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Fig. 7 Inhibition of Hh signaling impairs tenocyte regeneration. (A) Whole-mount mRNA in situ hybridization showing up-regulation of Hh target gene ptc2 at the injury site (asterisks) at 1 and 2 dpi. n = 10 of 10 (1 dpi) and 18 of 28 (2 dpi) embryos. (B) Images of ptc2:Kaede; col1a2NTR-mCherry embryos at 1 and 2 dpi showing Kaede+mCherry+ fibroblasts (arrowheads) near the injury site (asterisks). n = 17 of 27 (1 dpi) and 16 of 25 (2 dpi) embryos. (C) Experimental protocol for drug treatment and staining of injured embryos. Injured embryos were incubated in DMSO or cyclopamine (cyc.) before fixation and in situ hybridization for prelp at appropriate stages. (D) Tenocyte marker prelp expression in DMSO- and cyclopamine-treated embryos at 1 and 2 dpi. Ectopic prelp expression (arrowhead) outside the MTJ can be seen in DMSO-treated but not cyclopamine-treated fish at 2 dpi. (E and F) Quantification of staining area (E) and gap size (F) for drug-treated embryos as described in Fig. 2A. n = 16 (1 dpi) and 28 (2 dpi) DMSO-treated embryos; 13 (1 dpi) and 28 (2 dpi) cyclopamine-treated embryos. (G) Drug treatment protocol for live scxa:mCherry embryos in (H) to (J). (H) Representative images of injured MTJs from DMSO- and cyclopamine-treated embryos at 0 and 2 dpi. A notable gap between tenocytes (green) can be seen in cyclopamine- but not DMSO-treated embryos. (I and J) Number of new tenocytes generated at injured and uninjured MTJs (I) and mean gap between tenocytes at the injury site (J) from 0 to 2 dpi in DMSO- and cyclopamine-treated embryos. n = 32 (DMSO) and 28 (cyclopamine) embryos. All data are plotted as mean ± SEM. Statistics: Mann-Whitney U test [(E) and (F)]; Sidak’s multiple comparisons [(I) and (J)]. Significance: ns, P > 0.05; **P < 0.01; ****P < 0.0001. Asterisks denote injury site in all images. Scale bars, 100 μm (A), 50 μm [(B) and (D)], and 25 μm (H).