Fig. 5

Ostn regulates osteogenesis through Yap1/Wwtr1. (A) runx2 and sp7 promoter activity monitored by EGFP in the ps of Tg(Hsa.RUNX2:GAL4FF-2A-mCherry);(UAS:EGFP) larvae (top) and those of Tg(Ola.sp7:Gal4FF-2A-mCherry);(UAS:EGFP) (bottom) treated with vehicle (left) or ostn MO (right). Confocal images are of larvae stained with Alizarin Red at 5 dpf. (B) Illustration of how Tg fish enable detection of nuclear translocation of Yap1/Wwtr1 by EGFP expression in tissues where the sp7 promoter is active. (C) Confocal images of the ps of Tg larvae injected with vehicle (top) or ostn MO (bottom) and stained with Alizarin Red at 6 dpf. Arrows indicate EGFP-positive cells, which correspond to those in which Yap1/Wwtr1 enter the nucleus. (D) Quantitative analysis of C. The intensity of the EGFP-positive area in the ps is plotted. The number of EGFP-positive areas quantified is indicated at the top. (E) Representative images of Alizarin Red staining of Tg(Ola.sp7:Gal4FF-2A-mCherry) larvae at 7 dpf transiently transfected with the plasmids indicated on the left. Lateral view (left) and ventral view (right), anterior to the left. Arrows and dashed lines indicate the tip and the top of concave of the caudal part of the ps, respectively. (F) Quantitative analyses of ps length of the larvae in E. The length of ps was measured as in Fig. 3E. (G) Representative images of Alizarin Red staining of the ps of Tg(Ola.sp7:GAL4FF-2A-mCherry);(UAS:EGFP-ytip) larvae at 5 dpf. (H) Quantitative analyses of G. ps length relative to the mean length of the wild-type ps was plotted. *P<0.05, **P<0.01. Error bars indicate s.e.m. Scale bars: 50 µm in A,C,G; 200 µm in E.