FIGURE 4

Amacrine and horizontal cell competence factor ptf1a is upregulated in the light-damaged retina. (Aa–aj) Single z-plane confocal images of retinal sections from light-damaged Tg[ptf1a:EGFP]jh1 zebrafish (0, 48, 60, 72, 84, 96 hLT) immunolabeled for PCNA (Aa–f,As–ad), GFP (Ag–l,As–aj), HuC/D (Am–x,Aae–aj) and counterstained with DAPI (As–x). (Ay–aj) Regions outlined in panels (As–x) at higher magnification. Arrowhead, ptf1a:EGFP-positive ONL cell with an elongated morphology. Yellow arrows, ptf1a:EGFP, PCNA and HuC/D-triple positive cell. Red arrows, ptf1a:EGFP and HuC/D-double positive cell that is PCNA-negative. (B–D) Number of PCNA-positive, ptf1a:EGFP-positive and PCNA and ptf1a:EGFP-double positive cells in the INL (B), ONL (C), and GCL (D) over the light treatment timecourse. (E–G) Number of ptf1a:EGFP-positive, ptf1a:EGFP and PCNA−double positive cells in comparison to ptf1a:EGFP and HuC/D-double positive and PCNA and HuC/D-double positive cells in the INL (E), ONL (F), and GCL (G) of retinas exposed to constant intense light for 0, 36, 48, 60, 72, 84 and 96 h. Mean ± SE, n ≥ 10, *p_Tukey < 0.05 and ^#p < 0.05 indicate comparisons to 0 hLT for the different measures that were assessed. The symbols are color-coded according to the line that they represent in the corresponding graphs (p_ANOVA, see Table 1). Note, significance was not determined for PCNA in panels (B–D) and symbols indicating significance for ptf1a:EGFP and ptf1a:EGFP and PCNA-double-positive cells are not shown in panels (E-G), as they are indicated in panels (B–D). (Ha–c) Maximum projections of five confocal z-sections at the level of the inner plexiform/amacrine cell layer in light-damaged Tg[ptf1a:EGFP]jh1 dorsal retinal flatmounts at 96 hLT. Newly generated ptf1a:EGFP-positive amacrine cells (Ha,c) identified by EdU [(Hb,c); arrows], which was intraperitoneally injected during the proliferative phase, display neurite outgrowth (arrowheads). The images are representative of three independent experiments. (I) Higher magnification confocal images of the horizontally elongated ptf1a:EGFP-positive ONL cell at 96 hLT in panel (Al) (arrowhead), which potentially represents a newly generated horizontal cell. Confocal images also display HuC/D (Ia,b), PCNA (Ib) and DAPI (Ib). Scale bars, 20 μm (Aa, Ha) and 10 μm (Ay, Ia).