(a) In situ hybridization of Sox9 and Wwp2 in limb buds of wild-type, Sox9flox/floxPrx1-Cre (E12.5) and Sox9flox/floxCol2a1-Cre (E16.5) mice. Scale bar, 500 μm. (b) Analysis of Wwp2 mRNA expression levels by quantitative RT–PCR (qRT–PCR) in ATDC5 cells. ATDC5 cells were infected with human Sox9-expressing adenovirus (left panel) or transfected with 150 nM of siRNA for Sox9 (right panel; mean ± s.d.; *P < 0.05; **P < 0.01; n = 5). (c) Luciferase promoter analysis using truncated forms of the Wwp2 promoter in the presence or absence of the Sox9 expression vector. HEK293 cells were transfected with the pW−1172 (nt −1172 to −64), the pW−790 (nt −790 to −64), the pW−391 (nt −391 to −64) or the pWM−790 plasmid, which includes a 2 bp substitution mutation, in the presence (black bars) or absence (white bars) of Sox9 expression vector. The value in brackets represents the Sox9-dependent increase in ratio (mean ± s.d.; n = 4). (d) The specific binding of Sox9 to the Wwp2 promoter element detected by ChIP assay using ATDC5 cells. DNA–protein complex was immunoprecipitated by anti-Sox9 antibody. (e) EMSA revealed the specific binding of Sox9 to the Wwp2 promoter element. comp., competitor (unlabelled probe). (f) Small and fused palate in zebrafish carrying morpholino-mediated knockdown of wwp2 at 6 dpf. Zebrafish embryos were stained with alcian blue and alizarin red. UIC, uninjected control; MO, morpholino-treated animals. Scale bar, 200 μm. (g, h) RNA rescue experiments for wwp2 knockdown in zebrafish using sox9a and/or sox9b. (g) Palatal malformation induced by wwp2 knockdown was partially rescued by the co-injection of sox9a and sox9b. Scale bar, 100 μm. (h) Single injection of sox9a or sox9b did not rescue the palatal malformation induced by wwp2 knockdown. UIC; n = 51, wwp2 MO + GFP; n = 142, wwp2 MO + sox9a; n = 111, wwp2 MO + sox9b; n = 109, wwp2 MO + sox9a/b; n = 92 (white, normal; black, partial rescue; dark grey, fused and/or small; light grey, death).