ABSTRACT
In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that fully regenerate photoreceptors. MicroRNAs (miRNAs) regulate neurogenesis in the CNS, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miRNA miR- 18a regulates photoreceptor differentiation. The purpose of the current study was to determine in zebrafish the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization and immunohistochemistry showed that miR-18a expression increases throughout the retina by 1-day post-injury (dpi) and increases through 5 dpi. To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with bromodeoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess Müller glia-derived progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that during photoreceptor regeneration in zebrafish, miR-18a regulates proliferation and photoreceptor regeneration by regulating the inflammatory response.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1) A morpholino electroporation experiment has been used to knock down miR-18a and confirm the miR-18a mutant phenotype 2) A complete analysis of microglia behavior during photoreceptor regeneration in miR-18a mutant and wild type retinas has been performed The new results show that, in the absence of miR-18a, the microglia response to photoreceptor injury is prolonged, indicating that miR-18a indirectly regulates microglia.
