NR2E3 loss disrupts photoreceptor cell maturation and fate in human organoid models of retinal development

While dysfunction and/or death of light-detecting photoreceptor cells underlies most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation; up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify the specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than that observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation. Overall design: Three iPSC lines (ND [no disease] Control, NR2E3-null, and Isogenic Control) were differentiated to retinal organoids for 260 days. Samples were assayed by 3' scRNAseq at D40, D80, D120, D160 and D260 from all three lines. In a separate differentiation batch, NR2E3-null and Isogenic Control organoids were assayed by joint (RNA & ATAC) single-nucleus multimodal sequencing at D160 and D280. Multimodal runs yield paired scRNAseq and scATACseq libraries. For AAV experiments, NR2E3-null organoids were cultured for 120 days. Organoids were treated with AAV carrying NR2E3-T2A-GFP or GFP or left untransduced as described. Organoids were harvested at D120 or following treatment at D160 and assayed by 3' scRNAseq. For ESCS Patient 2 experiment, an iPSC line from ESCS Patient 2 was differentiated to retinal organoids and assayed at D80 and D160 by 3' scRNAseq. >>>Raw data were not provided for ATAC-seq Samples due to patient privacy concerns<<<