(a) The structure of the genomic Sox9 locus, targeting vector and targeted allele for Sox9-3′ EGFP knock-in mouse is indicated. Exons are depicted as closed boxes and intronic sequences are shown as solid lines. DNA fragments detected by Southern blot analysis are indicated by double arrows, with the restriction enzymes and the probe. The IRES-EGFP-pA/loxP-flanked PGK-neo-bpA cassette is depicted as open boxes. B, BamHI; P, Pst1; E, EcoRI; X, XbaI; Bg, BglII; H, HpaI. (b) Southern blot analysis of genomic DNA. Genomic DNA isolated from ES cell clones was digested with BglII and was then hybridized with the 3′ probe. The wild-type and mutant alleles were detected as 3 kb and 6.5 kb fragments, respectively. (c) EGFP expression in E9.5, E13.5 and E16.5 Sox9-3′ EGFP knock-in embryos. EGFP expression was detectable in a Sox9-specific pattern during embryogenesis. Scale bars, 500 μm (E9.5); 2 mm (E13.5) and 3 mm (E16.5). (d) The structure of the genomic Sox9 locus, targeting vector and targeted allele for Sox9-EGFP/EGFP null chimera is indicated. DNA fragments detected by Southern blot analysis are indicated by double arrows. (e) Southern blot analysis of genomic DNA. Genomic DNA isolated from ES cell clones was digested with EcoRI and was then hybridized with the 3′ probe. Wild-type and mutant alleles were detected as 7.1 kb and 5.8 kb fragments, respectively. (f) Southern blot analysis of the neodeleted Sox9 EGFP clones after Cre recombination. The neo-deleted Sox9 EGFP allele was detected as a 4.2 kb fragment. (g) Southern blot analysis of the neo-deleted Sox9 EGFP/EGFP-neo clones. (h) Dot plot cytograms show enrichment of EGFP-positive cells in Sox9-3′ EGFP knock-in embryos (top panel) and Sox9-EGFP/EGFP null chimeras (bottom panel). Only cells from a P1 population showing high green fluorescence were sorted. (i) RT–PCR analysis of Sox9 expression using total RNA extracted from sorted EGFP-positive cells of Sox9-3′ EGFP knock-in embryos and Sox9-EGFP/EGFP null chimeras.

(a) In situ hybridization of Sox9 and Wwp2 in limb buds of wild-type, Sox9^flox/floxPrx1-Cre (E12.5) and Sox9^flox/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).

(a) Wwp2 interacted physically with endogenous Sox9 as demonstrated by IP assay using ATDC5 cells. Myc-tagged Wwp2 was transfected into ATDC5 cells and total cell lysates were immunoprecipitated 48 h after transfection using rabbit IgG (negative control) or an anti-Sox9 antibody, followed by immunoblotting with an anti-myc antibody. (b) Schematic images of Sox9 deletion mutations. Sox9 contains a high-mobility group domain (HMG, residues 104–182); a proline, glutamine and alanine domain (PQA, residues 339–379); and a proline, glutamine and serine-rich domain (PQS-rich, residues 402–509). (c) Schematic images of Wwp2 deletion mutations. Wwp2 contains a calcium-dependent membrane targeting domain (C2, residues 19–115), four tryptophan-based WW domains (WW, residues 300–477) and a carboxy terminus domain that is homologous to that of E6-AP (HECT, residues 525–870). (d) Co-immunoprecipitation assays between myctagged Wwp2 and Flag-tagged deletion mutations of Sox9. GFP expression plasmid was used as a negative control. IB, immunoblotting; In, input. (e) Co-immunoprecipitation assays between HA-tagged Sox9 and V5-tagged deletion mutations of Wwp2. GFP expression plasmid was used as a negative control. (f) Immunohistochemical analysis of Sox9 and Wwp2 in wild-type mouse limbs at E16.5 (DAB staining). Pz, proliferative zone; Pre- Hz, prehypertrophic zone; Hz, hypertrophic zone. Scale bar, 100 μm. (g) Nuclear translocation of Wwp2 in the presence or absence of exogenously overexpressed Sox9 in C3H10T1/2 cells. The nuclear translocation of Wwp2 was associated with Sox9. Scale bar, 20 μm. (h) Luciferase reporter assay using the p89/4×48 Col2a1 reporter plasmid or Col11a2 reporter plasmid in C3H10T1/2 cells. The reporter activities increased in a Wwp2 dose-dependent manner (mean ± s.d.; n = 5).

(a) Verification of the physical interaction between Wwp2 and Med25 by a yeast two-hybrid assay. The AH109 yeast strain was co-transformed with the pGADT7 vector expressing full-length Med25 and the pGBKT7-Lam vector (negative control; left dish), or the pGBKT7 vector expressing full-length Wwp2 (right dish) was cultured on SD/ – Ade/ – His/ – Leu/ – Trp/X-α-Gal high-stringency plates. (b) Immunohistochemical analysis of Med25 in a wild-type mouse limb at E16.5 (DAB staining). Scale bar, 100 μm. (c) Whole-mount in situ hybridization of med25 in zebrafish at 1 dpf (top panel) and 2 dpf (bottom panel). Scale bar, 500 μm. (d) Morpholino-mediated knockdown of med25 in zebrafish induced palatal malformation. Three different kinds of palatal morphological malformations (med25 MOs1–3) were observed. Scale bar, 100 μm. (e) Med25 interacted physically with endogenous Sox9 detected by IP assay using ATDC5 cells. HA-tagged Med25 was transfected into ATDC5 cells and a total cell lysate was immunoprecipitated 48 h after transfection using rabbit IgG (negative control) or an anti-Sox9 antibody, followed by immunoblotting with an anti-HA antibody. (f) Schematic images of Med25 deletion mutations. Med25 contains a von Willebrand factor type A domain (VWA, residues 1–228), a synapsin I domain 1 (SD1, residues 229–381), a conserved region found in prostate tumour overexpressed protein 1 domain (PTOV, residues 395–545), an SD2 domain (residues 554–731) and an NR box (residues 646–650). (g–j) Co-immunoprecipitation assays using deletion mutations of Sox9, Wwp2 or Med25. GFP expression plasmid was used as a negative control. (g) Immunoprecipitation between HA-tagged full-length Med25 and V5-tagged deletion mutations of Wwp2. (h) Immunoprecipitation between V5-tagged full-length Wwp2 and HA-tagged deletion mutations of Med25. (i) Immunoprecipitation between Flag-tagged full-length Sox9 and HA-tagged deletion mutations of Med25. (j) Immunoprecipitation between HA-tagged full-length Med25 and Flag-tagged deletion mutations of Sox9.

(a) Luciferase reporter assay using the p89/4×48 Col2a1 reporter and Col11a2 reporter plasmid in C3H10T1/2 cells. Wwp2-CA includes a mutation in the HECT domain of Wwp2. (mean ± s.d.; n = 5). (b, c) Knockdown of Wwp2 (b) or Med25 (c) using siRNAs led to the downregulation of Col2a1 transcription in C3H10T1/2 cells detected by qRT–PCR. (mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001; n = 4). (d) Downregulation of Col2a1 induced by knockdown of Wwp2 or Med25 in C3H10T1/2 cells was not restored by exogenous Sox9 overexpression. (mean ± s.d.; Dunnett’s test, **P < 0.01; ***P < 0.001; n = 5). (e) EMSA using nuclear protein from C3H10T1/2 cells that were co-transfected with Flag-tagged Sox9, myc-tagged Wwp2 and HA-tagged Med25. The 48-bp Col2a1 enhancer oligonucleotide probe was used. Nuc-protein, nuclear protein. (f) ChIP assay detected recruitment of Sox9, Wwp2 and Med25 to the chondrocyte-specific enhancer region of the Col2a1 gene. Cell lysate extracted from C3H10T1/2 cells that were co-transfected with Flag-tagged Sox9, myc-tagged Wwp2 and HA-tagged Med25 was immunoprecipitated with antibodies for each tag. (g) Downregulation of Wwp2 led to the attenuation of the physical binding between Sox9 and Med25. The Wwp2 siRNA was initially transfected into ATDC5 cells and HA-tagged Med25 was additionally transfected 24 h after transfection with Wwp2 siRNA. Nuclear protein was immunoprecipitated with an anti-Sox9 antibody, followed by immunoblotting with an anti-HA antibody. (h) Model describing the mechanism underlying the physical and functional interaction between Sox9, Wwp2 and Med25. Sox9 regulates the transcription of Wwp2 and mediates the nuclear translocation of Wwp2, resulting in the formation of a transcriptional complex, including RNA polymerase II and Sox 5/6, which regulates the expression of the Col2a1 gene.