Figure 6.

PCNA immunolocalization in metronidazole-damaged Tg(zop:nfsB-EGFP)^nt19 fish. The regenerative response to rod photoreceptor ablation in the dorsal and ventral Tg(zop:nfsB-EGFP)^nt19 retina was examined by immunolabeling retinal cryosections for PCNA expression (magenta) with TO-PRO-3 counterstaining the nuclei (blue). A,I, B,J: Occasional PCNA expression was observed in the ONL of control retinas (A,I) and after 24 hours of metronidazole treatment (B,J), which corresponded to the rod precursor cells (arrowheads). C,K: After 24 hours of recovery, PCNA was detected in a small number of individual INL cells in the dorsal (C) and ventral (K) retina (arrows) and in the ONL. D,L, E,M, F,N: The number of proliferating INL cells increased at 48 (D,L) and 72 hours of recovery (E,M), with clusters of PCNA-positive neuronal progenitors visible at 72 hours (F) and 96 (N) hours of recovery. G,O: At 168 hours of recovery, the majority of the PCNA-positive cells had migrated to the ONL. H,P: By 28 days of recovery, the majority of PCNA-positive cells resided in the ONL at levels slightly above the undamaged control retinas. Q,R: The number of proliferating INL cells (Q) and cell clusters (R) were quantified over a 150-μm region lying approximately 250 μm from the dorsal (blue) and ventral (yellow) retinal margins. A repeated measures ANOVA analysis revealed that the effect of treatment was dependent on the time point. Therefore, we performed two-sample t-tests on individual time points and Bonferroni-corrected (0.05/ 10 tests = 0.005 as the effective α) to account for the multiple tests. Those time points that revealed a statistically significant difference were marked with an asterisk. Error bars represent the standard error of the mean. ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar = 20 μm in A (applies to A–P).