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Keywords:

  • Frizzled receptors;
  • DKK1;
  • LEF1;
  • CTNNB1;
  • FRZB1;
  • craniofacial;
  • chicken embryo;
  • in situ hybridization.

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

A comprehensive expression analysis of WNT signalling pathway genes during several stages of chicken facial development was performed. Thirty genes were surveyed including: WNT1, 2B, 3A, 4, 5A, 5B, 6, 7A, 7B, 8B, 8C, 9A, 9B, 11, 11B, 16, CTNNB1, LEF1, FRZB1, DKK1, DKK2, FZD1-8, FZD10. The strictly canonical WNTs (2B, 7A, 9B, and 16) in addition to WNT4 WNT6 (both canonical and non-canonical) are epithelially expressed, whereas WNT5A, 5B, 11 are limited to the mesenchyme. WNT16 is limited to the invaginating nasal pit, respiratory epithelium, and lip fusion zone. Antagonists DKK1 and FRZB1 are expressed in the fusing primary palate but then are decreased at stage 28 when fusion is beginning. This suggests that canonical WNT signalling may be active during lip fusion. Mediators of canonical signalling, CTNNB1, LEF1, and the majority of the FZD genes are expressed ubiquitously. These data show that activation of the canonical WNT pathway is feasible in all regions of the face; however, the localization of ligands and antagonists confers specificity. Developmental Dynamics 238:1150–1165, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The vertebrate face develops from the coordinated growth of the facial prominences, which initially consist of undifferentiated neural crest–derived mesenchyme covered by an epithelial layer. At first, the facial prominences (the frontonasal mass, maxillary, lateral nasal, and mandibular) are buds that surround the primitive oral cavity. In the avian embryo between stage 20 and 28 (Hamburger Hamilton,1951), there is considerable proliferation of the mesenchyme driving the outgrowth of the facial prominences (McGonnell et al.,1998; Wu et al.,2004,2006; Szabo-Rogers et al.,2008). This outgrowth culminates with contact of the frontonasal mass and maxillary prominence and fusion of the primary palate. If fusion is disrupted, cleft lip will ensue. During this period, the development of species-specific form also occurs. The formation of wide versus narrow beaks is dependent on the neural crest–derived mesenchyme (Schneider and Helms,2003). The spatial arrangement of proliferating mesenchymal cells and BMP signalling in the frontonasal mass (Abzhanov et al.,2004; Wu et al.,2004,2006) fine tune the morphogenesis of the upper beak. BMPs are also required in lip fusion (Ashique et al.,2002a,b). FGFs promote proliferation and outgrowth of facial mesenchyme (Richman et al.,1997; Mina et al.,2002; Szabo-Rogers et al.,2008) and Sonic Hedgehog is sufficient to induce secondary outgrowth axes in the frontonasal mass (Hu and Helms,1999) and mandibular prominences (Brito et al.,2008). When compared to other signalling pathways, much less is known about the role of WNTs during facial development.

The Wnt family (wingless-type MMTV integration site) of secreted glycosylated factors consists of 19 members in mouse. However, in chicken there are only 18 confirmed WNT ligands. The chicken genome does not appear to have WNT2, 4B, 7C, or 10B at the time of writing. The mouse genome also lacks Wnt4b and 7c. WNT proteins have a range of functions during various developmental processes, such as proliferation, asymmetric division, patterning, and cell fate determination (Veeman et al.,2003; Logan and Nusse,2004; Gordon and Nusse,2006; Karner et al.,2006a; Geetha-Loganathan et al.,2008a,b). WNTs signal through the canonical, β-catenin dependent pathway (Logan and Nusse,2004), or at least two different noncanonical pathways (Veeman et al.,2003; Karner et al.,2006a). Central to the canonical pathway is the regulation of β-catenin. Upon binding to frizzled (Fzd) receptors, Wnt ligands trigger an accumulation of β-catenin in the cytoplasm that is then translocated to the nucleus. There β-catenin binds to transcription factors of the LEF/TCF family, activating downstream gene transcription. One of the “non-canonical” pathways, the Wnt/Ca2+ pathway, leads to release of intracellular calcium, possibly via G-proteins (Sheldahl et al.,1999; Kuhl et al.,2000). This pathway also involves activation of phospholipase C and protein kinase C (PKC). The second “non-canonical” pathway is called the planar cell polarity pathway. Fzd activates JNK and directs asymmetric cytoskeletal organization and coordinated polarization of cells within the plane of epithelial sheets (Karner et al.,2006a,b). This pathway also controls convergent extension movements during gastrulation and has recently also been implicated in the directional migration of cells within the developing palatal shelves (He et al.,2008). Complex crosstalk between these “canonical” and “non-canonical” pathways may regulate the cellular readout of Wnt signalling (Weidinger and Moon,2003).

Mouse models in which Wnt pathway genes were targeted demonstrate the need for canonical and non-canonical WNT signalling in facial morphogenesis. Knockouts for Wnt1, 3a, 4, 5a, 7a, 7b, 9a, 9b, and 11 have been reported. Of these, only three have been shown to have roles in facial development. Targeted deletion of Wnt3a caused death upon birth due to mandibular defects. However, the cause of failure to feed (presumably a cleft palate) was not characterized (Yoshida et al.,2006). Targeted deletion of Wnt9b (Carroll et al.,2005) causes cleft lip in some of the embryos (Juriloff et al.,2006). Full deletion of Wnt5a in mice causes a striking truncation of the upper and lower jaws. Other molecules in the Wnt pathway that have resulted in craniofacial phenotypes when deleted are Ctnnb1 (β-catenin), Dkk1 (Dikkopf), Tcf4/Lef1 (T-cell factor/lymphoid enhancer factor) compound mutants, and Sfrp1/Sfrp2 (Secreted frizzled-related protein) compound mutants. Conditional deletion of Ctnnb1 in neural crest cells eliminates all intramembranous bones in the skull (Brault et al.,2001). The compound null Tcf4−/−/Lef1−/− mice, which have very little response to canonical Wnts, also have a facial phenotype consisting of poorly developed maxillas and shorter nasal septums (Brugmann et al.,2007). The deletion of Dkk1, which is predicted to cause an increase in canonical Wnt signaling, causes a dramatic loss of the entire facial complex due to a very early role in head induction (Mukhopadhyay et al.,2001). Sfrp1 and 2 compound mutants, which should lead to increased canonical and non-canonical signalling, also have defects in the face (Satoh et al.,2006) but far less severe than the Dkk1 knockouts. Thus, regulating the level of WNT signalling is important for several aspects of facial morphogenesis.

Surprisingly, descriptions of WNTs, FZD receptors, or signalling mediators in the developing face are difficult to find in the literature. Furthermore, only partial data on the avian face is available from a handful of studies including expression of SFRP2 and FRZB1 (Ladher et al.,2000), WNT7A and WNT5A (Dealy et al.,1993). During in situ analyses, the head is often presented in sagittal views, if at all. Even the recent study using optical projection tomography on E11.5 mouse embryos is not at high enough resolution to determine which genes are expressed in the different facial prominences and whether expression is epithelial or mesenchymal or both (Summerhurst et al.,2008). Furthermore, expression is very dynamic during facial morphogenesis so several consecutive stages need to be studied. We chose to study the expression of the WNT pathway genes in avian embryos because ultimately it will be convenient to manipulate WNT signalling directly in the developing face. Expression was mapped in stage-15 to -28 embryos thereby covering the critical stages of facial development. These stages include: establishment of jaw identity (Lee et al.,2001), growth of facial prominences (Richman and Tickle,1989), and induction of intramembranous bone (Tyler and Hall,1977; Hall,1978,1980; Tyler,1978) and fusion of the facial prominences (Ashique et al.,2002a; Szabo-Rogers et al.,2008).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

To determine potential roles of Wnt signalling during facial development, we have analyzed the expression patterns of WNTs- 1, 2B, 3A, 4, 5A, 5B, 6, 7A, 7B, 8B, 8C, 9A, 9B, 11, 11B, and 16; transducers of WNT signalling pathway CTNNB1 and LEF1; WNT antagonists FRZB1, DKK1, DKK2; and WNT receptors FZD1-8, FZD10 by in situ hybridization and compared their expression patterns (Tables 1, 2). Radioactive in situ hybridization on sections was also performed for some genes to clarify the expression patterns. The genes studied with both methods include WNT3A, 4, 5A, 6, 7A, 11, 16, DKK1, CTNNB1, LEF1.

Table 1. Summary of Expression Pattern of WNT Genes, Antagonists, and Intracellular Mediators of Canonical Signaling in the Facial Prominences
 Gene nameEpi and/or MesFrontonasal mass (fnm)Maxillary prominenceLateral nasal prominenceMandibular prominence
  • a

    These Wnts have never been shown to act via non-canonical pathways.

  • b

    These Wnts can act in the canonical or non-canonical pathway.

  • c

    There is also expression in the mandibular and maxillary branches of the trigeminal nerve.

  • d

    There is also expression in the trigeminal ganglion.

Canonical WNTsaWNT2BENo expression.Lateral epithelium starting at stage 17 expanding medially, decreased at stage 2B.No expression.Lateral edge starting at stage 15.
 WNT9BEContinuous across fnm stage 24–28.Lateral epithelium starting at stage 17, decreased at stage 2B.Present from stage 18–28.Present from stage 15–28.
 WNT16EcLateral edges stage 20–28.Cranial edge nearest to fusion zone beginning at stage 20–28.Medial edge lining nasal slit from stage 21–28.Mandibular nerve only, absent from epithelium and mesenchyme.
 WNT7AENo expression.No expression.No expression.Present in the first and second pharyngeal arch clefts.
Non-canonical WNTsWNT4bEGeneral expression continuous across fnm and in stomodeal epithelium stage 20–25.Both extraoral and intraoral epithelium, stage 20–25.Expression in surface ectoderm but not in nasal slit stage 20–25.Expression in oral and aboral ectoderm stage 20–25.
 WNT6bEVery low signal at stage 20. At stage 24 in the caudal and lateral edge epithelium.Very low signal at stage 20. Extraoral epithelium only, not intraoral stage 24.Very low signal at stage 20. Expression in surface ectoderm but not in nasal slit stage 24.Very low signal at stage 20. Expression in oral and aboral ectoderm stage 24.
 WNT5AbMStarting stage 20 at lateral edges, then spreading across.Lateral extra/oral starting at stage 15, then caudal third at stage 21–28.Low expression from stage 21.Entire first arch at stage 15, then midline stage 20–28.
 WNT5BMNo expression until stage 24, then in lateral edges.Caudal two-thirds at stage 24–28.Low expression.Present at stage 15–18. No expression in older stages.
 WNT11bMNo expression.Cranial third at stage 20–24, shifting laterally close to the eye at stage 28.No expression.Craniolateral edge.
WNT antagonistsDKK1Ed + MEpithelium only. Increased in nasal pit at stage 20–21. Present in caudal edge of the fnm stage 24 and 28.Epithelium and mesenchyme. Present from stage 15, resolves into two discrete medial domains at stage 24. Decreases at stage 28 but is still high in areas of fusion.Epithelium and mesenchyme. Along lateral edge of nasal slit stage 20–24, decreased at stage 28.Epithelium and mesenchyme. Strong at stage 15–21, then decreased.
 FRZB1MStage 18–23 high, then decreased.Diffuse expression stage 15–23, then decreased.Present at stage 18–24.Strongest until stage 24 near midline, decreased at stage 28.
Canonical WNT signal transductionCTNNB1E+MStage 15–28, higher in the nasal pit epithelium.Ubiquitous and high expression in epithelium and mesenchyme stage 15–28.Ubiquitous in mesenchyme, higher in nasal slit epithelium.Ubiquitous and high expression in epithelium and mesenchyme.
 LEF1Ed + MStage 24–28 across fnm.Stage 15–28, higher in medial mesenchyme.Ubiquitous, present stage 24–28.Slightly higher in midline than other regions between stages 24–28.
Table 2. Summary of Expression Pattern of Frizzled Receptor Expression in the Stage-24 Facial Prominences
Gene nameEpithelial/mesenchymalFrontonasal massMaxillary prominenceLateral nasal prominenceMandibular prominence
Frizzled receptors     
 FZD1MDeeper, ventral to telencephalon and in periocular mesenchyme.Proximal, cranial.Ubiquitous, lowUbiquitous, low.
 FZD2MContinuous across fnm.Ubiquitous, high.Ubiquitous, high.Ubiquitous, high.
 FZD3E,MHigher in globular process.Cranial edge nearest to fusion zone. Proximally expression is ubiquitous.Low expression in mesenchyme higher in nasal slit epithelium.Ubiquitous, low.
 FZD4E,MHigher in the caudal edge mesenchyme.Medial expression.Low expression in mesenchyme higher in nasal slit epithelium.Ubiquitous.
 FZD5E,MSlightly higher in globular processes, and in stomodeal epithelium.Ubiquitous, low.Ubiquitous, low.Ubiquitous, low.
 FZD6E,MMesenchyme has low expression, stomodeal epithelium has light signal.Ubiquitous, low in mesenchyme epithelium has light signal.No data due to deep sections.Ubiquitous, low.
 FZD7MCaudal edge has higher expression.Caudal medial edge distally and throughout mesenchyme proximally.Ubiquitous, high.Ubiquitous, high, except in mesodermal core.
 FZD8E,MSignal in stomodeal epithelium.Ubiquitous, lowNo data due to deep sections.Ubiquitous, low.
 FZD10E,MOnly deep sections through telencephalon studied, no expression.Lateral epithelium and mesenchyme proximally.No data due to deep sections.Medial, oral edge in epithelium and mesenchyme, also in dorsal tongue.

Of the 30 genes surveyed, there were 8 genes with no detectable expression in the face as determined with whole-mount in situ hybridization (Supp. Fig. S1A–J, which is available online). These negative genes included: WNT1, 3A, 7B, 8B, 8C, 9A, 11B, DKK2. The apparent lack of expression could be due to levels of transcript being below the level for detection for at least some of the genes. WNT3A was further examined in detail in radioactive in situ. However, no expression in the face was observed (Supp. Fig. S1B, B'). WNT7A was also studied carefully in radioactive in situ since a previous study showed expression in the pharyngeal arches (Dealy et al.,1993). We also found radioactive probes bound to the first and second pharyngeal arch clefts but not to the facial prominence epithelium (data not shown but see Supp. Fig. 2SD). Further radioactive in situ studies, and RT-PCR analyses, would be necessary to determine conclusively whether facial expression exists for the remaining genes.

WNT2B

WNT2B has been reported to be expressed in the ectoderm overlying the midbrain and in ectoderm of the developing eye in the chick (Jasoni et al.,1999; Fokina and Frolova,2006). Between stages 15 and 18, we also found exclusively ectodermal expression for this gene. WNT2B was initially restricted to the ectoderm dorsal to the eye but later extends ventrally to cover the maxillary prominence (Fig. 1A, Supp. Fig. S2A). There is also expression in the pharyngeal arches (Fig. 1A, Supp. Fig. S2A) At stage 24, expression is mainly restricted to the extra-oral surface of the maxillary prominence, lateral edges of the mandibular prominence, and second pharyngeal arch (Fig. 1C). Sections confirm that signal is localized to the ectoderm (Fig. 2A, A'). The expression of WNT2B is not detected in the frontonasal mass at any stage except for faint expression at the tip of the globular processes visible at stage 24 (Fig. 1C). At stage 28, expression is lost in the maxillary prominences and is only retained in the lateral mandibular prominences (Fig. 1D).

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Figure 1. Expression of WNT ligands during chicken craniofacial development. Whole mount in situ hybridization with digoxygenin-labeled probes. A, E, I, M, Q are lateral views; all others are frontal views of the facial prominences. A–C:WNT2B expression is seen in the dorsal retina and lateral ectoderm covering the maxillary prominences and pharyngeal arches. At stage 24 (C), expression is also seen in the lateral mandibular prominence and faint expression in the globular processes (arrows). D: Expression is only present in the lateral mandibular prominences at stage 28. E, F:WNT5A expression is seen throughout the mesenchyme of maxillary prominence and in the mandibular prominence (E). Expression is also seen in the lateral nasal prominences and lateral edges of the frontonasal mass at stage 21 (F). G: Restriction of WNT5A expression to a band midway across the rostral-caudal axis of the maxillary prominence, the midline of the mandibular prominence, and across the frontonasal mass. H: Relatively lower expression of WNT5A is seen in the midline of the frontonasal mass, overlapping the future chondrogenic region. I, J:WNT5B is present in the pharyngeal arches (black arrowheads) of stage-18 (I) and -20 (J) embryos. K:WNT5B expression in the maxillary prominences and weak expression is seen in the lateral edges of the frontonasal mass. L:WNT5B expression is present in the caudal maxillary prominences and lateral frontonasal mass mesenchyme similar to WNT5A. M, N: Expression of WNT9B in the lateral head ectoderm around the eye, the maxillary, and first pharyngeal arch of stage-18 (M) and stage-21 (N) embryos. O:WNT9B is strongly expressed in the ectoderm of all the facial prominences but the peripheral borders of the frontonasal mass and maxillary prominence lack expression (black arrowheads). P: Expression of WNT9B is decreased in the maxillary prominences at stage 28. Note increased expression in the centre and lateral edges of the frontonasal mass. Q:WNT11 expression is seen as a small spot in the maxillary region at stage 18 (black arrowhead). R:WNT11 transcripts are visible in the mesenchyme of the cranial maxillary prominences of stage-21 embryo. Note two faint bands of expression in each of the cranial mandibular prominences (black arrowhead). There is also expression in the extraocular mesenchyme. S: Extension of WNT11 expression to the anterior maxillary and craniolateral mandibular prominences of stage-24 embryo. No expression in the frontonasal mass. T: Expression of WNT11 restricted to the lateral maxillary prominence (black arrowheads) and to the cranial edge of the mandibular prominences. e, eye; f, frontonasal mass; gp, globular process; l, lateral nasal prominence; md, mandibular prominence; mx, maxillary prominence; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2. Scale bar = 500 μm.

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Figure 2. Tissue localization of WNT2B, 5A, 5B, 9B, 11, 16, DKK1, FRZB1, LEF1, and CTNNB1. Frontal sections of stage-24 embryos previously hybridized in whole-mount with digoxygenin-labeled probes. A, A': Expression of WNT2B in the lateral ectoderm of maxillary and mandibular prominences, second pharyngeal arch. Magnified image (A') of the region within the box in A showing clear restriction of WNT2B expression to the epithelium (white arrowhead). B, B': Superficial (B) and deeper (B') sections showing WNT5A expression in the mesenchyme of the frontonasal mass, lateral nasal, maxillary, and cranial mandibular as well as second arch mesenchyme (black arrowheads). C:WNT5B signal in the mesenchyme of the maxillary prominences and second arch (black arrowheads). D–D”':WNT9B expression in the epithelium of all the facial prominences in shallow (D) and deeper (D') sections. Clear localization of WNT9B expression in the epithelium of the frontonasal mass (D”), lateral maxillary, and mandibular prominences (D”', white arrowheads). E, E', E”:WNT11 expression is evident in the lateral maxillary mesenchyme (E, black arrowhead), craniolateral mandibular mesenchyme (E', black arrowhead), and lateral pharyngeal second arch mesenchyme (E”, black arrowhead). F: Expression of WNT16 in the caudal epithelium of lateral nasal prominence and lateral edge of frontonasal mass (white arrowheads). G:FRZB1 is in the mesenchyme of the maxillary prominences and cranio-medial mandibular mesenchyme (black arrowheads). H–H”':DKK1 is restricted to the epithelium of the frontonasal mass (H, H”, white arrowhead). In the maxillary prominences (H', H”'), expression is seen in both in the epithelium (white arrowhead) and mesenchyme (black arrowhead). I, I': Strong localization of CTNNB1 in the epithelium (white arrowhead) and diffuse expression in the mesenchyme (black arrowhead) of all the facial prominences. J, J':LEF1 expression in the epithelium (white arrowhead) and mesenchyme (black arrowhead) of the frontonasal mass. Expression is also seen in the maxillary, mandibular, and second arch mesenchyme (J', black arrowheads). e, eye; f, frontonasal mass; l, lateral nasal prominence; md, mandibular prominence; mx, maxillary prominence; pa2, pharyngeal arch 2. Scale bars = 500 μm (A, B–D', E–H', I, J, J'), 250 μm (A', D”, D”', H”, H”', I').

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WNT5A

WNT5A is exclusively expressed in the mesenchyme. At stage 15, there is strong expression in post-optic region (presumptive maxillary prominence) and first pharyngeal arch (Supp. Fig. S2B) and this pattern is continued to stage 18 (Fig. 1E). At stage 21, the WNT5A signal becomes stronger throughout the mesenchyme of the maxillary and mandibular prominences. Expression is also observed in the lateral nasal prominences and lateral edges of the frontonasal mass (Fig. 1F). By stage 24, WNT5A is predominantly confined to a band of cells midway along the caudal-rostral axis of the maxillary prominence, midline of the mandibular prominence, and is also observed across the frontonasal mass (Fig. 1G, 2B,B'). In the maxillary prominence, the expression of WNT5A shifts to the caudal half by stage 28. By this stage, there is less expression in the midline of the frontonasal mass where the midline cartilages and egg tooth will ultimately form (Fig. 1H).

WNT5B

WNT5B is closely related to WNT5A and expression patterns coincide to a large extent in the maxillary prominence and frontonasal mass. Weak expression in the pharyngeal arches is observed at stage 15 (Supp. Fig. S2C) and 18 (Fig. 1I). There is low expression of WNT5B throughout the facial prominences (Fig. 1J) until stage 24. Then weak WNT5B signal is observed in the lateral edge of the frontonasal mass but relatively stronger expression is present in the maxillary prominences (Figs. 1K, 2C). At stage 28, there is expression in the frontonasal mass and maxillary prominences overlapping with WNT5A and thus some redundancy in function is possible. In contrast to WNT5A, there is no WNT5B expression in the mandible between stages 24–28 (Fig. 1K,L).

WNT9B

WNT9B is expressed exclusively in the ectoderm, similar to WNT2B. In general, however, WNT9B signal is more widespread. At stage 15, there is strong expression in the ectoderm dorsal to the eye and first pharyngeal arch but the signal does not extend to cover the presumptive maxillary prominence (Supp. Fig. S2D). During the later stages (18–21), expression in the extraoral ectoderm of the maxillary and mandibular prominences is present with no detectable signal in the frontonasal mass (Fig. 1M–N). At stage 24, there is increased expression of WNT9B in the frontonasal mass ectoderm and thus all facial prominences become positive for WNT9B (Figs. 1O, 2D–D”'). Interestingly, there is a sharp boundary to WNT9B expression that leaves a band of non-expressing epithelium at the periphery of all the facial prominences. WNT9B overlaps exactly with WNT2B in the maxillary prominences at stage 24. At stage 28, expression of WNT9B is present but at lower levels in the maxillary prominences (Fig. 1P) whereas WNT2B is completely lost (Fig. 1D). WNT9B becomes concentrated in the centre of the frontonasal mass where the egg tooth will later form (Fig. 1P). Our data extend the recent results of another study (Hu and Marcucio,2009) in which stage-24 data were presented.

WNT11

Like the WNT5 genes, WNT11 is exclusively expressed in the mesenchyme. The expression of WNT11 is undetectable in the face prior to stage 18 (Supp. Fig. S2E, Fig. 1Q). We found that WNT11 is first expressed as a small spot in the maxillary region of the face at stage 18 (Fig. 1Q). At stage 21, WNT11 transcripts are clearly detectable in maxillary prominences and also two faint bands of expression are seen in each of the cranial mandibular prominences (Fig. 1R). Later these domains broaden, covering the anterior maxillary and craniolateral mandibular prominences (Figs. 1S, 2E,E'). WNT11 is also expressed in the lateral mesenchyme of the second pharyngeal arch (Fig. 2E”). At stage 28, the expression is shifted laterally in the maxillary prominence and towards the craniolateral edge of the mandibular prominence and is found in the periocular mesenchyme (Fig. 1T). It is also interesting that WNT11 is not detected in the frontonasal mass at any of the developmental stages. Radioactive in situs showed an identical distribution of transcripts to the whole-mounts, confirming the lack of expression in the frontonasal mass at stages 20 and 24 (data not shown).

WNT16

Like WNT11, the expression of WNT16 is not detected in the face until stage 18 (Supp. Fig. S2F, Fig. 3A). The first signal appears as a small spot in the maxillary, caudal mandibular, and cranial second pharyngeal arch clefts (Fig. 3A). At stage 20–21, increased expression of WNT16 is seen in the epithelium of lateral nasal prominence and there is an interesting restriction of expression to the caudal nasal pit epithelium, which includes the lateral edge of the frontonasal mass (Figs. 3B, 4A–B'). Expression of WNT16 is also seen in the mandibular branches of the trigeminal nerve (Fig. 3B). There is also faint expression in the anterior epithelium of maxillary prominences, also confirmed by radioactive in situ (Figs. 3B, 4B,B'). At stage 24, paired expression domains in the mandibular prominence corresponding to the mandibular nerve continue to be present on either side of the midline (Fig. 4C'). In saggital sections of stage-24 and -28 embyos, both the maxillary and mandibular branches of the trigeminal nerve are positive for WNT16 (Fig. 4D,D',F,F'). At stage 28, WNT16 is expressed in a subregion of the epithelium lining the nasal passages (Fig. 4E–F') that appears to exclude the specialized olfactory epithelium. It is likely that WNT16 marks the respiratory epithelium (Croucher and Tickle,1989).

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Figure 3. Expression of WNT16 and components of the WNT pathway. Whole mount in situ hybridization with digoxygenin-labelled probes. A, E, I, M, Q are lateral views, all others are frontal views. A: Expression of WNT16 seen as a small spot in the maxillo-mandibular cleft, first and second pharyngeal arch clefts. B: Strong expression of WNT16 is observed in the caudo-lateral nasal prominence and caudo-lateral edge of frontonasal mass (black arrow) at stage 21. The cranial half of the nasal pit does not express WNT16 (dashed line). Note weak expression in the anterior maxillary prominence (black arrowhead) and in the medial mandibular prominence (white arrowhead). C, D: Expression seen in the epithelium lining the nasal slit, extending slightly down to the maxillary prominence. E: Expression of FRZB1 seen in maxillary prominence and arches. There is also expression surrounding the nasal pit (black arrowhead). F: Expression becomes strong in the whole maxillary prominence and in lateral thirds of the frontonasal mass (black arrowhead) at stage 21. G, H: Weaker expression of FRZB1 in the frontonasal mass and maxillary prominences (black arrowhead) of stage-25 (G) and -28 (H) embryos. Strong expression seen until stage 25 in the medial portion of the mandibular prominences gets restricted to the distal tips at stage 28 (H, black arrowhead). I, J: Expression of DKK1 in the face of stage-18 (I) and -21 (J) chick embryo. Expression is seen in the maxillary prominences, pharyngeal arches, around the nasal pits and the eye. At stage 21, DKK1 is expressed around the nasal slit (J, white arrowhead) and along the caudal edge of the maxillary prominences. K, L: Expression disappears in the mandibular prominences but remains in the cranial (black arrow) and caudomedial (black arrowhead) portion of maxillary prominences. Expression is seen in the edges of the frontonasal mass at stage 24 (K, white arrowhead) and surrounding the nasal slit. By stage 28 (L), expression is restricted to the caudal edge of the frontonasal mass (white arrowhead) and medial edge of the maxillary prominence (black arrowhead). M: Expression of CTNNB1 around the nasal pit at stage 18. N: A stage-21 embryo showing strong expression around the nasal pit (white arrowhead). Note faint band of expression in the caudal maxillary prominences (black arrowhead). O, P: Expression of CTNNB1 seen in the facial prominences at stage 24 (O) and stage 28 (P). Relatively stronger expression is seen around the nasal slits. Q, R:LEF1 expression is seen in the maxillary, mandibular, and pharyngeal arch 2 prominences (black arrowheads) of stage-17 (Q) and -21 (R) embryos. Note strong expression in the trigeminal and geniculate ganglia (top and bottom asterisks, respectively). S, T: Expression of LEF1 seen across the frontonasal mass (white arrowhead) of stage-24 (S) and -28 (T) embryos. Expression is mainly found in the midline of the mandibular prominences and medial edge of the maxillary prominences (black arrowhead). There is also expression in the groove between the lateral nasal and maxillary prominences. e, eye; f, frontonasal mass; l, lateral nasal prominence; md, mandibular prominence; mx, maxillary prominence; np, nasal pit; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2. Scale bar = 500 μm.

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Figure 4. Epithelial localization of WNT16. Radiolabelled probes were used. A–F are bright field views, A'–F' are dark field views of the same sections so silver grains appear white on a black background. A, A', C, C', E, E' are frontal sections and B, B', D, D', F, F' are parasaggital sections. A, A':WNT16 is localized to epithelium surrounding the nasal pit (black arrowhead) but does not extend to the deepest part of the invagination. B, B': Expression in the anterior maxillary prominence epithelium continues into the nasal pit (black arrowhead). C, C': Expression in the medial nasal pit epithelium and in the mandibular branches of the trigeminal nerve on either side of the midline (black arrowhead). D, D': Localization of signal to the anterior maxillary epithelium as it becomes continuous with nasal pit epithelium (black arrowhead), maxillary and mandibular branches of trigeminal nerve (white arrowheads). E–F': Expression of WNT16 in the caudal, nasal slit epithelium (black arrowheads). There is also expression in the maxillary and mandibular branch of trigeminal nerve (white arrowhead). The retina appears birefringent under dark field illumination. e, eye; f, frontonasal mass; md, mandibular prominence; mn, mandibular branch of trigeminal ganglia; mx, maxillary prominence; mxn, maxillary branch of trigeminal ganglia; np, nasal pit. Scale bar = 250 μm.

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WNT4, WNT6

WNT4 and WNT6 have recently been shown to act by both canonical and noncanonical pathways. When bound to Fzd6, Wnt4 activates canonical signalling in kidney cells (Lyons et al.,2004). However Wnt4 has also been shown to activate the novel non-canonical pathway, p38 MAPK, in the context of osteogenesis (Chang et al.,2007). WNT6 activates the canonical (Linker et al.,2005) and non-canonical pathways (Schmidt et al.,2007), depending on the context. WNT4 is expressed in the external and stomodeal/oral epithelium of the entire head between stages 20 and 25 (Fig. 5A–D). There is no expression in the nasal pit at stage 20 (Fig. 5A) or in the nasal slit at stage 25 (Fig. 5C). In contrast, WNT6 is barely detectable at stage 20 (Fig. 5E) and is increased at stage 24 in the external head ectoderm. There is additional expression near the lateral edges of the frontonasal mass but not extending up into the nasal slit (Fig. 5F). The intraoral or medial surfaces of the maxillary prominences also express WNT6 distally (Fig. 5G) but not proximally (Fig. 5H). Very low levels of WNT4 and WNT6 were recently detected in the nasal slit epithelium with radioactive in situ hybridization by others (Hu and Marcucio,2009). It is possible that this is because the expression was very restricted in their study and that we may not have included this small region in our sections. Interestingly, unlike the other epithelially expressed WNT genes, it was not possible to observe signals in whole-mount in situ hybridization, perhaps indicating lower abundance of these transcripts.

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Figure 5. Epithelial expression of WNT4 and WNT6. All are frontal sections hybridized with radiolabelled probes and photographed in darkfield. A–D:WNT4 is expressed throughout the external head epithelium and epithelium lining the oral cavity (white arrows). There is no expression in the nasal pit at stage 20 (arrowhead, A) or in the nasal slit epithelium at stage 25 (C). E–H:WNT6 is barely detectable in the external head ectoderm at stage 20 (arrow, E). Stronger expression is seen in the head ectoderm, the maxillary prominence, and lateral edges of the frontonasal mass at stage 24 (white arrows in F,G,H). There is no expression in the nasal slit (F). The oral surface of the mandibular prominence also expresses WNT6 (H). e, eye; f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence; ns, nasal slit; pa2, second pharyngeal arch. Scale bar = 500 μm.

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Expression of WNT Antagonists

FRZB1.

FRZB antagonizes both canonical and non-canonical WNT signalling by direct binding of its cysteine-rich domain to the WNT ligands (Bhanot et al.,1996; Bafico et al.,1999). FRZB1 is first expressed in the cranial neural crest (Ladher et al.,2000). Our data at later stages agree with that originally published (Ladher et al.,2000). However, we were able to resolve more clearly the expression around the nasal placode at stage 18 (Fig. 3E). There is also medially restricted expression in the maxillary prominences and relatively higher expression in the midline of the mandibular prominence at stage 25 (Fig. 3G). We also confirmed mesenchymal expression in sections through the facial prominences (Fig. 2G). Until stage 23, FRZB1 transcripts are abundant throughout all facial prominences (Supp. Fig. S3A). From stage 25, mesenchymal expression of maxillary and frontonasal prominences is down-regulated compared to mandibular prominences where it is strongly expressed in the medial portion (Figs. 2G, 3G,H, Supp. Fig. S3B). This striking down-regulation is similar to previous published results (Ladher et al.,2000). At stage 28, there is a small area of expression at the cranial edge of the mandibular prominence and caudal edge of the frontonasal mass (Fig. 3H).

DKK1.

DKK1 specifically antagonizes canonical WNT function by preventing WNT ligands from interacting with LRPs (low-density lipoprotein receptor-like proteins; Niehrs,2006). In the mouse, DKK1 is expressed in the first pharyngeal arch (Monaghan et al.,1999) similar to what we have observed in the chicken at stage 15 (Fig. 6A,A', Supp. Fig. S2H). There is no expression in the frontonasal region until stage 17–18 when expression is seen around the nasal pit (Fig. 3I). The first maxillary expression is also observed at stage 17–18. At stage 21, DKK1 is expressed both in the epithelium and mesenchyme of the maxillary and mandibular prominences (Figs. 3J, 6B,B'). In contrast, in the frontonasal mass expression is restricted to the epithelium surrounding the nasal slit (Figs. 3J, 6B,B'). Stage-24 embryo sections confirmed that DKK1 is restricted to the ectoderm of the nasal slit and caudal edge of the frontonasal mass (Fig. 2H, H”). This trend of exclusively epithelial expression for DKK1 in the frontonasal mass is continued at stage 28 where the caudal edge or frontonasal epithelial zone is positive (Fig. 3L, 6D,D' (Hu et al.,2003). However, the nasal slit epithelium is no longer expressing DKK1. At stage 24, expression in the maxillary prominences is restricted to a cranial and caudal domain on the medial side of the prominence (Figs. 2H”',3K, 6C,C') and at stage 28 a narrow band of mesenchyme subadjacent to the medial epithelium continues to express DKK1 (Fig. 3L, 6D,D'). In the mandibular prominence, there is a reduction of expression from stage 24–28 compared to stage 20–21, although in saggital sections some mesenchymal expression can still be detected (Fig. 6C,C'). This suggests DKK1 can modulate canonical WNT signalling in a temporally and spatially specific manner within each of the facial prominences.

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Figure 6. Tissue localization of DKK1. A, A', C, C' are parasaggital sections; B, B', D, D' are frontal sections. A–D are photographed in bright field and A'–D' are the same sections photographed in dark field. A, A': Strong expression of DKK1 in the pharyngeal arch mesenchyme (black arrowheads). B, B': Expression is mainly found in the epithelia surrounding the nasal pits (black arrows) and in the mesenchyme of the mandibular prominences (black arrowhead). C, C': Expression of DKK1 is found in the mesenchyme of maxillary (white arrowheads) and mandibular (black arrowhead) prominences. Expression in the maxillary is more concentrated in the cranial and caudal mesenchyme (white arrowheads). Note the expression in the nasal slit epithelium (black arrow) and in the trigeminal ganglia (V, white arrow). D, D': Localization of DKK1 in the caudal edge epithelium borders the frontonasal mass (black arrows) whereas in the maxillary prominences there is expression in both the epithelium and mesenchyme (white arrowhead). Note the loss of expression surrounding the nasal slit. e, eye; f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence; np, nasal pit; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2; V, trigeminal ganglia. Scale bar = 250 μm.

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Expression of Canonical WNT Pathway Genes

CTNNB1.

Between stages 15–18, CTNNB1 expression is highest around the nasal pits (Supp. Fig. S2I). From stage 21, there is enriched expression around the nasal pits and faint bands appeared at the caudal edge of the maxillary prominences (Fig. 3N, Supp. Fig. S4B,B'). At stage 24 and 28, expression of CTNNBI appeared to be more ubiquitous in the face with the strongest signal being localized to the nasal slits (Fig. 3O,P). Expression is also observed in the mesenchyme; however, it was hard to detect the signal above the background. These results are clarified by radioactive in situs, which showed that in fact CTNNB1 expression is very abundant in both the epithelium and mesenchyme at all stages (Supp. Fig. S4A–F). Thus, this important mediator of canonical WNT signaling is expressed in all regions of the developing face.

LEF1.

We found the nuclear mediator of canonical WNT signalling, LEF1, is expressed in the mesenchyme of the maxillary and pharyngeal arches at stage 15 (Fig. 7A,A', Supp. Fig. S2J). Similar patterns are observed at stages 17 to 28 except that maxillary expression becomes more concentrated in the medial edge (Figs. 3Q–T, 7C',D',F'). In the mandibular prominence, expression is throughout at stage 20 but then becomes more abundant near the midline by stage 24 (Figs. 3R–T, 7C',E'). In the frontonasal mass, expression is detected in the midline at stage 24 and this expands across the medial-lateral axis by stage 28. LEF1 transcripts were also found in the lateral edges of the lateral nasal processes between stages 24 and 28 (Fig. 3S,T). Sections confirmed that LEF1 is also expressed in the pharyngeal endoderm of the first pharyngeal pouch (Fig. 7B′,C′,E′).

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Figure 7. Tissue localization of LEF1. A–B', D, D', F, F' are parasaggital sections. C, C′, E, E′ are frontal sections. A–C, D–F are photographed in brightfield and A'–C', D'–F' are the same sections photographed in darkfield. A, A': Expression of LEF1 in the mesenchyme of maxillary region (white arrowhead) and pharyngeal arches (black arrowhead). B–C': Expression of LEF1 in both the epithelium and mesenchyme of maxillary (white arrowhead) and mandibular (black arrowheads) prominences. Note strong expression in the trigeminal ganglia (B', black arrow). D–E': Stage-24 embryo shows enriched signal in the distal maxillary prominence (D', white arrowhead) and frontonasal mass (D', black arrow). In the frontal plane (E'), there is slightly more signal in the medial maxillary prominences (white arrowhead), caudal and lateral edges of frontonasal mass (black arrows), and medial mandibular mesenchyme (black arrowheads). Note the strong expression in the caudal side of the mandibular prominence (white arrow). F, F': Expression is strongest in the mesenchyme subadjacent to the epithelium in the frontonasal (black arrow), maxillary (white arrowhead) and mandibular prominences (black arrowhead). e, eye; f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2; V, trigeminal ganglia. Scale bar = 250 μm.

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Expression of FZD Receptors

Expression data were published for several FZD genes in HH stages 3–20 (Stark et al.,2000). Therefore, we focused on older stages. Diffuse mesenchymal expression in ectomesenchymal cells of the maxillary and mandibular prominences was observed for FZD1,2,3,4,5,6 and 7. FZD1, 2, 4, and 7 are also expressed in the frontonasal mass. FZD1,4,7 were also highly expressed in the periocular mesenchyme whilst FZD4 and 7 were highly expressed in the medial maxillary mesenchyme. FZD2 and 7 were the only receptors noticeably lower in the mesodermal core of the mandibular prominence. To confirm this, we hybridized adjacent sections to MYOD, a muscle-specific marker expressed in the mesodermal core. Expression patterns of MYOD, FZD7, and FZD2 were exactly complementary (Fig. 8G”, G”', and data not shown). The most striking expression was noted for FZD10 where medial mandibular prominence had intense expression in the mesenchyme just beneath the epithelium (Fig. 8I, I'). In addition to mesenchymal expression, several of the genes had signal in the epithelium including FZD3, 4, 5, 6, 8, and 10 (Fig. 8E,E',F,F',H,H',I,I').

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Figure 8. Expression of Frizzled receptors. All panels are frontal sections of stage-24 embryos at shallow and deeper planes photographed in darkfield. A,A':FZD1 is ubiquitously expressed in facial mesenchyme with stronger expression in the periocular mesenchyme (arrow). B,B':FZD2 and (C,C') FZD3 are ubiquitously expressed in all facial mesenchyme. FZD3 has slightly higher expression in the fusion point between the frontonasal mass and maxillary prominence (arrowhead in C). FZD2 is absent from the mesodermal core of the mandibular prominence. D,D':FZD4 has higher expression along the caudal edge of the frontonasal mass (D, arrow) and medial maxillary prominence (D', arrow). E,E':FZD5, (F, F') FZD6, (H,H') FZD8 have ubiquitous expression in the mesenchyme at low levels and expression in the epithelium (arrowheads). G,G',G”':FZD7 is expressed in the mesenchyme throughout the face, however, is absent from the core mesoderm in the mandibular prominence (G”', arrowhead). G”:MYOD expression in the mesodermal core, complementary to the adjacent section hybridized to FZD7. I, I':FZD10 is restricted to the medial mandibular prominence epithelium and mesenchyme (arrows) and in the epithelium of the head (arrowheads). More proximal sections show expression in the dorsum of the tongue (I', arrow shows mesenchyme, arrowheads indicated epithelial expression). c, mesodermal core; e, eye; f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence; t, tongue. Scale bar = 500 μm

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Our study provides a comprehensive overview of the expression patterns of WNT ligands, the major effector molecules, antagonists, and receptors in the developing face. The data suggest where, when, and which endogenous ligands are controlling different aspects of facial morphogenesis. These basic descriptive studies are necessary before we can go on to test the functions of WNT signalling in craniofacial development.

Multiple FZD Receptors Are Present in the Developing Face

All of the facial prominences express several of the FZD genes. Therefore, there would be multiple receptors capable of binding to the WNT ligands. The expression of FZD10 is intriguing and it may be that only a restricted set of WNTs is capable of activating this receptor. At the present, the binding specificities of the FZD receptors are not well understood. However, it does appear that choice of receptor, in part, determines whether some WNTs activate canonical or non-canonical pathways (van Amerongen et al.,2008). It is, therefore, not possible to predict on the basis of gene expression whether ligands act via canonical or non-canonical WNT pathways. For example, both WNT5A and WNT11 have been shown to activate the canonical pathway in some experimental systems depending on which FZD receptor is present (Tao et al.,2005; Mikels and Nusse,2006). Therefore, one needs to perform functional experiments to determine which of the ligands act via the canonical or non-canonical pathways.

Canonical WNTs Are Localized to the Epithelium

An important overarching observation is that all of the WNTs for which only canonical signalling has been experimentally proven are ectodermally restricted. Since both CTNNB1 and LEF1 and numerous FZD receptors are present throughout the facial mesenchyme, canonical WNT signalling could take place in areas where the ligands WNT- 2B, 9B, 16, and 7A are expressed. Others have determined with reporter assays in mouse and chicken that canonical WNT activity is present in the face. In the chicken, the delivery of the reporter has been conducted with a lentivirus (Brugmann et al.,2007) or with electroporation (Hu and Marcucio,2009). The former study found a higher level of activity in the midline of the frontonasal mass while the latter found more ubiquitous expression in all areas that took up the plasmid including those lateral to the midline. The authors (Hu and Marcucio,2009) attributed the differences in reporter activity to the DNA delivery method and we agree with this interpretation. Thus, it is not clear yet whether all stages or areas of the chicken face have active WNT signalling.

There is generally good correspondence between our chicken WNT ligand expression and the mouse Wnt reporter data (TOPGAL, BATgal; Lan et al.,2006; Brugmann et al.,2007). For example, there is initially very low to absent activity in the frontonasal mass at E9.5, a stage equivalent to stage 15 in the chicken embryo, which is consistent with the fact that none of the WNT ligands could be detected in the frontonasal mass at stage 15. The mouse β-gal staining is strongest in the ectoderm and in the subadjacent mesenchyme in the medial nasal, maxillary, and mandibular prominences. Although there are two WNT ligands present in the ectoderm that could activate non-canonical signalling (WNT4 and WNT6), the expression of the mouse reporter argues in favour of canonical activity taking place at sites of epithelial-mesenchymal interactions in the face. Furthermore, the injection of DKK1 adenovirus, which only blocks the canonical pathway, leads to severe reduction in growth of the frontonasal mass prominence (Brugmann et al.,2007).

Interestingly, several of the WNTs with non-canonical activity, WNT5A, 5B, and 11 are expressed only in the mesenchyme. One of these, Wnt5a, is essential for outgrowth (Yamaguchi et al.,1999; He et al.,2008). These Wnts may also be essential for skeletogenesis and myogenesis as in other regions of the body (Anakwe et al.,2003; Church et al.,2003; Gros et al.,2009). Further experiments designed to selectively block the non-canonical pathways are needed to see whether facial growth and cell differentiation are affected.

Finally, there are overlapping patterns of expression between the canonical WNTs and non-canonical WNTs. Thus, there may be cross-talk between the pathways. Wnt5a has, for example, been shown to antagonize canonical signalling via the Ror2 receptor (Mikels and Nusse,2006). Such mutual antagonism may limit the extent of canonical signalling taking place in the mesenchyme.

WNT Activity During Fusion of the Lip and Palate

The contact of the frontonasal mass (medial nasal prominences in mice) and maxillary prominences to form the lip is also likely to involve WNT signalling. There is already evidence that Wnt9b is involved in lip fusion as there is cleft lip in some of the mutant mice (Carroll et al.,2005; Juriloff et al.,2006). There is high expression of LacZ precisely in the fusing tips of the medial nasal and maxillary prominences at the time of fusion (Lan et al.,2006; Brugmann et al.,2007). Further evidence that canonical Wnt signalling is essential for lip fusion is that Dkk1 overexpression in mice causes cleft lip (Brugmann et al.,2007). Fusion of the facial prominences requires growth such that the prominences can make contact and permit the fusion of competent epithelia. Our data shows that WNT9B is not expressed in the bilayered epithelial seam or mesenchymal bridge, arguing that if the expression patterns are conserved in mice, WNT9B has a crucial role in outgrowth of the facial prominence mesenchyme, thereby facilitating contact.

Additional Wnts may also be involved and from our data we now suggest that WNT16, which also signals via the canonical pathway, could be important in forming the epithelial seam between the frontonasal mass and maxillary prominences. WNT16 is the only WNT ligand expressed in these fusing epithelia. In addition, it is expressed prior to (stage 20–24) and during fusion (stage 28). There are very few other signals (BMP2, 4, 7; Ashique et al.,2002a) expressed on both sides of the fusion zone in the frontonasal mass and maxillary epithelia. Thus far, no detailed descriptions of Wnt16 in the mouse have been reported so it will be interesting to see whether the patterns are conserved and whether this gene is required for lip fusion. WNT11 is also potentially involved in fusion as it is present in the mesenchyme of the cranial maxillary prominence at stage 24. However, expression is shifted laterally at stage 28 just prior to fusion. Therefore, it is possible that WNT11 may initially control outgrowth but not participate actively in fusion of the lip.

The expression of the two WNT antagonists, FRZB1 and DKK1, in our study is very dynamic in the fusion zone. Both are present at high levels at stage 24 but are dramatically decreased at stage 28 when fusion is beginning. A similar pattern has been reported for SFRP2 (Ladher et al.,2000). These data, together with the above, suggest that canonical WNT activity is carefully controlled and is only allowed to increase at the moment when fusion is taking place. A similar observation was made by us previously with the BMP antagonist, NOGGIN (Ashique et al.,2002a). Decreased expression of NOG occurred in the globular processes just prior to fusion, allowing an increase in BMP activity.

Another potentially important role for WNT signalling is in formation of the secondary palate. Here we can point to the regionally restricted expression of non-canonical WNTs in the maxillary mesenchyme as potential players in palatogenesis. Recent analysis of the Wnt5a−/− mice (He et al.,2008) shows there are clefts of the secondary palate. Wnt5a is needed for outgrowth and directional cell migration within the palatal shelves (He et al.,2008). These data and the lack of WNT reporter activity in the palatal shelves (He et al.,2008) point to a likely predominance of non-canonical signalling in palatal fusion.

In summary, there is much to learn about WNT signalling during facial morphogenesis. Our studies have helped to identify the main WNT ligands that are likely to be involved in facial patterning, lip fusion, and nasal morphogenesis. Future work will test the hypotheses we have raised and thereby build a more complete picture of molecular signalling during craniofacial development.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In Situ Hybridization

Fertilized chicken eggs were incubated at 38°C. Embryos were fixed overnight at 4°C in 4% paraformaldehyde. Embryos were washed twice in phosphate buffered saline with 0.1% Tween-20, dehydrated in methanol, and stored at −20°C. Whole mount in situ hybridization was performed, as previously described, but using an Intavis in situ hybridization robot (Song et al.,2004). Selected stained embryos were processed through isopropanol, isopropanol:paraffin (50:50) and then embedded in paraffin. Embryos were sectioned at a thickness of 7μm and counterstained with eosin. Section in situ hybridization was also performed with antisense 35S-labeled antisense probes using published protocols (Wilke et al.,1997).

The following individuals provided gallus cDNAs for this study: C. Marcelle, WNT1, 3A 5B, 11, CTNNB1 (Marcelle et al.,1997; Geetha-Loganathan et al.,2005); C. Hurle, DKK1 (Grotewold and Ruther,2002), C.J. Tabin, LEF1 (Gavin et al.,1990; Bergstein et al.,1997; Kengaku et al.,1998; Jasoni et al.,1999); S. Chapman, WNT8B (Chapman et al.,2004); K.G. Storey, WNT8C (Olivera-Martinez and Storey,2007); E. Frolova, WNT5A, WNT9B (Fokina and Frolova,2006); P.A. Krieg, WNT11B (Hardy et al.,2008); P. Francis-West, FRZB1 (Ladher et al.,2000); T. Nohno, WNT4, FRZD 1, 2, 3, 4, 6, 7, 8. 10 (Kengaku et al.,1997; Kawakami et al.,2000); and L. Burrus, FRZD5 (Cauthen et al.,2001). DKK2 was purchased from MRC Geneservice (833 bp, chEST339h24). We independently cloned a 758-bp fragment of WNT16 using published sequence (GenBank accession number: AY753296). For non-radioactive in situs, we used WNT6 (1,500 bp) as described (Rodriguez-Niedenfuhr et al.,2003) and for radioactive in situs we used the WNT6 probe from A. McMahon (Hollyday et al.,1995).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

This work was funded through grants from the CIHR to J.M.R. and BBSRC to P.F.W. P.G.-L. and S.N. are supported by MSFHR post-doctoral fellowships. We also thank GR. Handrigan for helping in cloning WNT16. We extend our gratitude to all those who provided us with in situ hybridization probes.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_21934_sm_SupplFigS1.tif5680KFig. S1. Expression of WNT1, 3A, 4, 7A, 8B, 8C, 9A, 11B, DKK2. Whole-mount in situ hybridization was used for all panels except B', which is a darkfield image with radioactive probe. A: Stage-16 embryo with expression of WNT1 is in the brain and dorsal neural tube (black arrowhead). B: Stage-20 chick embryo with WNT3A expression in the dorsal neural tube (black arrowhead) and in the apical ectodermal ridges of the limb buds (white arrowhead). B': Parasaggital section of a stage-15 embryo shows expression of WNT3A in the midbrain but not elsewhere in the head. C: Strong expression of WNT4 in the dorsal neural tube (black arrowhead) of stage-19 embryo. D, D': WNT7A is found in first (white arrowhead) and second (black arrowhead) pharyngeal arch clefts but not in the facial prominences. Note strong expression in the dorsal ectoderm of the developing limb buds (arrows). E: Expression of WNT7B in dorsal telencephalon (black arrowhead) and eye (white arrowhead) of a stage-17 embryo. F: A stage-18 embryo, showing strong expression of WNT8B in the dorsal telecephalon and diencephalon (black arrowhead). G: Dorsal view of a stage-11 embryo with strong expression of WNT8C exclusively in the segmental plate region (black arrowhead). H, H': Expression of WNT9A is seen in the hepatic epithelium (white arrowhead), dorsal telecephalon, and diencephalon (black arrowhead) of a stage-18 embryo. H': WNT9B is localized to the interdigital and presumptive joint regions of the hind limb of a stage-28 embryo. I: WNT11B is expressed in the isthmus between the telencephalon and diencephalon (black arrowhead) of a stage-15 embryo. J: At stage 25, expression of DKK2 is observed in the apical ectodermal ridges (black arrowheads), in the proximal mesenchyme proximal to the limb buds, and in the interlimb lateral plate mesoderm. The head was removed since there was no expression of DKK2. m, mesencephalon. Scale bars = 1 mm except for B', which is 250 μm.
DVDY_21934_sm_SupplFigS2.tif2026KFig. S2. Expression of WNT pathway genes in stage-15 embryos. All are lateral views of the heads of embryos that underwent whole-mount in situ hybridization. A: WNT2B signal is strongest dorsal to the eye and does not yet extend into the maxillary prominence. There is also signal in the first and second arches. B: Expression of WNT5A is strongest dorsal to the eye, the dorsal retina, the diencephalon, and the pharyngeal arches. There is also expression in the post-optic maxillary mesenchyme (arrowhead). C: Weak expression of WNT5B is seen in the pharyngeal arches (black arrowheads). D: Localization of WNT9B in the post-optic ectoderm, ectoderm around the eye, and in pharyngeal arches but not extending into the presumptive maxillary prominence. E: No expression of WNT11 except in the optic stalks (arrowhead). F: WNT16 has no expression in the face or pharyngeal arches. G: Expression of FRZB1 seen in maxillary region and pharyngeal arches. H: DKK1 is abundant in the pharyngeal arches and relatively lower in the eye and maxillary region. I: Strong expression of CTNNB1 seen around the nasal pits, in the telencephalon and frontonasal mass. The whole embryo has some expression above background. J: Localization of LEF1 in the maxillary region, proximal first arch, and trigeminal ganglion (arrowhead). e, eye; f, frontonasal mass; mx, presumptive maxillary prominence; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2. Scale bar = 500 μm.
DVDY_21934_sm_SupplFigS3.tif934KFig. S3. Expression of the antagonist FZB1. A: At stage 23, FRZB1 transcripts are observed in the corners of the frontonasal mass, throughout the lateral nasal process, maxillary and mandibular prominences. B: At stage 25, expression in the maxillary and frontonasal mass prominences is decreased whereas the expression in the mandibular prominence remains stronger. f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence. Scale bar = 500 μm.
DVDY_21934_sm_SupplFigS4.tif4981KFig. S4. Tissue localization of CTNNB1. Due to very abundant signal, sections were photographed in bright field. Signal appears as black silver grains. A: A parasaggital section showing signal in the mesenchyme surrounding the eye, maxillary and pharyngeal arch mesenchyme. B: Parasaggital section with strong expression of CTNNB1 in both the epithelium and mesenchyme of all facial prominences. B': Frontal view showing especially high signal in the nasal pit epithelium (black arrowhead). C, D: Frontal (C) and parasaggital (D) sections showing ubiquitous expression of CTNNB1 in all the facial prominences. E, F: Frontal (E) and parasagittal (F) sections of stage-28 embryo showing expression in both the epithelium and mesenchyme of all facial prominences. The expression is most concentrated along the nasal slits and in the medial maxillary mesenchyme (black arrowheads). e, eye; f, frontonasal mass; md, mandibular prominence; mx, maxillary prominence; np, nasal pit; pa1, pharyngeal arch 1; pa2, pharyngeal arch 2. Scale bar = 250 μm.

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