NR2E3-deficient progenitors commit to a rod fate yet fail to form mature rod photoreceptors in an induced pluripotent stem cell-based model of human retinal development

Abstract

Purpose :
Photoreceptor cell development is controlled by a cascade of transcriptional regulatory events. Mutations in the genes encoding the transcription factors that participate in photoreceptor specification are known to cause vision loss in several inherited retinal diseases. However, the precise molecular aberrations of photoreceptor cells born of this pathologic specification process are not fully understood. As a result, prognosis and treatment of this class of inherited retinal disease is limited. Based on the clinical phenotype of night blindness in Enhanced S-Cone Syndrome patients carrying mutations in the NR2E3 gene, we hypothesized that photoreceptor precursors lacking functional NR2E3 would exhibit a failure in rod photoreceptor development.

Methods :
We used induced pluripotent stem cells (iPSC) to model Enhanced S-Cone Syndrome and single-cell RNA sequencing to identify the transcriptional changes undertaken by abnormal photoreceptors during retinal development. We generated retinal cells in vitro from three iPSC lines; one homozygous for a loss-of-function mutation in NR2E3, a CRISPR-corrected isogenic control, and an unrelated healthy control line. We sampled developing retinal cells across a 260-day time course with single-cell RNA sequencing to understand how and when NR2E3 acts in human photoreceptor development.

Results :
Using gene expression profiles of over 79,000 individual cells across five timepoints, we identified a unique developmental detour away from normal rod photoreceptor maturation taken by cells lacking functional NR2E3. Differential gene expression analysis shows that the resulting abnormal cells lack expression of some, but not all, genes present in normally functioning rod photoreceptors while remaining transcriptionally distinct from normal cone photoreceptors.

Conclusions :
These results show that NR2E3 loss damages normal rod photoreceptor development through misexpression of a specific subset of rod genes, yielding a divergent rod photoreceptor lineage unique to the disease state.

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

 

Retinal cells generated from NR2E3 patient and control iPSCs across five differentiation timepoints.