Fig. S7

Fig. S7

BMP signaling is essential for bone formation. (A-D) Immunostaining of a 48 hpa fin section with pSmad 1/5/8 (green) and Runx2 (red) antibodies. The red arrow points to Runx2+/pSmad- pre-osteoblasts localized in the distal blastema. Yellow arrows indicate Runx2^low/pSmad+ cells. Nuclei are stained with Hoechst (blue) and the dashed line is the amputation plane. The scale bar is 50 μm. (E-H) Fin regeneration in Tg(sp7:EGFP) animals treated with DMSO (E and F) or BMPRi (G and H, 5 μM) from 0-4 dpa. Shown are representative whole-mount Rotterman contrast (E and G) and epifluorescent images (F and H) from one of the three animals in each group. Yellow arrows mark osteoblasts and the amputation site is indicated with a dashed line. (I-L) Von Kossa staining of frozen sections to detect calcified bone in control (I and J) and BMPRi-treated (K and L) fish. Black arrows point to calcified tissue (stained black) distal to the site of amputation (dashed yellow line); blue arrows point to regions rich in mucopolysaccharides (stained blue). The regions bounded by boxes are shown in higher magnification in the adjacent panel. Shown are representative examples from one of three fish for each treatment. (M and N) col10a1 in situ hybridization on DMSO (M) and BMPRi treated (N) fins. Red arrows point to regions of gene expression stained blue. (O and P) Immunostaining of sections from Tg(sp7:EGFP) animals treated with DMSO (O) or 5 μM BMPRi (P) from 0-72 hpa) to demonstrate Runx2 (white) and sp7:EGFP (green) expression and levels of pSmad1/5/8 (red). Hoechst-stained nuclei are shown in blue. White arrows indicate Runx2+ cells and yellow arrows show sp7:EGFP+/pSmad+ cells. The dashed yellow line indicates the amputation site. A representative section from one of three fish in each group is shown. Three or more rays were examined for each animal. (Q) EdU incorporation in osteoblast sub-types under DMSO or BMPRi conditions. No significant difference was observed (p > 0.05, two-tailed Student’s t-tests). (R-U) Runx2 (red) and pSmad1/5/8 (green) immunostaining of primary cultured fin osteoblasts. Hoechst stained nuclei are shown in blue and the yellow arrows point to Runx2+ osteoblasts displaying pSmad1/5/8 immunoreactivity. For each treatment, at least 400 osteoblasts were examined from more than six comparable sections compiled from three different animals. (V) qRT-PCR analysis demonstrating relative transcript levels of bmp2a, bmp2b, bmp4 and bmp6 in cultured fin osteoblasts. Equal amounts of template were used for each reaction and relative expression levels were determined by normalizing to rpl8 expression. Mean relative expression levels from three independent cultures are shown and the error bars indicate one standard deviation.