Abstract
Purpose :
The purpose of this study was to characterize retinal gene expression changes that occur in CRISPR-induced rhodopsin zebrafish mutant, rhofl9 which contains a non-sense mutation at codon 347 (pS347*), eliminating the conserved VXPX C-terminal targeting signal. (Zelinka et al., 2018). We previously showed that zebrafish heterozygous for the rhofl9allele display opsin mislocalization, rapid rod degeneration but no secondary effect on cones — a similar phenomenon was observed in XOPS-mCFP transgenic zebrafish (Morris et al., 2011). We predicted that the gene expression profile in rhofl9 would reflect dramatic downregulation of rod phototransduction genes but upregulation of genes associated with rod regeneration.
Methods :
Total RNA was extracted from retinas of age-matched wild-type and rhofl9 adult zebrafish. Gene expression profiles produced from RNAseq were used to determine differentially expressed genes, and pathway analysis was used to identify the relevant biological pathways. Cell proliferation was assessed by EdU labeling.
Results :
RNAseq counts yielded 15,401 genes; expression of 1,413 genes was significantly different from wild-type. As expected, these data show decreased expression of rod-associated genes, including many disease-associated genes. As anticipated, increased expression of cell cycle-associated genes was observed, but no changes in genes expressed by cones were observed. Initial pathway analyses indicated that significantly down- and upregulated genes are involved in regulating phototransduction, cell mitosis, and photoreceptor specification. When compared to expression data from XOPS-mCFP zebrafish, the pathway outputs were nearly identical. For both mutants, the top downregulated genes consisted of rod-associated genes, while the top upregulated were involved in cell cycle control and neuronal differentiation. EdU immunolabeling on rhofl9 adults confirmed the increase of mitosis of rod progenitor cells, but no Müller glia were labeled.
Conclusions :
These data characterize gene expression changes that occur in the rhofl9 retina. The similarities found between two distinct rod degeneration models suggest that genetic manipulations leading to rod degeneration evoke a predictable pattern of gene expression consistent with a persistent cycle of rod degeneration, regeneration and degeneration. These tools provide genetic models to better understand persistent rod death and degeneration-induced regeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.