Fig. 8

S-phase progression in wild-type, shh^-/- and p53^-/-shh^-/- mutants was analysed at 49 and 57 hpg after one-hour labelling with BrdU. Confocal sections stained with anti-BrdU antibodies and DAPI are shown with the anterior side to the top. (A) Anti-BrdU/DAPI staining of wild-type, shh^-/- and p53^-/-shh^-/- mutant retinas at 49 hpf identified a greater extent of cell-cycle exit (fewer cells labelled) in the wild-type and p53^-/-shh^-/- mutant than in the shh^-/- retina. The data from these stainings were then used for quantification in (D). (B) At 57 hpf in the wild-type retina, BrdU-positive cells were found only in the ciliary marginal zone (CMZ). However, in the shh^-/- mutant retina, cells still failed to exit the cell cycle. Like in the wild-type retina, cells of p53^-/-shh^-/- mutant mostly exited the cell cycle and proliferative cells were located only in the CMZ. (C) At 38 hpf, p57kip2 expression was at a high level in both wild-type and p53^-/-shh^-/- mutant retinas, whereas no p57kip2 was present in the shh^-/- mutant retina. By contrast, p53 expression was high in the shh^-/- mutant retina and very low in the wild-type and p53^-/-shh^-/- mutant retinas. (D) Proportions of BrdU-positive cells (S-phase indices) in wild-type, shh^-/- and p53^-/-shh^-/- retinas at 49 hpf were obtained from confocal sections such as presented in (A). The S-phase indices in wild-type (34,1%, SD = 4,4%) and p53^-/-shh^-/- (39%, SD = 2,8%) were each significantly lower than in the shh^-/- mutant retina (53,8%, SD = 7%) (t-test, P-value<0,001), the difference between them still being significant (t-test, P-value<0,01). (E) S-phase indices in wild-type, shh^-/- and p53^-/-shh^-/- retinas at 57 hpf were obtained from confocal sections such as presented in (B). The S-phase indices in wild-type (17,1%, SD = 1,3%) and p53^-/-shh^-/- (19%, SD = 2,8%) were not significantly different (t-test, P-value>0,05), but were each significantly lower than in the shh^-/- mutant retina (55,8%, SD = 5%) (t-test, P-value<0,001). Total 10 sections from 5 embryos (2 retinal sections per embryo) were analysed for each genotype and mean +/- standard deviation are indicated above the graph bars. Statistical differences were evaluated using t-test with P-values<0,01 (**) or<0,001 (***).