Molecular basis of retinal remodeling in a zebrafish model of retinitis pigmentosa

Abstract

A hallmark of inherited retinal degenerative diseases such as retinitis pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult zebrafish retina of wild type AB strain (WT) and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. Retinas from both female and male fish were pooled to generate each library, combining data from both sexes. We provide a benchmark atlas of retinal cell type transcriptomes in zebrafish and insight into how each retinal cell type is affected in the P23H model. Oxidative stress is found throughout the retina, with increases in reliance on oxidative metabolism and glycolysis in the affected rods as well as cones, bipolar cells, and retinal ganglion cells. There is also transcriptional evidence for widespread synaptic remodeling and enhancement of glutamatergic transmission in the inner retina. Notably, changes in circadian rhythm regulation are detected in cones, bipolar cells, and retinal pigmented epithelium. We also identify the transcriptomic signatures of retinal progenitor cells and newly formed rods essential for the regenerative process. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.

Cost-effective strategies to knock down genes of interest in the retinas of adult zebrafish

“…We previously performed single-cell RNA sequencing (SC RNA-Seq) analysis on retinas from adult WT and P23H transgenic zebrafish (expressing Flag-tagged P23H mutant rhodopsin in rods) to examine how the retinal environment changed during retinal degeneration and regeneration ( Santhanam et al, 2023 ). WT and P23H SC RNA-Seq data were integrated to allow for cells in the same cluster to be compared between conditions ( Figure 2A ).…”

“…WT and P23H SC RNA-Seq data were integrated to allow for cells in the same cluster to be compared between conditions ( Figure 2A ). We identified 24 different clusters and many transcriptional differences between WT and P23H within each cluster [ Figure 2B ; data from Santhanam et al (2023) ]. Analysis of the integrated data identified that PCNA expression was predominantly found to be in cluster 14, retinal progenitor cells, and at a much lower level in a few other cell types ( Figure 2C ).…”

“…PKCα is abundant in bipolar cells, providing a readily visible marker for the “rod” bipolar cell, and is important for normal cell signaling in the retina ( Haug et al, 2019 ). Analysis of single-cell RNA sequencing data from Zf retina (data from Santhanam et al, 2023 ) 1 revealed that prkcaa is expressed predominantly in bipolar cells, but is also the only isoform highly expressed in rod bipolar cells ( Figure 5A ). Prkcab is expressed predominantly in bipolar cell populations ( Figure 5B ).…”

“…We perform knockdowns in a P23H mutant rhodopsin transgenic zebrafish model developed in our laboratory (Santhanam et al, 2020). This line has been characterized to have continuous degeneration and regeneration of rod photoreceptors throughout adulthood (Santhanam et al, 2020(Santhanam et al, , 2023. Single-cell RNA sequencing and immunohistochemistry have shown PCNA to be a highly expressed gene unique to proliferating cells that eventually become rod photoreceptors in our P23H transgenic zebrafish line (Santhanam et al, 2020(Santhanam et al, , 2023.…”

“…This line has been characterized to have continuous degeneration and regeneration of rod photoreceptors throughout adulthood (Santhanam et al, 2020(Santhanam et al, , 2023. Single-cell RNA sequencing and immunohistochemistry have shown PCNA to be a highly expressed gene unique to proliferating cells that eventually become rod photoreceptors in our P23H transgenic zebrafish line (Santhanam et al, 2020(Santhanam et al, , 2023. By targeting PCNA, we are able to directly assess the extent of knockdown as well as whether or not there are any unintended impacts from the technique.…”