Background: Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs were examined for their potential roles in regulating zebrafish retinal regeneration. Results: To investigate the requirement of miRNAs during zebrafish retinal regeneration, we knocked down the expression of Dicer in retinas prior to light-induced damage. Reduced Dicer expression significantly decreased the number of proliferating Müller glia-derived neuronal progenitor cells during regeneration. To identify individual miRNAs with roles in neuronal progenitor cell proliferation, we collected retinas at different stages of light damage and performed small RNA high-throughput sequencing. We identified subsets of miRNAs that were differentially expressed during active regeneration but returned to basal levels once regeneration was completed. We then knocked down five different miRNAs that increased in expression and assessed the effects on retina regeneration. Reduction of miR-142b and miR-146a expression significantly reduced INL proliferation at 51 hours of light treatment, while knockdown of miR-7a, miR-27c and miR-31 expression significantly reduced INL proliferation at 72 hours of constant light. Conclusions: miRNAs exhibit dynamic expression profiles during retinal regeneration and are necessary for neuronal progenitor cell proliferation.

Damage of the zebrafish retina triggers a spontaneous regeneration response that is initiated by Müller Glia (MG) dedifferentiation and asymmetric cell division to produce multipotent progenitor cells. Subsequent expansion of the progenitor pool by proliferation is critical for retina regeneration. Pax6b expression in the progenitor cells is necessary for their proliferation, but exact regulation of its expression is unclear. Here, we show that miR-203 is downregulated during regeneration in proliferating progenitor cells. Elevated miR-203 levels inhibit progenitor cell expansion without affecting MG dedifferentiation or progenitor cell generation. Using GFP-reporter assays and gain and loss of function experiments in the retina, we show that miR-203 expression must be suppressed to allow pax6b expression and subsequent progenitor cell proliferation.

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SUMMARYUnlike the adult mammalian retina, Müller glia (MG) in the adult zebrafish retina are able to dedifferentiate into a ‘‘stem cell’’-like state and give rise to multipotent progenitor cells upon retinal damage. We show that miR-216a is downregulated in MG after constant intense light lesioning and that miR-216a suppression is necessary and sufficient for MG dedifferentiation and proliferation during retina regeneration. miR-216a targets the H3K79 methyltransferase Dot1l, which is upregulated in proliferating MG after retinal damage. Loss-of-function experiments show that Dot1l is necessary for MG reprogramming and mediates MG proliferation downstream of miR-216a. We further demonstrate that miR-216a and Dot1l regulate MG-mediated retina regeneration through canonical Wnt signaling. This article reports a regulatory mechanism upstream of Wnt signaling during retina regeneration and provides potential targets for enhancing regeneration in the adult mammalian retina.