Highly-enriched retinal pigment epithelium and photoreceptor genes without circadian variability

Abstract

Purpose : A number of physiological processes in retinal pigment epithelium (RPE) and photoreceptors (PR) are regulated by the circadian clock. In this study, we tested the hypothesis that circadian clock-regulated genes in RPE and PR overlap with the current lists of known “signature” RPE and PR genes. We defined new lists of highly-enriched RPE and PR genes by using arrhythmic clock gene mutant Per1^-/- Per2^Brdm1 mice.

Methods : We performed RNA-sequencing on laser-capture microdissected (LCM) RPE and PR from WT and Per1^-/- Per2^Brdm1 mouse eyes obtained under constant darkness at 4 time-points over 24h. We compared time-affected genes from WT RPE and PR with current “signature” RPE and PR gene lists. To define the new list of non-rhythmic highly-enriched RPE and PR genes, we first averaged gene expression levels obtained at all time points from RPE and PR genes obtained from Per1^-/- Per2^Brdm1 mice. We ranked the top 10% genes based on expression levels, subtracted overlapping and time-affected genes. Functional annotation was performed using g:Profiler.

Results : RNA sequencing of LCM samples revealed that 594 genes (~3% total) in the RPE and 2,372 genes (~10% total) in PR showed temporal variations in WT mice. In contrast, only 2 genes were time-affected in Per1^-/- Per2^Brdm1 PR and none in RPE. We found that 2 (out of 64) RPE and 12 (out of 65) PR “signature” genes were time-affected. Unexpectedly, we found overlap between “signature” genes of PR and time-affected RPE genes (and vice versa) suggesting that sampling time affected the compiling of current “signature” genes. By using transcriptomes obtained from Per1^-/- Per2^Brdm1mice, we defined 828 non-rhythmic highly-enriched RPE and 641 PR genes. We found that highly-enriched RPE genes are enriched in MAPK signaling, focal adhesion and oxidative phosphorylation, whereas PR genes are enriched in mTOR signaling and GTPase activity, among others. Finally, we found that 37 RPE and 9 PR genes overlap between “signature” and our new highly-enriched gene lists.

Conclusions : Our findings indicate that sampling time substantially affected the compiling of “signature” RPE and PR genes. Our new lists will be useful in a number of applications such as extrapolating RPE and PR data from whole eye transcriptomics and for assessing the molecular characteristics of stem-cell derived RPE and PR.

This is a 2021 ARVO Annual Meeting abstract.