Single-cell transcriptomic analysis of human and murine NRL-null retinas

Abstract

Purpose : Retinal degenerative diseases (RD) remain largely untreatable, partly due to limited understanding of the complex gene-expression patterns directing photoreceptor (PR) development. By performing single-cell transcriptomic analysis of human in vitro and mouse in vivo models of retinal development in the presence and absence of Neural Retina-specific Leucine zipper protein (NRL), a transcription factor vital to rod PR development and degeneration, we are working to systematically define the gene networks involved in PR differentiation. Pathways that have been previously unappreciated as being important for PR differentiation and maintenance could potentially be therapeutic targets for RD.

Methods : We used two microfluidic-based single-cell RNA capturing platforms, Dropseq and 10X Genomics, to obtain transcriptomic data for retinal cells. Human induced pluripotent stem cell control and NRL-null lines were differentiated into retinal organoids and samples were collected at time points for single cell analysis. We performed parallel analyses in wild type (WT) and Nrl-null mice at postnatal days 7, 11, 18 and 25. We analyzed the transcriptomic data by principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) to cluster cells by variably expressed genes. Using marker genes to determine cluster identity, we compared PR populations across WT and NRL-null datasets.

Results : WT human stem cell-derived retinal organoids yielded characteristic retinal cell populations, including rod and cone PRs. The NRL-null models produced only cone PR populations, with S-cones as the dominant subtype. Consistent with the human datasets, Nrl-null mouse retinas contained only cone PRs with S-opsin more highly expressed than M-opsin. Comparative analysis of the murine and human cells is providing additional insights into retinal cell type-specific expression patterns.

Conclusions : Consistent with the function of NRL in directing PRs toward a rod fate, human and murine retinas lacking NRL led to development of cone-dominant PR populations. Further analysis comparing the nature of PRs which develop with and without NRL will better define the regulatory networks involved in PR differentiation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

t-SNE representation of 5,514 cells from human NRL-null retinal organoids demonstrating absence of rod PRs.

 

t-SNE representation of 3,504 cells from human control retinal organoids demonstrating presence of rod PRs.