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Nrl-dependent and independent rod photoreceptor development in zebrafish [scRNA-Seq]

ABSTRACT: NRL (Neural retina leucine zipper) is a key regulator of the fate determination and gene expression of rod photoreceptors in the retina of many vertebrates. In this study, we observed a unique retinal phenotype in the nrl knockout zebrafish model characterized by reduced rods with shortened outer segments and gradually increased green-cones in adulthood. By tracing and comparing the developmental processes of WT and nrl knockout rods, we found there might be two waves of rod genesis distinguished as nrl-dependent and independent with different starting times. To reveal the underlying cellular and molecular mechanisms, bulk and single-cell RNA-seq were performed. The changes in gene expression and cell proportion of rods and cones agreed well with our histological and immunofluorescence studies. Interestingly, we found that rods exhibited noticeable heterogeneities in the gene expression patterns, and a part of rods in nrl knockout zebrafish misexpressed the green-cone genes, reflecting a hybrid status of rod and green-cone. Furthermore, we identified mafba as a novel regulator of rod genesis, which was responsible for the development of rods in nrl knockout zebrafish. Our study will largely improve the current understanding of the developmental processes and regulatory mechanisms of rods in zebrafish and probably other species, and may facilitate future studies in the fields of retinal development and retinal degeneration.

ORGANISM(S): Danio rerio

PROVIDER: GSE160140 | GEO | 2020/10/27

REPOSITORIES: GEO

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Project description:Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors. NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation. Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans. To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease. Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional. Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo. Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells. The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression. Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis. Experiment Overall Design: We mated the Crx::Nr2e3/Nrlko mice with the Nrl::GFP transgenic mice, in which the expression of GFP is driven by an Nrl promoter. Mouse retinas were dissected at 4 wk. GFP+ photoreceptors were enriched by FACS (FACSAria, BD Biosciences, Franklin Lakes, NJ). RNA was extracted from 1~5x105 flow-sorted cells using Trizol (Invitrogen). Total RNA (40-60 ng) was used for linear amplification with Ovation Biotin labeling system (Nugen), and 2.75 ug of biotin-labeled fragmented cDNA was hybridized to mouse GeneChips MOE430.2.0 (Affymetrix) having 45,101 probesets (corresponding to over 39,000 transcripts, and 34,000 annotated mouse genes). Experiment Overall Design: Four independent samples were used at 4 weeks. We normalized Experiment Overall Design: Crx::Nr2e3/Nrl-ko-Gfp data along with Nrl-ko-Gfp 4 weeks samples ( 4 replicates, refer to Series submission GSE4051). The normalized data was then subjected to two stage analysis based on False Discovery Rate Confidence Interval (FDR-CI) for screening differentially expressed genes (24, 27) with a minimum fold change of 4.

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Nrl-dependent and independent rod photoreceptor development in zebrafish [scRNA-Seq]

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