Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing or the relative production of alternative transcripts. Modulation of alternative splicing in the retina is also instrumental in the design of novel therapeutic approaches for retinal dystrophies, since it enables precision medicine for specific mutations.

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 electrophysiological dysfunction that resembles the ESCS phenotype. The ΔE8 mutant exhibits progressive retinal degeneration, as occurs in human RP patients. Our mutants suggest a role for Nr2e3 as a cone-patterning regulator and provide valuable models for studying mechanisms of NR2E3-associated retinal dystrophies and evaluating potential therapies.

SUMMARY: Mutations in NR2E3 cause two retinal dystrophies with a distinct phenotype. NR2E3 encodes an orphan nuclear transcription factor that contributes to photoreceptor cell fate determination by repressing cone while activating rod genes. To dissect NR2E3 function, we performed scRNA-seq in the retinas of wild type and two different Nr2e3 mouse models that show phenotypes similar to patients carrying NR2E3 mutations. Our results reveal that rod and cone populations are not homogeneous and can be separated into different sub- classes. We identify a previously unreported cone pathway that generates hybrid cones that co-express both cone- and rod-related genes. In mutant retinas, this hybrid cone subpopulation is more abundant, as it includes a subpopulation of rods transitioning towards a cone cell fate. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Overall, our results shed light on the role of NR2E3 in modulating photoreceptor differentiation towards cone and rod fates and explain how mutations in NR2E3 lead to different visual disorders in humans. GRAPHICAL SYNOPSIS: Mutations in the gene encoding the retinal transcription factor NR2E3 cause two different inherited retinal dystrophies: retinitis pigmentosa and enhanced S-cone syndrome. scRNA-seq in retinas of wild type and two Nr2e3 mouse models reveal that rod and cone populations are not homogeneous and can be classified into different sub-classes. A previously unreported cone pathway that generates hybrid cones co- expressing both cone- and rod-related genes is identified. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Expression of rod and cone signature genes change in response to different NR2E3 mutations thus providing a frame to understand the molecular basis of distinct NR2E3-associated diseases.

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, whereas allele ΔE8 (full deletion of exon 8), produces only the short isoform that lacks the dimerization and repressor domains. The Δ27 mutant shows developmental alterations and a non-progressive electrophysiological dysfunction that resembles the ESCS phenotype. The ΔE8 mutant exhibits progressive retinal degeneration, as occurs in human RP patients. Interestingly, the mutant retinas show invaginations similar to fovea-like pits. Our mutants suggest a role of Nr2e3 as a cone-patterning regulator and provide valuable models for studying mechanisms of NR2E3 -associated retinal dystrophies and evaluating potential therapies. Abstract Figure: Highlights: - Nr2e3 mouse models were generated by exon 8 deletion using CRISPR/Cas9 D10A nickase. - New Nr2e3 mRNA retaining intron 7 encodes a short protein expressed in adult retina. - Deletion of 9 aa of the NR2E3 dimerization domain causes enhanced S-cone syndrome. - Deletion of exon 8 produces a phenotype similar to Retinitis Pigmentosa in mouse.