Abstract
Purpose :
In humans, many retinal diseases destroy photoreceptors causing permanent blindness. Human photoreceptors are never replaced but, in the injured zebrafish retina, photoreceptors fully regenerate 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 :
Following light-induced photoreceptor injury, qPCR and in situ hybridization were used to analyze expression of pre-miR-18a and mature miR-18a, respectively. To determine the function of miR-18a, cellular responses to photoreceptor injury were compared between wild type (WT) and miR-18a [CRISPR] mutant retinas. Fish were immersed in 5 mM Bromodeoxyuridine (BrdU) for one hour prior to sacrifice and eyes from different fish were prepared for histology at 3, 7, and 10 days post injury (dpi). Immunolabeling for BrdU and/or PCNA was used to compare cell proliferation and in situ hybridization was used to compare expression of the inflammatory molecules nfkb1 and nfkb2.
Results :
qPCR showed that expression of pre-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 showed that mature miR-18a expression follows a similar pattern. Immunolabeling for BrdU and PCNA showed that at 3 and 5 dpi, miR-18a mutant and WT retinas have similar numbers of proliferating cells but at 7 and 10 dpi, when regenerated photoreceptors have typically differentiated, miR-18a mutant retinas have 200% more proliferating cells. In zebrafish, inflammatory regulation is critical for neural regeneration. To determine if miR-18a regulates inflammation, in situ hybridization was used to compare expression of nfkb1and nfkb2 between mutant and WT retinas. Expression did not differ at 3 dpi but at 7 dpi, mutant retinas had greater expression of both molecules, indicating prolonged inflammation.
Conclusions :
These data indicate that following photoreceptor injury, miR-18a regulates the cell cycle in photoreceptor progenitors and suggest that miR-18a functions by suppressing inflammation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.