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Fig. S1. Palatogenesis in Wnt9b−/− mice. (A-C) No cell proliferation defect in the palatal shelves of Wnt9b−/− embryos at E13.5. BrdU-positive cells within the palatal shelves (dotted boxes) were counted in three independent embryos. (D,E) Vertical growth of the palatal shelves in E13.5 embryos. No obvious defect was detected in Wnt9b−/− mice. (F-J) In vitro palatal shelf fusion assay. (F) Schematic of palatal shelf explant culture. (G-J) Wild-type (n=4) and Wnt9b−/− (n=4) palatal shelves isolated from E13.5-14.0 embryos were cultured for up to 48 hours. All palatal shelves from Wnt9b−/− mice fused normally (n=4/4), similar to wild-type shelves (n=4/4). Complete disappearance of epithelium on the midline (red dotted lines) was observed. High-magnification images of G and H are shown in I and J, respectively. mes, mesenchyme; ps, palatal shelf; se, surface ectoderm; t, tongue.
Fig. S2. Measurement of skull dimensions. The length (A) and width (B) of the skull and the lengths of the upper jaw (C) and mandible (D) are presented as mean±s.e.m. Statistical significances were determined by Student�s t-test. N.S., not significant.
Fig. S3. No apparent defects in cell survival and fusion in the NPs and MxP of Wnt9b−/− embryos. (A,B) Cell apoptosis was examined by immunostaining for active caspase 3 at E10.5. Arrows indicate apoptotic cells in the epithelial seam in wild-type but not in mutant embryos. (C,D) Coronal section of wild-type and Wnt9b−/− upper facial tissue at E11.5. Whereas the NPs and MxP fused completely in wild-type embryos, the LNP and MNP remained separated in Wnt9b−/− mutants. (E) The NP/MxP tissues isolated from Wnt9b−/− embryos were successfully fused after 36 hours in explant culture. Note the absence of epithelial cells (red arrows) within the fusion area, indicating that fusion occurs normally. lnp, lateral nasal process; mnp, medial nasal process; mxp, maxillary process.
Fig. S4. Gene expression analysis by qRT-PCR. (A) Ex vivo activation of WNT/β-catenin signaling in the NP and MxP explants by WNT9B protein. The NP/MxP explants were cultured in the presence of WNT9B protein for 24 hours before RNA isolation. (B) In vivo activation of WNT/β-catenin signaling by LiCl in wild-type and Wnt9b−/− embryos. The NP and MxP tissues were dissected from embryos and processed for RNA isolation. Statistical significances were determined by Student�s t-test (*P<0.05).
Fig. S5. Bmp4 and Shh expression in the face of Wnt9b−/− embryos. (A-F) No significant differences in Bmp4 and Shh gene expression were detected in both wild-type and Wnt9b−/− embryos. mdp, mandibular process of branchial arch 1; lnp, lateral nasal process; mnp, medial nasal process; mxp, maxillary process.
Fig. S6. Mesenchymal cell migration. Wound-healing (A-D) and Transwell (E) cell migration assays. Cell migration was measured at 16 hours (wound healing assay) and 8 hours (Transwell assay), respectively, after recombinant WNT9B and FGF proteins were added to the culture.