NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors

Abstract

SUMMARY Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. As the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal day 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously un-annotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor.

Pias3 is necessary for dorso-ventral patterning and visual response of retinal cones but is not required for rod photoreceptor differentiation

“…6C). Using Nrl −/− and flow-sorted rod photoreceptor RNA-Seq data (Kim et al, 2016b), we observed several rod- and cone-enriched genes (38% and 16%, respectively) among the P21 Pias3 −/− DEGs (Fig. 6D).…”

“…In Pias3 −/− retina, the expression of Pias genes and other E3 SUMO ligases was unchanged, indicating that compensation is not due to increased expression at the transcriptional level but rather through functional redundancy (). In addition, analysis of published RNA-Seq data from flow-sorted photoreceptors of wild-type and cone-only retina showed that multiple E3 SUMO ligases are expressed in both rod and cone photoreceptors () (Kim et al, 2016b). Such functional redundancy has been described previously.…”

Molecular dissection of cone photoreceptor‐enriched genes encoding transmembrane and secretory proteins

“…Taking advantage of the published transcriptomes of Nrl‐GFP:Nrl +/+ rods and Nrl‐GFP: Nrl −/− SCLs (Kim et al, ) at distinct stages of photoreceptor maturation (Postnatal day (P)6, P10, P14, P28), we identified 2662 differentially‐expressed genes with higher expression (fold change ≥2, false discovery rate ≤0.01) in at least one time point in SCLs (Figure a). We further filtered for genes encoding plasma membrane or secreted proteins applying the Gene Ontology (GO) term annotations plasma membrane (GO: 0005886) and extracellular region (GO: 0005576) and selected genes with human homologs (Figure a,b).…”

“…Transcriptomes of Nrl:GFP;Nrl +/+ and Nrl:GFP;Nrl −/− flow‐sorted photoreceptors (Kim et al, ) (GEO accession # GSE74660). Selection of transmembrane and secreted genes was performed by Ensembl BioMart (Kinsella et al, ) (https://www.ensembl.org, RRID:SCR_002344), using Ensembl genes 91 database and the GRCm38.p5 genome.…”

“…Instead of rods, photoreceptors in the Nrl −/− retina acquire molecular and functional characteristics of S‐cones (Akimoto et al, ; Daniele et al, ; Nikonov et al, ; Yoshida & Cepko, ). By comparing the transcriptomes of Nrl −/− S‐cone‐like photoreceptors (SCLs) and wildtype (WT) rods (Kim et al, ), we identified cone‐enriched genes encoding plasma membrane and secretory proteins and performed in situ hybridization studies for seven of them. In vivo knockdown of one of the genes, Ephrin type‐A receptor 10 ( Epha10 ), by electroporation of shRNA in newborn mouse retina, provided strong evidence in support of its physiological relevance and validated our strategy for investigating molecular components involved in intercellular interactions.…”