Fluorescence visualization of ultraviolet‐sensitive cone photoreceptor development in living zebrafish

Journal of Biological Chemistry

2008

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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...

Section: Methodsmentioning

confidence: 99%

“…Whereas the ϩϩϩ and ϩϩ were taken as a significant sign of the expression induction, the ϩ was not. The selection of transgenic lines in the subsequent generations was performed as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

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

Takechi¹,

Seno

Journal of Biological Chemistry

2008

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“…The RH2-LCR was further tested for its ability to induce gene expression in the SDCs by using the promoter regions of a non-SDC, UV (SWS1) opsin (17), and a nonretinal, keratin 8 (18) gene. The 854-and 564-bp adjacent upstream regions of the SWS1 and keratin 8 genes, designated SWS1up854bp and krt8up564bp, respectively, were sandwiched between the RH2-LCR and the GFP reporter and these resulting constructs were referred to as LCR:SWS1 and LCR:krt8, respectively (Fig.…”

Section: A Distal Regulatory Region Rh2-lcr Was Found In the Rh2-pamentioning

confidence: 99%

Identification of a locus control region for quadruplicated green-sensitive opsin genes in zebrafish

Tsujimura

Chinen

Proc. Natl. Acad. Sci. U.S.A.

2007

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113

Duplication of opsin genes has a crucial role in the evolution of visual system. Zebrafish have four green-sensitive (RH2) opsin genes (RH2-1, RH2-2, RH2-3, and RH2-4) arrayed in tandem. They are expressed in the short member of the double cones (SDC) but differ in expression areas in the retina and absorption spectra of their encoding photopigments. The shortest and the second shortest wavelength subtypes, RH2-1 and RH2-2, are expressed in the central-to-dorsal retina. The longer wavelength subtype, RH2-3, is expressed circumscribing the RH2-1/RH2-2 area, and the longest subtype, RH2-4, is expressed further circumscribing the RH2-3 area and mainly occupying the ventral retina. The present report shows that a 0.5-kb region located 15 kb upstream of the RH2 gene array is an essential regulator for their expression. When the 0.5-kb region was deleted from a P1-artificial chromosome (PAC) clone encompassing the four RH2 genes and when one of these genes was replaced with a reporter GFP gene, the GFP expression in SDCs was abolished in the zebrafish to which a series of the modified PAC clones were introduced. Transgenic studies also showed that the 0.5-kb region conferred the SDC-specific expression for promoters of a non-SDC (UV opsin) and a nonretinal (keratin 8) gene. Changing the location of the 0.5-kb region in the PAC clone conferred the highest expression for its proximal gene. The 0.5-kb region was thus designated as RH2-LCR analogous to the locus control region of the L-M opsin genes of primates.gene duplication ͉ GFP reporter ͉ transgenesis F unctional differentiation of duplicated genes is important for organismal evolution and is achieved through modifications of not only coding regions but also regulatory mechanisms of the gene expression in the duplicates. The regulatory mechanism for the spatial and the temporal coordination of gene expression among duplicated genes has been a subject of intense interest (1, 2). Gene duplications have an essential role in the evolution of the visual system. The visual opsins in vertebrates are classified into five phylogenetic types that originated before the vertebrate radiation: rod opsin or rhodopsin (RH1); RH1-like, or green cone opsin (RH2); short wavelength-sensitive type 1, or UV-blue cone opsin (SWS1); short wavelength-sensitive type 2, or blue cone opsin (SWS2); and middle-to long-wavelength-sensitive, or redgreen cone opsin (M/LWS) (3). However, the understanding of the regulatory mechanisms for the differential expression among the five types is still fragmentary. Subtypes of the visual opsins, created by more recent gene duplications, provide an excellent model to study the regulatory evolution of duplicated genes. A well documented example is the locus control region (LCR) of the L-M opsin genes (subtypes of M/LWS) arrayed on the X chromosome in the catarrhine primates (Old World monkeys, apes, and humans) (4).The primate L-M opsin LCR is located at Ϸ3.5 kb upstream of the gene array and interacts with only the most proximal or the second proximal gene o...

“…The restoration of the central expression of RH2-2 occurred between 1-2·months pf. The hybridization signal of RH2-2 was much stronger than that of RH2-1, which was consistent with our previous observation in real-time RT-PCR, in that the expression level of RH2-2 in adult retina is hybridized with the RH2-1 CDR probe, where progress of the gene expression is staged as follows: stage 1, expression is detected in a patch of ventronasal retina; stage 2-3, another patch appears in nasal retina and the two fuse to fill the ventronasal retina; stage 4, expression extends from the ventronasal region to the central region and, less extensively, in dorsal and ventrotemporal directions; stage 5-6, expression is detected in more than three-fourths of the retina (Raymond et al, 1995;Takechi et al, 2003). Dotted line indicates rim of the eye.…”

Section: Rh2 Opsin Expression In Adult Zebrafish Retinamentioning

confidence: 99%

Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development

Takechi

Journal of Experimental Biology

2005

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148

185

SUMMARY Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4)in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.