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CHI SUN, Deborah L Stenkamp; Study of Selected Rod Photoreceptor-Enriched Transcripts in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):573.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of the current study is to determine in-depth functions of selected transcripts that are enriched in rod photoreceptors, in order to gain insights into intrinsic mechanisms underlying rod development and regeneration.
We used a transgenic zebrafish line (XOPS:eGFP) in which rod photoreceptors express green fluorescent protein (GFP) as a model organism for this study. RNA-seq of FACS-sorted dissociated retinal cells was performed to identify differentially expressed (in GFP+ vs. GFP- cells) transcripts with FDR < 0.01. Selected rod-specific genes were prioritized for further qRT-PCR and in situ hybridization studies at different life stages of the zebrafish, and for qRT-PCR studies of rods that regenerated after widespread chemical lesioning of the retina. The rxrγa gene was of particular interest as its transcript was enriched in rods, while previous studies in mouse indicated roles in cone determination. Therefore, a new rxrγa mutant line was created by CRISPR/CAS9 technology and used to perform a loss-of-function study.
The list of differentially expressed (GFP+ vs. GFP-) transcripts consisted of 615 entries. 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 mef2ca, nr1d4a, rxrγa, and rxrγb. Expression levels of these genes were verified by qRT-PCR as rod-enriched at both 5 days post-fertilization (dpf) and adult age. The rxrγa mutants displayed reduced expression of the following opsin genes at 5% p-value threshold: rhodopsin (△△CT=-4.6); rhodopsin-like (-5.1); lws1 (the first member of the tandemly-duplicated long wavelength-sensitive cone opsin array) (-6.1); and the final three genes of the tandemly-quadriplicated rh2 (green-sensitive cone opsin) array (-3.1 for rh2-2, -4.6 for rh2-3, -8.8 for rh2-4). There were no significant differences in expression of lws2, rh2-1, or sws1 as compared with controls at 14dpf.
RNA-seq and subsequent analysis identified numerous transcripts differentially expressed in rod photoreceptors as compared to other retinal cells. Rxrγa may be an important regulator of expression of rod opsins, and of differential expression of tandemly-replicated cone opsins. qRT-PCR studies are underway to verify differential expressions of selected transcripts in FACS-isolated regenerated rods.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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