Fig. 6

Fig. 6 Wnt2bb signaling induces Yap expression to regulate chondrogenic precursor proliferation as well as chondrocyte maturation during pharyngeal cartilage development. A?C: Yap levels in pharyngeal arch 1 of WT and wnt2bb?/? embryos with a Tg(fli1:EGFP) background at 3 dpf. Fixed embryos were stained with anti-YAP1 and anti-GFP antibodies. Nuclei were labeled with DAPI. The relative fluorescence intensity of Yap in PA1 was quantified in B (n = 5). The nucleocytoplasmic (n/c) ratio of Yap in PA1 was shown in C (n = 6). D?F: Yap levels in PA2 at 3 dpf. The relative fluorescence intensity of Yap in PA2 of WT (n = 4) and wnt2bb?/? (n = 5) embryos was quantified in (E). The nucleocytoplasmic (n/c) ratio of Yap in PA2 was displayed in F (n = 6). G: Expression profile of yap1 in WT and wnt2bb?/? embryos at 36 hpf and 3 dpf. Numbers at the bottom right corner indicate the number of embryos with phenotypes shown out of the total number of embryos examined. H and I: WT embryos with a Tg(fli1:EGFP) background were exposed to 10 ?M verteporfin from 28 hpf to 5 dpf, when they were collected for live imaging (H). The chondrocyte volume for each group was quantified in I (n = 6). J?M: wnt2bb?/? embryos with a Tg(fli1:EGFP) background were treated with 10 ?M XMU-MP-1 or 2 ?M PY-60 from 28 hpf and then harvested for cell proliferation assay at 36 hpf (J) or live imaging at 5 dpf (L). Virtual puncta for BrdU incorporated cells in PA1 and PA2, and 3D reconstruction for individual chondrocytes in ceratohyal cartilages was generated using the Imaris software. The dotted lines and asterisks refer to individual cells subjected to in-depth analysis. The number of BrdU incorporated chondroprogenitors was quantified in K (n = 4). The chondrocyte volume for each group including WT (n = 7), untreated wnt2bb?/? (n = 7), wnt2bb?/? treated with XMU-MP-1 (n = 7), and wnt2bb?/? treated with PY-60 (n = 5) was quantified in M. ???, P < 0.001; ns, not significant (student's t-test). WT, wild-type; PA, pharyngeal arch; dpf, days post fertilization; hpf, hours post fertilization. Scale bars, 20 ?m (A, D, H, J, L); 100 ?m (G).