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Deborah L Stenkamp, Craig B Stevens, Ruth A Frey, Shoji Kawamura; Retinoic acid signaling regulates expression of the tandemly duplicated LWS1 and LWS2 genes in zebrafish.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4032.
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© ARVO (1962-2015); The Authors (2016-present)
Differentiation of diverse photoreceptor phenotypes in the vertebrate retina requires multiple signaling pathways that activate cascades of gene expression. The signaling molecule retinoic acid (RA) is known to regulate rod and cone cell fate, differentiation, and survival. The purpose of the current study is to identify photoreceptor genes controlled by RA signaling in the embryonic retina of the zebrafish.
We treated embryos with RA at 48 hours post-fertilization (hpf) and isolated total RNA from eyes for microarray analysis at 75 hpf in order to identify genes responding to RA over the period of photoreceptor differentiation. Differentially expressed genes were validated by quantitative RT-PCR (qRT-PCR) and in situ hybridization. Wild-type zebrafish and those carrying an RA signaling reporter transgene (RARE:YFP) were used.
We identified 180 genes with significantly altered gene expression. Of interest was the long wavelength sensitive opsin 1 (LWS1) gene, which was upregulated in eyes of RA-treated embryos. LWS1 is the upstream member of the tandemly duplicated LWS gene array, and is normally expressed in red-sensitive cones of ventral retina, but not until larval stages. qRT-PCR verified significant upregulation of LWS1, but not LWS2, in eyes of RA-treated embryos. In situ hybridization using probes specific for the 3’ UTR of each LWS gene revealed that nearly no control retinas expressed LWS1, while those of embryos treated with (9-cis or all-trans) RA from 48 hpf to 5 dpf all expressed LWS1, predominantly in the ventral half of the retina. Control embryos all expressed LWS2 throughout their retinas, while RA-treated embryos showed a dramatic reduction in the number of cones expressing LWS2. The expression domain of LWS1 in the photoreceptor layer of RA-treated embryos matched the expression domain of a YFP reporter gene driven by a series of RA response elements.
RA signaling regulates numerous molecular targets in the developing eye, including the tandemly-duplicated LWS1 and LWS2 genes. It is possible that the level of RA signaling within a developing red-sensitive cone acts as a molecular toggle to favor the expression of LWS1 and/or suppress the expression of LWS2. This is the first evidence that an extracellular signal may regulate expression of opsins in a tandemly duplicated gene array.
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