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First published online 15 August 2007
doi: 10.1242/dev.005132

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Wnt signaling mediates regional specification in the vertebrate face

¹ Department of Plastic and Reconstructive Surgery, Stanford University, Stanford, CA 94305, USA.
² Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
³ Howard Hughes Medical Institution, Stanford University, Stanford, CA 94305, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Comparisons between X-Gal staining patterns in Wnt reporter mice. (A-D) X-Gal staining for Wnt reporter activity at E9.5. (A) Staining is evident in the midbrain (mb) and anterior neural folds (nf) of BATgal embryos. (B) A lateral view shows reporter activity in the first pharyngeal arch (p1). (C) In TOPgal embryos, Wnt reporter activity is evident in the first pharyngeal arch (p1) as well as the midbrain (mb) and hindbrain (hb). No Wnt reporter activity is detectable in the anterior region of the face (f). (D) Wnt reporter activity in the midbrain (mb) and anterior neural folds (nf) of TOPgal embryos generally mirrors that seen in BATgal embryos. Note that the BATgal and TOPgal embryos are at slightly different stages of development, as gauged by the extent of neural tube fusion (dotted white lines, A and D). (E-H) X-Gal staining at E10.5. (E) Wnt reporter activity is evident in the midbrain (mb) and forebrain (fb) of BATgal embryos. (F) Reporter activity is also evident in the distal regions of the maxillary (mx) and mandibular (mn) prominences, the aboral surface of the mandible (red arrow), the proximal first arch (red arrowhead), the lateral nasal (l) prominences, and around the developing eye (e). (G) A nearly identical pattern of reporter activity is seen in the TOPgal face. (H) One subtle difference in reporter activity at this stage is in the forebrain region, where X-Gal staining appears more restricted than that observed in BATgal embryos (compare E with H). (I-L) X-Gal staining at E14.5. (I) Wnt reporter activity is evident in surface ectoderm overlying the dermal papilla, signaling the site of whisker development. (J) At the same stage, Wnt reporter activity is abundant in the maxillary (mx) prominences but absent in the frontonasal (f) prominence. (K,L) The pattern of Wnt reporter activity in the TOPgal face and whisker primordia mirrors that seen in BATgal embryos at the same stage.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Mapping the spatiotemporal pattern of ß-gal activity in TOPgal Wnt reporter embryos. (A,B) At E8.5, reporter activity is restricted to the lateral (L) neural folds and is absent from the medial (M) midline region near the anterior neural ridge (anr). (C) X-Gal staining of a transverse section reveals reporter activity is confined to lateral neuroectoderm of the neural fold (nf). (D) Frontal and (E) lateral views at E9.5 illustrate reporter activity in cranial neural crest cells migrating into the first pharyngeal arch (p1) and the prosencephalon (pros). Cranial neural crest cells covering the midbrain (mb) are also ß-gal-positive. (F) A transverse section through p1 show that at this stage, X-Gal staining is found in neural crest (nc) cells but not surface ectoderm (se) or neuroectoderm (ne). (G) Frontal and (H) lateral views at E10.5 indicate a distinct boundary between reporter-positive and reporter-negative cells. The frontonasal prominence (f) is devoid of staining, whereas some staining is evident in the median nasal prominence (m). The lateral nasal (l) and maxillary (mx) prominences show strong X-Gal staining. (I) Transverse sections reveal that surface ectoderm and mesenchyme of the median (m) and lateral nasal (l) prominences are sites of X-Gal staining, but neither surface ectoderm or mesenchyme in the frontonasal (f) prominence is positive for the reporter. (J) Frontal and (K) lateral views at E11.5 show an increase in X-Gal staining in the median nasal (m) prominence, whereas the frontonasal (f) prominence remains devoid of staining. At this stage, the median nasal prominence and maxillary prominence have fused (red asterisk). (L) Transverse sections show X-Gal staining disappears from surface ectoderm, but persists in maxillary mesenchyme as the prominences fuse (red asterisk). (M) Although reduced in size, the median nasal prominence remains free of X-Gal staining. (N) Transverse sections show a boundary between ß-gal-positive and ß-gal-negative regions in the frontonasal mesenchyme (red arrows). At this stage, X-Gal staining is restricted to mesenchyme (inset, red arrow). (O) At E12.5, the frontonasal (f) prominence is compressed by the growth of the maxillary (mx) prominences and (P) transverse sections show that the frontonasal region lacks reporter activity. The median nasal (m) prominence exhibits reporter activity. (Q) At E13.5, the mouse muzzle is fully formed and ß-gal activity is evident in the whisker primordia. The continued growth of the maxillae (mx) relative to the compressed frontonasal (f) prominence produces the infranasal depression (ind, red arrow). (R,S) Transverse sections show the general lack of X-Gal staining in the frontonasal prominence relative to strong X-Gal staining in the maxillary prominences (mx, red dotted line). (T,U) Low- and high-power magnifications at E14.5 reveal frontonasal (f) prominence-derived tissues are reduced to a thin stripe of midline tissue (dotted red line) that generally maintains its ß-gal-negative status. The maxillary prominences (mx) remain ß-gal-positive. (V) A ventral view reveals the general lack of X-Gal staining in frontonasal prominence-derived primary (1o) palate. The boundary of X-Gal staining follows the demarcation between structures derived from the maxillary and frontonasal prominences (dotted red lines). White dotted circle, incisive foramen. Scale bars: white, 250 µm; black, 100 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Between E10.5 and E15.0, the expression of downstream targets and mediators of Wnt signaling correlates with sites of Wnt reporter activity. (A) At E11.5, Msx1 is robustly expressed throughout the mouse maxillary (mx), lateral (l) and median (m) nasal prominences, whereas the frontonasal (f) prominence lacks Msx1 expression (red arrow). (B) At E11.5, Msx2 is also expressed in the maxillary (mx), lateral (l) and median (m) nasal prominences but is absent from the frontonasal (f) prominence (red arrow). (C) At E11.5, Lef1 is expressed in the maxillary (mx) and, to a lesser extent, in the lateral nasal (l) prominences. Expression is largely absent from the frontonasal (f) prominence (red arrow). (D) Tcf4 expression is confined to domains within the lateral (l) and median nasal (m) prominences, whereas the frontonasal (f) lacks Tcf4 expression (red arrow). (E,F) Ventral view at E15.5 shows Msx1 and Msx2 expression limited to the maxillary (mx) prominences (dotted white line) and absent from frontonasal (f) prominence and the primary (1o) palate. (G) At E13.5, Lef1 transcripts are detected in mesenchyme and ectoderm of the maxillary (mx) prominences, and at lower levels in frontonasal (f) mesenchyme. (H) Tcf4 transcripts are evident through the frontonasal (f) and maxillary (mx) mesenchyme. (I) At E10.5, Nmyc transcripts localize to maxillary (mx) mesenchyme and are largely absent from frontonasal (f) tissues (red dotted line). (J) At E13.5, Nmyc is expressed in all surface ectoderm and continues to be expressed in maxillary mesenchyme (mx) but is absent from frontonasal (f) mesenchyme (red dotted line). (K) At E15.5, Nmyc maintains its same general expression pattern (red dotted line). (L) At E10.5, PCNA immunostaining on transverse sections shows evidence of cell proliferation in frontonasal (f) ectoderm and maxillary (mx) mesenchyme. (M) Co-staining for BrdU and ß-gal shows that at E13.5, cell proliferation and reporter activity are colocalized to maxillary (mx) mesenchyme, whereas decreased levels of cell proliferation and reporter activity are detected in frontonasal (f) mesenchyme (dotted red line). Scale bars: 100 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Tcf4-/-; Lef1-/- mutant mice have disrupted midfacial development and malformed teeth and tastebuds. (A-D) Whole-mount view of E17.0 embryos with varying dosages of Tcf4 and Lef1. (A) Tcf4 heterozygote embryos (Tcf4+/-/Lef1+/+) are unaffected, and epithelial specializations such as whiskers are present (black arrows), and eyelid fusion occurs normally (yellow arrow). (B) Tcf4+/+; Lef1-/- mutants have disrupted whisker pattern (asterisk) and exhibit hypoplastic maxillae. (C) Tcf4-/-; Lef1+/- mutants lack eyelids (yellow arrow). (D) Tcf4-/-; Lef1-/- embryos show evidence of a severely reduced maxillae, in addition to their lack of eyelids and disrupted whisker primordia (yellow arrow and black asterisk). (E-H) In a comparison of E16.0 wild type and Tcf4-/-; Lef1-/- compound mutants, wild-type embryos (E,F) show fully developed maxillae, an infranasal depression (dotted white line), and correctly spaced nostrils (dotted red line). Note organized whisker primordia. By contrast, Tcf4-/-; Lef1-/- embryos (G,H) have a malformed frontonasal prominence and underdeveloped maxillae, which results in an infranasal depression that looks more like a human philtrum (dotted white line, red arrows). The nostrils are displaced laterally as a consequence (dotted red line). Note disorganized whisker primordia and absence of fused eyelids. (I-L) In a comparison of E15.0 wild-type and Tcf4-/-; Lef1-/- embryos, wild-type embryos (l,J) show fully developed maxillae and organized whisker primordia; the infranasal depression (white dotted line, ind) is evident. Tcf4-/-; Lef1-/- embryos (K,L) exhibit hypoplastic maxillae and lack the infranasal depression (dotted white line); note the absence of whisker primordia. Scale bar: 1 mm.

View larger version (97K): [in this window] [in a new window]   Fig. 5. Skeletal and molecular analyses of Tcf4-/-; Lef1-/- compound mutants. Histological staining on transverse sections through E15.5 wild-type (Tcf4+/+/Lef1+/+) mouse embryos shows (A) the normal length of the cartilaginous nasal septum (ns) and (B) the curvature of the nasal cartilage (nc) and condensations of the whisker buds (black arrows). The nasal epithelium (ne) is correctly organized. (C) In compound null-mutant embryos nasal cartilage (nc) growth is truncated and whisker buds are absent (black asterisks). Despite this dramatic alteration in shape, the cartilage is still well developed and the nasal epithelium (ne) is organized. (D) In contrast to the wild-type nasal septum shown in A, the mutant nasal septum is dramatically foreshortened. (E,F) Ventral view of Alcian Blue/Alizarin Red skeletal preparation (mandibles removed) shows that in wild-type embryos (E), the basisphenoid (bs), premaxillae (pmx, white dotted line), maxillae (mx), palatine bones (pal) and the nasal septum have formed normally and have their proper orientation relative to one another. (F) Tcf4-/-; Lef1-/- mutants exhibit grossly underdeveloped premaxillae (pmx, dotted white line), which fail to make contact and fuse across the midline. The major skeletal elements of the posterior palate appear normal. (G) Pentachrome staining of transverse sections through E15.5 jaws reveal tooth primordia at the bell stage, where the dental epithelium (de) has invaginated and dental mesenchyme (dm) has condensed in response to signals from the ectoderm. (H) Tcf4-/-; Lef1-/- dental epithelium (de) fails to invaginate properly. Note, however, that maturation of Meckel's cartilage (mk) and the bone of the mandible (mn) are unaffected by the loss of Tcf4 and Lef1. (I,J) Pentachrome staining of transverse sections through E15.5 wild-type embryos reveals the characteristic `Y' shape (yellow dotted line) of the anterior nasal septum, which correlates to the location of the infranasal depression (ind). In an adjacent tissue section, the osteogenic condensation of the maxillae is evident (dotted white line), as well as the tooth (t) and epithelial seam (dotted white box) of the palatal shelves, which is dissolving on an appropriate time scale. (K,L) In E15.5 Tcf4-/-; Lef1-/- littermates, the osteogenic condensation of the maxillae is reduced (dotted white line), the tooth (t) is developmentally delayed and the epithelial seam (dotted white box) of the palatal shelves remains evident. The anterior nasal septum exhibits a dysmorphic `T' shape (yellow dotted line), corresponding to the malformed midface seen earlier (Fig. 4). (M,N) In situ hybridization on transverse sections of E15.5 wild-type embryos shows that collagen II (Col II) is expressed throughout the cartilaginous nasal septum (ns) and Msx1 transcripts are detected in surface ectoderm (se, dotted red line), nasal epithelium (ne) and undifferentiated mesenchyme. (O,P) In E15.5 Tcf4-/-; Lef1-/- littermates, Msx1 expression is specifically lost in surface ectoderm (dotted red line) but maintained in nasal epithelium and underlying mesenchyme. Collagen II transcripts persist in the dysmorphic nasal septum (ns), indicating normal chondrocyte differentiation. Scale bars: white, 1 mm; black, 100 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 6. In utero gene transfer of a Wnt inhibitor phenocopies the Tcf4-/-; Lef1-/- facial defect. (A-C) Mouse embryos were injected at E10.5 with a control adenovirus encoding lacZ then collected 24 hours later and stained for ß-gal activity. Lateral and frontal views show widespread X-Gal staining, indicating widespread, unifrom adenoviral infection in the lateral nasal (l), frontonasal (f), maxillary (mx) and mandibular (mn) prominences. (D,E) Non-injected or (F,G) Ad-Dkk1-injected embryos were collected at E13.5 (i.e. 96 hours after injection at E9.5). (D,E) Control embryos show characteristic Wnt reporter activity in the face and limb buds; note activity in the apical ectodermal ridge (aer) and skeletal condensations of the forelimb (yellow asterisks). (F,G) Ad-Dkk1 treatment truncates Wnt-dependent forelimb and digit growth (inset); note corresponding reduction in Wnt reporter activity. (H-K') Comparison between control and Ad-Dkk1-treated faces. (H,H') Control embryos exhibit normal facial morphology and undisturbed boundaries of Wnt reporter activity in the face and whisker buds. (I-K') Ad-Dkk1-treated embryos show an increasingly severe facial malformation that parallels the reduction in Wnt reporter activity. For example, the width of the frontonasal prominence is variably expanded (compare red brackets) owing to a reduction in growth of the maxillae. The more severe phenotypes correspond to the most dramatic reduction in Wnt reporter activity, so that the maxillary (mx) prominences are smaller and the frontonasal (f) is concomitantly larger. The reduction in X-Gal staining is clearly visible in Ad-Dkk1-treated whisker primordia. (L,M) Transverse sections through embryos collected at E13.5 (96 hours after injection at E9.5). Control (L, non-injected) nasal capsules show normal fusion of the facial prominences. In Ad-Dkk1-treated embryos (M), the reduction in maxillary growth is sometimes associated with facial clefting. Scale bars: white, 1 mm; black, 500 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 7. Wnt responsiveness predicts differential growth of the facial prominences. (A) In the E12.0 mouse face, growth of the frontonasal prominence relative to the lateral nasal (l) and maxillary (mx) prominences produces a midline furrow (dotted yellow line). (B) In chick, the converse is seen, where the frontonasal prominence grows at a faster rate than the maxillary and lateral nasal prominences, thus producing a pointed beak. (C,D) Adipose-derived mouse mesenchymal cells infected with a 7xTcf-eGFP lentivirus and incubated in control medium do not express the GFP reporter (C), but addition of purified Wnt3a protein activates GFP expression (D). (E,F) In chick, injection of a control lenti-eGFP at St. 13 results in widespread infection by St. 34. GFP expression is scattered throughout the upper beak; E, frontal view; F, lateral view. et, egg tooth; np, nasal pit; e, eye. (G) In chick, injection with 7xTcf-eGFP at St.13 results in a robust, spatially restricted pattern of GFP expression in the midline of the frontonasal prominence by St. 25. (H) At St. 29 in chick, this same pattern of GFP expression is seen in the frontonasal midline. (I,J) The frontonasal midline of avian embryos is a site of continued growth that eventually results in the elongated upper beak. When Wnt signaling is inhibited in avians by Ad-Dkk1 injection at St. 13, the growth of the frontonasal midline is dramatically impeded (compare dotted yellow line in I and J). The lateral nasal and maxillary prominences are unaffected. Scale bars: 250 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 8. Model of Wnt-mediated signaling regulating species-specific facial morphogenesis. (A) At the phylotypic stage of development, murine and avian embryos have very similar facial features. (B,C) Our data suggest that species-specific patterns of Wnt responsiveness pre-date and predict regional growth within the facial prominences. For example, in embryos with an elongated frontonasal prominence (B), Wnt responsiveness (green) predominates in the midline, whereas in embryos with a compressed frontonasal region and expanded maxillary prominences (C), Wnt signaling (blue) dominates in the lateral regions and is absent from the midline. (D,E) Wnt responsiveness is predictive of areas of greater outgrowth. In avians (D), Wnt signaling is dominant in the frontonasal midline but in animals with muzzles or snouts (E), Wnt signaling is largely confined to the expanding lateral facial prominences. (F,G) When the facial prominences have assumed their species-specific morphology, Wnt signaling is maintained in regions of outgrowth. In avians (F), this area correlates to the apex of the upper beak, whereas in mice (G) it correlates to the expanding maxillary prominences.