Abstract
Purpose :
Inherited retinal degenerative diseases such as Retinitis Pigmentosa (RP) involve progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Each regenerating system contains a complex network of cell types with different circadian mechanisms contributing to regeneration. Understanding the molecular underpinnings of this circadian regulation of tissue regeneration would accelerate the development of new regenerative therapies. Hence, we performed analyses of single-cell (SC) transcriptome data from adult Zebrafish retina of wild-type and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration (deg/reg).
Methods :
SC RNA sequencing analysis was performed on WT AB and P23H rhodopsin transgenic Zf retinas. TSNE clusters and transcriptomic analysis of specific targets were generated through Seurat. ClueGo, and STRING pathway analysis tools were used to identify functional networks. Hiplex was used to validate gene expression by in situ hybridization.
Results :
The transcriptome profile of the circadian regulators cry1a, cry1aa, cry1bb, per1b, per2, and tefa that belong to the negative arm of the transcriptional regulation network of the circadian rhythm were increased in the P23H cones and RPE compared to the WT. To validate the single-cell transcriptome data, we examined the expression of per1, per2, and tefa using multiplex in situ hybridization. The results demonstrate the increased expression of per1, per2, and tefa in the outer nuclear layer (ONL) of the P23H retina compared to the WT suggesting the change is coordinated between RPE and photoreceptors. Further analysis revealed increased expression of cry and per genes in amacrine and bipolar cell clusters suggesting circadian perturbations throughout the deg/reg retina.
Conclusions :
Unexpected significant increases in expression of some circadian clock genes associated with one phase of the circadian cycle are noted in several cell types in the retina. Anecdotally we observed that P23H zebrafish lay eggs several hours after lights are on rather than in the first hour compared to WT. Studies have shown the possibility to treat RP through circadian synchronization and our study provides the lead to understand the circadian changes during deg/reg conditions.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.