Generation of all neuronal cell types and expression of cell type specific developmental competence factors in the light-damaged retina. (Aa–d) Single z-plane confocal images of retinal sections from light-damaged EdU-injected albino zebrafish (Aa,c,d) at 7 days of recovery (drec) immunolabeled for the amacrine/ganglion cell marker, HuC/D (Ab–d), and counterstained with the nuclear dye, DAPI (Ad). Yellow arrowhead, GCL EdU-positive ganglion or amacrine cell; yellow arrow, INL EdU-positive amacrine cell; white arrowhead, EdU-positive and HuC/D-negative cell in the apical INL, the region where bipolar cells reside; white arrow, EdU-positive cell in the cone nuclear layer. Insets represent the EdU-positive cells in panels (Aa–d) (arrows; yellow arrowhead) displayed at higher magnification. Scale bar, 20 μm in panel (Aa). (B) Schematic of the experimental paradigm: albino zebrafish were exposed to constant intense light for 96 h and subsequently recovered under normal light conditions until 7 drec. Fish were intraperitoneally injected with EdU at 24, 36, 48, 60, 72, 84, 96 hLT and at 1 and 2 drec (red arrows). AC, amacrine cell; C, cone photoreceptor cell; GC, ganglion cell; GCL, ganglion cell layer; hLT, hours of light treatment; INL, inner nuclear layer; ONL, outer nuclear layer; R, rod photoreceptor cell. (C–I) Line plots displaying mRNA expression levels as log2-fold changes relative to 0 h controls for proliferation marker, pcna(C) and genes required for the developmental specification of retinal neurons (D–I) during light damage-induced retinal regeneration (0, 36, 48, 60, 72, 84, 96 hLT, 2, 5, 7 drec): (D)atoh7 (ganglion cells); (E)ptf1a (amacrine/horizontal cells); (F)otx2 (bipolar and photoreceptor cells); (G)vsx1 (bipolar cell); (H)prdm1a (photoreceptor cell); (I)nrl (gray line, rod photoreceptor cell specification) and mature rod photoreceptor cell gene, rhodopsin (black line).
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