Investigating cone photoreceptor development using patient-derived NRL null retinal organoids

Abstract

Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa. While murine Nrl loss has been characterized, studies of human NRL can identify important insights for human retinal development and disease. We utilized iPSC organoid models of retinal development to molecularly define developmental alterations in a human model of NRL loss. Consistent with the function of NRL in rod fate specification, human retinal organoids lacking NRL develop Sopsin dominant photoreceptor populations. We report generation of two distinct S-opsin expressing populations in NRL null retinal organoids and identify MEF2C as a candidate regulator of cone development.

Nr2e3functional domain ablation by CRISPR-Cas9D10A identifies a new isoform and generates Retinitis Pigmentosa and Enhanced S-cone Syndrome models

“…Note that although rhodopsin was detected in our mutant retinal sections, it may not indicate the presence of fully differentiated rods. Some authors have reported a rod/cone intermediate population, such "cods" have been recently characterized in retinal organoids from a NRL null patient showing an ESCS phenotype (Kallman et al, 2020). The observed phenotype of the D27 mutant could be the result of the short isoform overexpression plus the incapability of the long isoform to dimerize.…”

Boolean Implication Analysis Improves Prediction Accuracy of In Silico Gene Reporting of Retinal Cell Types

“…One of the most common methods to study the effect of key genes on retinal development is the use of genetically modified knocko murine models, which are frequently used to validate differentially expressed genes from microarray data. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Fluorescent gene reporter lines are widely used to check for gene expression in single cells, or purified populations of a single cell type. 2,[21][22][23][24][25] Bulk RNA sequencing (RNA-seq) has helped define the transcriptomes of larger populations of retinal cell types.…”

“…36,37 Single-cell RNA sequencing (scRNA-seq) is increasingly common today and is one the most detailed methods to profile transcriptomes of retinal cell types and subtypes. 2,8,13,22,[38][39][40][41][42][43][44][45][46][47][48] Most studies on retinal cell types have relied upon murine models, but many increasingly study human donor retinas 6,30,31,[48][49][50] , especially in order to profile retinal disease. 31,43,[50][51][52][53] Glaucoma, age-related macular dystrophy and retinal light damage have also been studied in murine models.…”

“…7,14,29,34,35,54,55 Some studies have grown cell lines in vitro from fetal retina 49, 56 , whereas other have used human pluripotent, induced pluripotent or embryonic stem cells to generate purified cell populations or retinal organoids. 2,3,8,28,38,[57][58][59] In order to study the development of retinal cell types over time, the lineage of stem cell progeny 58 and time course data from different time points (using PCR and RNA-seq) have been investigated. 39,41,54…”

“…63 Correlational methods for ranking gene expression are also widely used, bypassing the need to discover cell clusters and identifying co-expressed genes in complex cultures, including developing retinal organoids. 2,8,23,27,49,64 Identifying relationships between genes has led towards broader goals of graph 47,60 and networkbased analysis. 9,10,17,25,27,31,60,65 Gene expression networks can be used to identify transitions between phenotypes and disease states, paving the way for clinical target identification.…”