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CHI SUN, Carlos Galicia, Peter G Fuerst, Deborah L Stenkamp; Study of Selected Rod Photoreceptor Expressed Transcripts in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):367.
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© ARVO (1962-2015); The Authors (2016-present)
Rod photoreceptors are primarily affected in many hereditary retinal diseases in humans. In order to gain insights into intrinsic mechanisms underlying rod development and maintenance, we analyzed the rod photoreceptor transcriptome of the zebrafish using RNA-Seq, and carried out expression and/or functional analysis of selected transcripts.
We used as a model organism a transgenic zebrafish line (XOPS:eGFP), in which rod photoreceptors express green fluorescent protein (GFP). Dissociated, FACS-sorted retinal cells were subject to RNA-Seq. Selected rod-expressed genes were prioritized for further qRT-PCR, in situ hybridization, and functional studies. The dscamb and rxrγa genes were of particular interest. Retinal expression studies of the duplicated dscama and dscamb genes were performed; these encode cell adhesion molecules. Previous studies on rxrγ in mouse indicated roles in cone differentiation, while our RNA-seq and qRT-PCR data indicated that the rxrγa transcript was also expressed in zebrafish rods. Therefore, rxrγa mutants were created by CRISPR/CAS9 technology and used to perform a loss-of-function study.
1630 transcripts were detected as differentially expressed (in GFP+ rods vs. GFP- non-rod retinal cells) with FDR < 0.01, among which 597 were rod-enriched. These included known rod-specific transcripts such as rhodopsin and nr2e3, as well as transcripts not previously known to be enriched in rods, such as rhodopsin-like, insb, nr2f1b, and esrrd. Among these transcripts of interest, dscamb was enriched in rods, while dscama in contrast was enriched in the non-rod retinal cells. In situ hybridization studies of dscam genes were consistent with the RNA-Seq data. Preliminary analysis of genetically heterogeneous F1 progeny of rxrγa mutants at 7 days post-fertilization displayed up-regulated expression of nr2e3, nrl, thrb, and rxrγb (△△CT=5.52, 1.02, 1.28, and 3.63, respectively; p<0.05 for all), and down-regulated expression of several cone opsin genes, as well as both rhodopsin genes.
RNA-seq and subsequent analyses identified numerous transcripts differentially expressed in rod photoreceptors as compared to other retinal cells. The roles of dscama and dscamb appear to be subfunctionalized within the retina. Rxrγa may be an important regulator of expression of opsin genes in rods as well as cones, and may regulate other photoreceptor-associated transcripts.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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