Abstract
Purpose :
In humans, many retinal diseases destroy photoreceptors causing permanent blindness. In the injured zebrafish retina, however, injured photoreceptors are fully regenerated to restore vision. Understanding the mechanisms that govern photoreceptor regeneration in zebrafish may be key to understanding the requirements to therapeutically regenerate human photoreceptors. MicroRNAs (miRNAs) are small ~22 base molecules that regulate molecular expression and little is known about the roles of miRNAs in photoreceptor regeneration. The purpose of this study was to determine the role of the miRNA miR-18a in photoreceptor regeneration.
Methods :
At key time points following light-induced photoreceptor injury, in-situ hybridization was used on transgenic Tg(gfap:egfp) and Tg(mpeg1:egfp) fish lines to determine the cellular expression of miR-18a. This was used along with qPCR, immunolabeling for proliferating cells (BrdU and/or PCNA) and in situ hybridization for mature photoreceptors, in both wild type (WT) and miR-18a mutant retinas, to spatiotemporally determine miR-18a expression and function. In miR-18a mutants compared with WT, both inflammation and progenitor cell proliferation are prolonged. To determine if miR-18a functions through inflammatory regulation, dexamethasone (2.5mM in 0.1% DMSO) was used to block inflammation from 4-5 and 5-6 days post-injury (dpi) to attempt to rescue the miR-18a mutant phenotype.
Results :
In situ hybridization in Tg(gfap:egfp) and Tg(mpeg1:egfp) fish showed that by 1 dpi, miR-18a is expressed in Müller glia (MG) and by 3 dpi is expressed in both MG and MG-derived photoreceptor progenitors, but is not expressed in microglia. qPCR indicates that miR-18a increases 5-fold by 3 dpi and 7-fold by 5 dpi, corresponding to peak progenitor proliferation and the beginning of photoreceptor differentiation, respectively. In situ hybridization at 14 dpi showed that miR-18a mutants have more regenerated cones (but not rods) than WT. Finally, treatment of miR-18a mutants with dexamethasone partially rescues the mutant phenotype (excess progenitor proliferation), indicating that miR-18a functions, in part, by regulating inflammation.
Conclusions :
These data indicate that following photoreceptor injury among photoreceptor progenitors, miR-18a regulates the cell cycle and cone photoreceptor differentiation and indicate that miR-18a functions partially by suppressing inflammation.
This is a 2020 ARVO Annual Meeting abstract.