Fig. 6

Embryos from shh^+/- parent fish were injected with either dnPKA-GFP or EGFP mRNA, grown to 12-somite stage and in situ stained for patched1, target gene of Hh signaling, and cyclinG1, p53 target gene, and ApopTag staining was performed to characterize the level of apoptosis. (A) Injection of dnPKA-GFP led to a strongly increased and more widely spread expression of patched1 (category “high”) in 87,7+/-3,8% of injected embryos, the rest having normal wild-type or low shh^-/- mutant patched1 expression comparable to expression of patched1 in EGFP-injected embryos from shh^+/- parent fish (category “low”). (B) Expression of cyclinG1 in uninjected embryos from shh^+/- parent fish. Categories “high” and “low” indicate expression levels in shh^-/- mutant and wild-type embryos, respectively. (C) ApopTag staining of apoptotic cells in uninjected embryos from shh^+/- parent fish. Categories “high” and “low” indicate levels of apoptosis in shh^-/- mutant and wild-type embryos, respectively. (D) Statistical analysis of results from injections of dnPKA-GFP and EGFP mRNA into embryos from shh^+/- parent fish. All of EGFP-injected embryos had “low” expression of patched1, 25,3 +/- 1,15% (Mean +/- standard deviation) of them had “high” expression of cyclinG1 and 24,7 +/- 1,53% had “high” apoptosis levels. dnPKA-GFP mRNA injection led to “high” expression of patched1 in 87,7 +/- 3,8% of embryos and decreased expression of cyclinG1 and apoptosis levels: “high” proportions are 6 +/- 2% and 5,8 +/- 2,4%, respectively. The experiment was done 3 times and 60 embryos were used for each staining. Asterisks (***) on top of shh^-/- mutant bars indicate significant statistical differences of proportions (Fisher′s exact test, P-value<0,001).