Identification of cis-Acting Elements Repressing Blue Opsin Expression in Zebrafish UV Cones and Pineal Cells

Abstract

Opsin genes are expressed in a cell type-specific manner in the retina and the pineal organ for visual and nonvisual photoreceptive purposes, but the regulatory mechanism behind the tissue and cell selectivity is not well understood. In this study, we focus on the expression regulation of the blue-sensitive opsin gene SWS2 of zebrafish by taking a transgenic approach using the green fluorescence protein as an expression reporter. The zebrafish SWS2 is a single-copy gene and is expressed specifically in the "long single cones" in the retina. We found the following. 1) A 0.3-kb region between 0.6 and 0.3 kb 5 of the SWS2 initiation codon, encompassing four cone-rod homeobox-binding sites (OTX sequences), contains the region necessary and sufficient to drive gene expression in long single cones. 2) A 15-bp portion (؊341 to ؊327) in the 0.3-kb region represses the gene expression in the "short single cones," which are dedicated to the UV-sensitive opsin gene SWS1. 3) An 11-bp sequence TAACTGCCAGT (؊441 to ؊431) in the 0.3-kb region, with its adjacent OTX element, also works as a repressor for gene expression in the pineal cells. 4) Finally, this OTX site is necessary for expression repression in the bipolar cells in the retina. These findings open a way for understanding the complex interaction of positive and negative regulatory factors that govern the cell type specificity of the opsin gene expression in the photoreceptive cells in the retina and the pineal organ. We termed the novel 11-bp sequence as the pineal negative regulatory element, PINE.Vertebrate retina contains two types of visual photoreceptor cells, rods and cones, the former responsible for dim light vision and the latter for bright light and color vision. The vertebrate visual opsins are classified into the following five phylogenetic types that originated before the vertebrate radiation: one produced in rod cells (RH1 3 or rhodopsins) and the other four produced in cone cells with different spectral sensitivity, red (M/LWS), green (RH2), blue (SWS2), and UV types (SWS1) (1). The combined output from the different spectral types of the cones allows an animal to perceive color. Although the selective expression of an opsin gene in a given cone cell is a basis of forming color vision, its regulatory mechanism achieving the cell type specificity remains less understood compared with the mechanism of rod-specific expression studied for the RH1 type opsin gene (2, 3).Non-mammalian vertebrates have multiple extra-ocular photosensors, mainly localized in the pineal complex (4) that secretes melatonin under a regular day/night cycle, the phase of which can be shifted upon its photoreception (5). Several nonvisual opsin genes have been found expressed in pineal cells of non-mammalian vertebrates, such as pinopsin (6), exo-rhodopsin (exrho) (7), and parapinopin (8). It has been found, however, that some visual opsin genes are also expressed in these pineal cells (9 -11). A physiological study has shown that the M/LWS opsin is indeed involved in melatonin supp...

Transmission from the dominant input shapes the stereotypic ratio of photoreceptor inputs onto horizontal cells

“…We used a combination of transgenic lines to visualize UV and/or blue cones by fluorescent protein (FP) expression. Tg(sws1:GFP ) 47 labels UV cones, and Tg(sws2:GFP ) 48 labels blue cones. As an additional method to visualize blue cones, a transgenic line was engineered to express mCherry in blue cones Tg(sws2:mCherry ) 49 .…”

“…Cone type-specific promoters were excised from sws1:GFP 47 and sws2up3.2kb:EGFP plasmids 48 , and inserted into p5E plasmids. TeNT-CFP 45 was inserted into pME plasmid.…”

Genetics of photoreceptor degeneration and regeneration in zebrafish

“…The identification of promoters to express genes specifically in rod photoreceptors [26], all cones [27], and cone subtypes [28, 29] makes these studies imminent. As an initial example of this type of study, the rod photoreceptor-specific promoter was used to derive expression of membrane-tagged cyan fluorescent protein to rod photoreceptors resulting in their apoptosis [16].…”

Eye evolution: common use and independent recruitment of genetic components

“…Crx, strongly expressed in differentiated photoreceptor cells, directly regulates the phototransduction genes, rhodopsin, b-PDE, arrestin and guanylate cyclase, via binding the PCE element in the promoters (Chen et al 1997;Furukawa et al 1997;Qian et al 2005) (for a review, see Hennig et al 2008). The expression of ciliary-phototransduction cascade genes in the vertebrate pineal gland is mediated by the action of Otx genes as well (Appelbaum & Gothilf 2006;Takechi et al 2008). Besides the direct regulation of photoreceptor-specific genes, Otx genes are involved in the regulation of pigmentation.…”