Abstract
Purpose :
In humans, retinal disease can permanently destroy photoreceptors, but in zebrafish (Danio rerio), following retinal injury, functional photoreceptors are regenerated from intrinsic stem cells (Müller glia) in about 14 days. MicroRNAs (miRNAs) are short, non-coding RNA sequences, and recent studies show critical roles for miRNAs in photoreceptor development and regeneration. The miRNA miR-9 regulates developmental neurogenesis in the retina, but its roles in development and injury-induced regeneration of photoreceptors are unknown. This research will fill these gaps by determining the function of miR-9 in these processes in the zebrafish retina.
Methods :
In situ hybridization was used to determine temporal and spatial expression of miR-9 in the developing embryonic and adult regenerating retina followed by immunolabeling for proliferating cells and Müller glia. RT-qPCR was used to quantify expression of precursors for miR-9 family members (pre-miR9-1 through 7) at different time points after photoreceptor injury and Taqman RT-qPCR was used to quantify expression of mature miR-9. Morpholino oligonucleotides were used for miR-9 loss-of-function, first in the developing retina then in the regenerating adult retina followed by immunolabeling for proliferating cells and mature photoreceptors. All miR-9 knockdown fish were compared to standard control morpholino injected fish, and cell counts were done on retinal cross-sections.
Results :
In situ hybridization showed miR-9 is expressed throughout the developing retina from 48-72 hours post-fertilization (hpf) and in dividing Müller glia and progenitors at 3 days post lesion (dpl) during photoreceptor regeneration. RT-qPCR showed miR9-1 and miR9-2 precursors are most highly expressed in the injured retina, and miR-9 expression peaks between 3 and 5 dpl. In embryos, miR-9 knockdown resulted in fewer proliferating cells at 48 hpf and fewer mature photoreceptors at 72 hpf. In adults at 3 dpl, miR-9 knockdown increased numbers of proliferating cells in the outer nuclear layer (ONL) only. Experiments are currently being conducted on a newly established miR9-1 mutant line, and miR9-2 mutants are being produced.
Conclusions :
In the developing retina, miR-9 plays critical roles in cell proliferation and photoreceptor development. In the injured adult retina, miR-9 regulates mechanisms that control progenitor migration and/or the timing of photoreceptor regeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.