Abstract
Purpose :
The purpose of this study is to understand the regeneration mechanism of photoreceptors and identify biomarkers for crucial stages of regeneration in the diurnal, cone-dominant zebrafish retina. It will provide insight into the mechanisms of degeneration and will provide key insight into how natural regeneration occurs in an animal capable of regeneration.
Methods :
We developed a transgenic zebrafish model of Retinitis Pigmentosa expressing flag-tagged mouse rhodopsin carrying the P23H mutation driven by the zebrafish rhodopsin promoter. Cell turnover was assessed by IP injection of BrdU in adult fish. Adult retina was characterized using immunofluorescence and TUNEL studies. Transcriptome profiling was done in pooled adult retina using single-cell 3’ RNA sequencing. Principal component analysis was conducted using Seurat, an R toolkit for single-cell analysis. Clusters were manually annotated and classified into retinal cell types. Functional analysis was done using ClueGO.
Results :
Adult P23H zebrafish had 40% cells as wild-type (WT) fish at the outer nuclear layer (ONL) and outer segment lengths were greatly reduced (Fig. 1). The ONL contained TUNEL positive cells in the mutant suggesting cell death, as well as PCNA positive cells consistent with proliferation (Fig. 1). BrdU pulse-chase labeling revealed approximately 20x more newly born cells in the mutant than in WT. P23H mutant fish lost 56% of the newly-made cells in one week, while there was no loss of BrdU-labelled cells in WT retina at the same time. Single-cell analysis revealed enrichment of retinal progenitor cluster and early progenitor derived rods in the mutant compared to WT. Biomarkers for rod progenitors were identified through single-cell gene expression analysis. As a proof of concept, deoxyuridine triphosphatase (dut), a new marker identified for rod progenitor cells, is seen highly enriched in the ONL in the mutant compared to the WT fish (Fig. 2). Bioinformatic analysis shows molecular pathways responsible for cell proliferation are highly enriched the mutant progenitor cells.
Conclusions :
We have developed a zebrafish model of Retinitis Pigmentosa (RP) that displays continuous rod degeneration as well as regeneration throughout life, providing an elegant system to study the regeneration of neurons. We have identified markers for rod progenitors as well as functionally enriched molecular pathways during regeneration.
This is a 2020 ARVO Annual Meeting abstract.