Expression of developmental competence factors and generation of all neuronal cell types following rod photoreceptor cell death in a genetic ablation model. (A–E) Line plots displaying mRNA expression levels expressed as log2-fold changes relative to 0 h controls for pcna(A), atoh7(B), ptf1a(C), prdm1a(D), nrl (E, gray line), and rhodopsin (E, black line) following metronidazole-induced rod photoreceptor cell death in Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish (24, 48, 72, 96, and 120 h after metronidazole treatment onset). Mean ± SE, n ≥ 3. (F) Schematic of the experimental paradigm: Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish were either exposed to metronidazole (mtz) or system water (H2O) for 24 h and subsequently recovered in system water for 10 days (10 drec). Intraperitoneal EdU injections at the indicated timepoints (red arrows). (Ga–h,H,L) Single z-plane confocal images of retinal sections from metronidazole or water-exposed EdU-injected Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish (Ga,b,g,h) at 10 drec that were labeled for GFP (Gc,d,g,h), HuC/D (Ge–h), and DAPI (Gg,h). Arrowhead, GCL EdU-positive ganglion/amacrine cell; yellow arrow, INL EdU-positive amacrine cell; white arrow, EdU-positive cell in the cone nuclear layer. Scale bar, 20 μm (Ga). (H) EdU-positive cells in panel (Ab) (arrows, white arrowhead) at higher magnification. (I) Number of EdU-positive cells in the cone nuclear layer, apical and basal INL and those identified as rod photoreceptor cells by co-labeling with rho:Eco.NfsB-EGFP, as amacrine and ganglion cells based on the expression of HuC/D in the basal INL or GCL, respectively, at 10 drec following exposure of Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish to either system water (control) or metronidazole for 24 h. Mean ± SE, n ≥ 9, Student’s t-test, p < 0.05. (J,K) Single z-plane confocal images from water or metronidazole-exposed EdU-injected (Ja,b,e,f,Kc–f)Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish at 10 drec that were labeled for Zpr-1 (Jc–f) or PKCα (Ka–f) and counterstained with DAPI (Ke,f). Yellow arrowheads (J), Zpr-1 & EdU-double positive cells; white arrowhead (K), PKCα & EdU-positive cell. (L) EdU & HuC/D-double positive ONL cell (yellow arrowhead) in panel (G) at higher magnification. aINL, apical inner nuclear layer; bINL, basal inner nuclear layer; CL, cone nuclear layer; RL, rod nuclear layer; mtz, metronidazole.
|
