Fig. 3

Ihha promotes the efficient calcification of regenerated bone by non-canonical Hedgehog signaling. (A-F) Whole-mount Kaede fluorescence images of 5 dpa caudal fins from ptch2:Kaede (A-C) and ihha^−/−;ptch2:Kaede (D-F) fish 24 h after photoconversion. Photoconverted pre-existing Kaede is magenta; Kaede produced after photoconversion is green. Magenta arrows indicate extreme distal epidermal tissue exclusively expressing converted Kaede. The white brackets indicate the domain of newly expressed Kaede protein. (G,H) Whole-mount images showing GFP-expressing osteoblasts in fins from sp7:EGFP and ihha^−/−;sp7:EGFP fish at 11 dpa. Yellow arrows mark newly formed joints. Red lines and arrows denote points of ray bifurcation. (I,J) Bright-field images of Alizarin Red-stained sp7:EGFP(I) and ihha^−/−;sp7:EGFP (J) fins at 11 dpa. Black and red brackets show the total length of the regenerate and the extent of mineralization from the site of amputation, respectively. Yellow dashed lines in all panels show amputation positions. (K) Quantification of the relative extent of calcified regenerated bone in sp7:EGFP versus ihha^−/−;sp7:EGFP fish at 5, 8 and 11 dpa. Means and data points representing individual fish are shown. Significant differences between control and ihha-null fish (P<0.05) were determined by two-tailed Student's t-tests. Scale bars: 500 μm.