in patients with Pierre Robin (Benko et al., 2009), Stickler (Snead and Yates, 1999), Velocardiofacial syndrome (Goldberg et al., 1993), Nager syndrome (Lansinger and Rayan, 2015), and Treacher Collins syndrome (The Treacher Collins Syndrome Collaborative Group, 1996).

Here we provide evidence that the palate and the tongue depend on molecular signals that are integrated in the primary cilium. By deleting a component of the primary cilium encoded by the intraflagellar transport protein Kif3a (Lin et al., 2003), both Hh and Wnt signaling are disrupted. Other investigators have also reported that disrupted Hh signaling is associated with tongue anomalies including aglossia (Jeong et al., 2004). In these experiments, muscle progenitor cells were lost, but in the Kif3a mutants shown here, Nestin expressing myoblasts still form (Fig. 5). Clearly, there are multiple means by which tongue development can be perturbed.

Some—but not all—ciliopathies have palatal clefting and tongue defects as a hallmark. For example, patients with well-known ciliopathies including oro-facial-digital (Toriello and Franco, 1993) and Meckel-Gruber (Barisic et al., 2015) syndromes exhibit palatal clefting. In addition, patients with lesser-known Kif7-related ciliopathies including hydrolethalus and acrocallosal syndromes also exhibit palatal clefting (Putoux et al., 2011; Walsh et al., 2013). It should be emphasized, however, that not all Joubert-related disorders are associated with palatal clefting (see Parisi and Glass [1993] for an updated Internet review). The phenotypic spectrum represented by the ciliopathies attests to both the importance and incomplete knowledge we have about the roles of the primary cilium in integrating Hh and Wnt signals during development.

We gained mechanistic insights into the basis for the combination of aglossia and cleft palate. Normally, Hh and Wnt signals originate from epithelia (ectoderm or endoderm) and signal to adjacent mesenchyme (Fig. 5S, step 1). In response to the Hh signal, mesenchymal cells increase the expression of Hh target genes including Patched and Gli1 (Fig. 5S, step 2, green), and in response to the Wnt signal, they increase the expression of Wnt target genes, including Axin2 (Fig. 5X, step 2, yellow). Mesenchymal cells respond to producing other signals (Cobourne et al., 2004; Rice et al., 2006) that travel back to the ectoderm/endoderm (Fig. 5S, step 3, green arrow) and ultimately elicit the proliferation and invagination of the epithelial tissue to form epithelial specializations such as taste buds (Liu et al., 2007; Castillo et al., 2014).

In Kif3a mutant embryos, step 1 proceeds normally; both Hh and Wnt signals in the epithelia are intact (Figs. 4 and 5). The mesenchymal response to these ectodermal signals, however, is disrupted (step 2): Deciliated mesenchymal cells no longer respond to a Hh signal, as shown by the loss of both Ptc and Gli1 expression (Fig. 4B and 4H). Deciliated mesenchymal cells also express Axin2 at aberrantly high levels (Fig. 5B, 5D, and 5F).

The lack of a Hh-dependent response from the mesenchyme leads to elevated Hh signaling in the ciliated ectoderm (Fig. 5S, step 3, purple); simultaneously, the Wnt-dependent hyper-response of the mesenchyme causes a downregulation in Wnt signaling in the ciliated ectoderm (Fig. 5F and 5H; and see S, step 3, blue). Consequently, outgrowths such as the palate and the tongue as well as epithelial specializations that depend on a balance between Hh and Wnt no longer form. Analyses of other cleft palate phenotypes, such as Lef1^-/-;Tcf4^-/- embryos shown here (Fig. 5) and those from other groups (Iwatsuki et al., 2007; Kurosaka et al., 2014), support this interpretation.

In previous work (Liu et al., 2014), we evaluated how Kif3a deletion affected tooth development and found that the same two pathways, mediated by Hh and Wnt ligands, were responsible for the arrest in the cap stage of odontogenic differentiation. Similar to the data shown here (Figs. 4 and 5), Hh signaling was lost while Wnt signaling was increased in dental mesenchyme (Liu et al., 2014), which resulted in hyper-proliferation in the odontogenic ectoderm. These data underscore the critical roles played by Hh and Wnt signaling in mediating epithelial-mesenchymal interactions in craniofacial development (reviewed in Santosh and Jones [2014]).

We compared the phenotypes exhibited here with phenotypes resulting from the deletion of Gpr177 (also known as Wntless) in cranial neural crest cells (Liu et al., 2015). Based on the function of Gpr177 as an essential chaperone protein that escorts the lipidated Wnt ligand from the Golgi to the cell surface (Zhong et al., 2012), we found the comparison between these mutants and the Kif3a mutants to be especially informative. The same Wnt1Cre deleter line was employed for both studies, therefore differences in the phenotypes could be attributed to the molecular pathways that were disrupted by the gene mutations.

Gpr177 mutants exhibited secondary cleft palate but no tongue defects (Liu et al., 2015) caused by a loss of Wnt signaling that subsequently led to a pathological increase in Shh expression. Although the same molecular changes were observed in our study (i.e., an increase in Wnt signaling and a reduction in Hh signaling), the effect on cell proliferation was completely different: Liu and colleagues report a reduction in cell proliferation, whereas we show that simultaneous gain-in-Wnt (Fig. 5) and loss-in-Hh (Fig. 4) signaling causes cell hyperproliferation (Fig. 3). Although the two mutant strains share one common feature, cleft palate, the other manifestations of gene disruption were quite distinct. We attribute these differences to two important features: first, timing, and second, specificity. With regard to timing, the Gpr177 mutant clearly exhibits Wnt pathway inhibition first; the loss of Hh signaling occurs subsequently. In our Kif3 mutant, both Wnt and Hh pathways were simultaneously disrupted. Whether that difference in timing is sufficient to explain the differences in cell proliferation and the lack of a tongue phenotype in the Gpr177 mutant is not clear.

The second difference is specificity: Mutants in Gpr177 adversely affect both cells that are the targets of the Wnt signal as well as the cells producing the Wnt ligand (Zhong et al., 2012). Therefore, both the cranial neural crest and the ectoderm would be affected in the Gpr177 mutant. In our study, deletion of Kif3a only affected cranial neural crest cells; the primary cilium remained intact in the adjacent ectoderm (Liu et al., 2014). Again, it remains to be determined whether this difference in specificity is sufficient to explain phenotypic differences between the Gpr177 and Kif3a mutants.

Collectively, data shown here pinpoint the molecular basis for an arrest during tongue and palate development and lend further support for the primary cilia as an integrating center for Hh and Wnt signaling in the facial prominences. The importance of these two pathways for proper craniofacial development cannot be overemphasized. For example, loss-of-Hh function causes holoprosencephaly in humans (Solomon et al., 1993) and palatal clefting is a common feature in milder forms of holoprosencephaly (Heyne et al., 2015). Gain-in-Hh mutations cause Greig cephalopolysyndactyly (Ondrey et al., 2000), and at least in animal models of the human disease, cleft palate is a common feature (Huang et al., 2008). Wnt signals regulate multiple aspects of craniofacial development (e.g., Brugmann et al., 2006; Liu et al., 2010; Yin et al., 2015), and mutations in Wnt pathway components are also implicated in the etiology of human facial clefting (Menezes et al., 2010).

Supplemental Materials

SUPPLEMENTAL Figure 1 Deciliated mesenchyme is hyper-responsive to Wnt signals. (A) Coronal tissue sections stained with Xgal from e18.5 (n = 6) control and (B) mutant embryos; genotypes as indicated. Boxed areas of the palatal region are shown in (C) at higher magnification. (D) Mutant midline tissues. oc, oral cavity, mc, Meckel's cartilage; mes, mesenchyme; gg, genioglossus. Scale bars = 100 μm.

Footnotes

Acknowledgments

We thank Jingtao Li tor helpful comments on the manuscript.

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Cleft Palate and Aglossia Result from Perturbations in Wnt and Hedgehog Signaling

Abstract

Objective

Design

Results

Conclusion

Keywords

Methods and Materials

Collection of Embryos

Sample Preparation, Tissue Processing, Histology, and Bone and Cartilage Staining

Xgal Staining

Immunostaining and BrdU Staining

Results

The Axin2LacZ/+ Strain of Wnt Reporter Mice Represents the Status of Endogenous Wnt Signaling in the Facial Prominences

Kif3a Mutant Embryos Exhibit Palate and Tongue Defects

Neural Crest Proliferation Requires a Functional Primary Cilium

Hedgehog Responsiveness Is Obliterated by Kif3a Deletion

Deciliated Cells Show Potentiated Wnt Responsiveness

Integrated Wnt Signaling Is Required for Taste Bud Development

Discussion

Supplemental Materials

Footnotes

Acknowledgments

References

The Axin2^LacZ/+ Strain of Wnt Reporter Mice Represents the Status of Endogenous Wnt Signaling in the Facial Prominences