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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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