Abstract
Purpose :
A cellular hallmark of inherited retinal degenerative diseases is progressive loss of photoreceptors until one is completely blind. Unlike mammals, Zebrafish (Zf) have the capacity to regenerate neurons following retinal insult. We have previously generated and characterized a Zf model (P23H rho) with continuous degeneration and regeneration of rods. The goal of this study is to understand the mechanisms of rod regeneration and successful integration in Zf retina by identifying genes crucial for these processes. We hypothesize that genes differentially expressed in retinal progenitor cells (RPCs) and new rods in P23H Zf can reveal factors important for rod regeneration and integration.
Methods :
SC RNA sequencing analysis was performed on WT and P23H rhodopsin transgenic Zf retinas. UMAP and trajectory analyses were performed in Seurat and Monocle 3. Differentially expressed genes (DEG’s) were analyzed through DrivAER and GO Enrichment analyses to identify candidate genes involved in rod regeneration and synapse formation necessary for new rod integration in the retina. Morpholinos and siRNAs were used to knock down candidate genes. Immunohistochemistry was performed to assess impact on regeneration and integration of rods.
Results :
DrivAER analysis in the P23H dataset revealed master regulator genes at each stage of regeneration. Knockdown of prdm1a reduced differentiation of RPCs into rods while not affecting proliferation. GO Enrichment analysis revealed DEGs involved in synapse formation of rods. Knockdown of dscamb, ncam1b, and auts2b combined increased the length of bipolar cell (BPC) axons while knockdown of ncam1b alone increased the number of co-localizations found between rod and BPC dendrites.
Conclusions :
DEGs found in RPCs and new rods of the P23H Zf dataset appear to play important roles in the regeneration and integration of new rods in the retina. Prdm1a plays an important role in the differentiation of RPCs into rods. Knockdown of dscamb, ncam1b, and auts2b increased BPC axon length while knockdown of ncam1b alone increased the number of co-localizations found between rods and BPC dendrites. Such increases suggest that these genes play a role in inhibiting synapse formation. The upregulation of these genes may be a consequence of retinal degeneration and will have to be taken into account in therapeutic strategies trying to restore vision in the blind.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.