Exploring the role of NR2E3 in photoreceptor cell fate determination

Abstract

The transcription factor NR2E3 participates in photoreceptor cell fate determination as it functions as both transcriptional repressor of cone genes and activator of rod genes. Mutations in NR2E3 cause retinitis pigmentosa (RP) and enhanced S-cone syndrome (ESCS) in humans. This gene produces a large isoform encoded in 8 exons and a previously unreported shorter isoform of 7 exons, whose function is unknown. We generated two mouse models by targeting exon 8 of Nr2e3 using CRISPR/Cas9- D10A nickase. Allele Δ27 is an in-frame deletion of 27 bp that ablates the dimerization domain H10, whereas allele ΔE8 (full deletion of exon 8) produces only the short isoform, which lacks the C-terminal part of the ligand binding domain (LBD) that encodes both H10 and the AF2 domain involved in the Nr2e3 repressor activity. The Δ27 mutant shows developmental alterations and a non-progressive retinal dysfunction that resembles the ESCS patient’s phenotype. Early ∆27 affectation is caused by alterations in rod and cone differentiation, but expression of ∆27 long isoform in the adult retina slows down the degenerative process. On the contrary, the ΔE8 mutant exhibits progressive retinal degeneration, with a milder phenotype at early stages and severe dysfunction at old stages, as occurs in human RP patients. scRNA- seq of wildtype and Nr2e3 mutant retinas reveals that rod and cone populations are not homogeneous, and identifies a previously unreported pathway of cone differentiation, in which a subpopulation of cones displays traits of a hybrid intermediate stage of differentiation and co-expresses cone and rod genes. This subpopulation of mis-differentiated cones is highly increased in the Nr2e3 mutant retinas as well as a subpopulation of rods transitioning towards the cone fate. Hybrid photoreceptors with high mis-expression of both cone and rod genes are prone to regulated necrosis. Our findings suggest that the NR2E3 short isoform regulates the long isoform by competing for the binding to target sites and partners of the NR2E3 regulatory complex, as well as by reducing NR2E3 long isoform stability. Overall, our results (i) propose a role for NR2E3 as a cone-patterning and differentiation regulator, (ii) shed light on the role of NR2E3 in the modulation of the photoreceptor precursor differentiation towards cone and rod fates, and (iii) explain why mutations in NR2E3 lead to different visual disorders in humans, providing valuable models for studying mechanisms of NR2E3-associated retinal dystrophies and evaluating potential therapies.