Photoreceptors participate in the development of the deep vascular plexus through Otx2/Crx gene regulatory network.

Abstract

Purpose : Crx^Rip is a mouse mutant carrying a dominant mutation in the photoreceptor-expressed transcription factor Crx, causing congenital blindness due to arrested photoreceptor differentiation. Photoreceptors degenerate in Crx^Rip/Rip mice while they are preserved for an extended period in Crx^Rip/+. We initiated a comparative transcriptomic analysis between these two genotypes to understand why the lack of functional CRX causes photoreceptor degeneration.

Methods : Cell death was quantified following TUNEL assays. RNA-Seq analysis was performed at P21 and P30 in Crx^Rip/Rip and Crx^Rip/+ mice. The vasculature was observed by angiography and on flat-mounted retinas. Gene expression was assessed by qPCR and RNAscope in situ hybridization.

Results : RNA-seq analysis at P30 revealed 475 differentially expressed genes between Crx^Rip/Rip and Crx^Rip/+ retina. Pathways analysis identified the biological process “negative regulation of vascular development” among deregulated pathways, suggesting a potential alteration of retinal vasculature in Crx^Rip/Rip mouse retina. Angiography showed blood leakage in Crx^Rip/Rip as early as P30 despite limited thinning of the ONL at this stage. Further analysis on flat-mounted retinas and retinal sections showed a profound reduction of the deep plexus as early as P12 in Crx^Rip/Rip mouse retinas. Among the genes deregulated related to vascular development, Vasohibin-1 (Vash1) was of first interest for its known antiangiogenic function. We confirmed Vash1 upregulation in Crx^Rip/Rip specifically in photoreceptors. qPCR analysis showed that Vash1 expression decreased during normal retinal development following the decrease of Otx2 expression and the development of the retinal vasculature. In the Crx^Rip/Rip retina, Otx2 expression was maintained as shown for Vash1. Since Crx and Otx2 binding sites were found in Vash1, we hypothesized that lack of Crx prevents Otx2 downregulation in photoreceptors keeping Vash1 expression and preventing the deep plexus formation.

Conclusions : Our results suggest a non-autonomous function of CRX in photoreceptors to regulate the formation of the deep vasculature plexus, highlighting their critical role in vasculature development. We thus propose a model in which the deep plexus vasculature defects observed in Crx^Rip/Rip retinas would be the main cause of subsequent photoreceptor cell degeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.