Figure 1. Suppression of miR-216a Is Required for MG Dedifferentiation and Proliferation during Retina Regeneration.

(A) Schematic for post-mitotic and dedifferentiated MG sorting. Adult zebrafish were dark adapted for 2 weeks and then exposed to constant intense light lesioning for 45 h. For post-mitotic MG isolation, GFP⁺ cells were sorted from dark adapted Tg(gfap:gfp) retinas. For dedifferentiated MG isolation, GFP⁺ cells were isolated from 45 h light lesioned Tg(1016tuba1a:GFP) retinas.

(B) Fold changes in miR-216a levels in FACS-purified MG were determined by qPCR. miR-216a is enriched in post-mitotic MG (GFP⁺) from undamaged retinas in Tg(gfap:gfp) fish. After 45 h of light damage, miR-216a is downregulated ~5-fold in dedifferentiated MG (GFP⁺) in Tg(1016tuba1a:gfp) fish. miR-216a expression did not change in non-MG cells (GFP⁻) during regeneration. Data are from 5 independent experiments with 3 technical replicates of qPCR. MG were purified from 18 and 20 light-damaged fish in each experiment. Error bars represent the SEMs. *p < 0.05 and **p < 0.01 using 2-way ANOVA with Fisher’s least significant difference (LSD) post hoc test.

(C) Control miRNA (siRNA against luciferase [siLuc]) or miR-216a was injected and electroporated into the left eyes of Tg(1016tuba1a:gfp) zebrafish before intense light exposure (0 h). After 45 h, retinas were collected, sectioned, and immunostained using antibodies against GFP and PCNA. Nuclei were counterstained with TOPRO (blue). miR-216a gain of function abolished tuba1a:GFP transgene expression and significantly reduced the number of INL PCNA⁺ proliferating cells.

(D) Quantification of PCNA⁺ cells in the INL and ONL. Error bars represent the SEMs (n = 5–6 fish); **p < 0.01 using Student’s t test.

(E) Overexpression of miR-216a reduced the number of GFP⁺/PCNA⁺ proliferating progenitor cells after 60 h of intense light damage using Tg(1016tuba1a:gfp) zebrafish.

(F) Quantification of total GFP⁺ and PCNA⁺ cells. Error bars represent the SEMs (n = 10 fish); *p < 0.03 and **p < 0.003 using Student’s t test.

GCL, ganglion cell layer; INL, inner nuclear layer; ns, not significant; ONL, outer nuclear layer.

Scale bars, 50 µm.