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A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

ABSTRACT: A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio) have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs) in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC) clones encompassing the two genes and identified a 0.6-kb "LWS-activating region" (LAR) upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins.

SUBMITTER: Tsujimura T

PROVIDER: S-EPMC3002997 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

Tsujimura Taro T Hosoya Tomohiro T Kawamura Shoji S

PLoS genetics 20101216 12

A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio) have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the ...[more]

PMID: 21187910

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Project description:BACKGROUND:Long wave-sensitive (LWS) opsin genes have undergone multiple lineage-specific duplication events throughout the evolution of teleost fishes. LWS repertoire expansions in live-bearing fishes (family Poeciliidae) have equipped multiple species in this family with up to four LWS genes. Given that color vision, especially attraction to orange male coloration, is important to mate choice within poeciliids, LWS opsins have been proposed as candidate genes driving sexual selection in this family. To date the genomic organization of these genes has not been described in the family Poeciliidae, and little is known about the mechanisms regulating the expression of LWS opsins in any teleost. RESULTS:Two BAC clones containing the complete genomic repertoire of LWS opsin genes in the green swordtail fish, Xiphophorus helleri, were identified and sequenced. Three of the four LWS loci identified here were linked in a tandem array downstream of two tightly linked short wave-sensitive 2 (SWS2) opsin genes. The fourth LWS opsin gene, containing only a single intron, was not linked to the other three and is the product of a retrotransposition event. Genomic and phylogenetic results demonstrate that the LWS genes described here share a common evolutionary origin with those previously characterized in other poeciliids. Using qualitative RT-PCR and MSP we showed that each of the LWS and SWS2 opsins, as well as three other cone opsin genes and a single rod opsin gene, were expressed in the eyes of adult female and male X. helleri, contributing to six separate classes of adult retinal cone and rod cells with average lambdamax values of 365 nm, 405 nm, 459 nm, 499 nm, 534 nm and 568 nm. Comparative genomic analysis identified two candidate teleost opsin regulatory regions containing putative CRX binding sites and hormone response elements in upstream sequences of LWS gene regions of seven teleost species, including X. helleri. CONCLUSIONS:We report the first complete genomic description of LWS and SWS2 genes in poeciliids. These data will serve as a reference for future work seeking to understand the relationship between LWS opsin genomic organization, gene expression, gene family evolution, sexual selection and speciation in this fish family.

Project description:BackgroundIn the Duplication-Degeneration-Complementation (DDC) model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps) genes by dietary fatty acids (FAs) in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid), sunflower oil (12% lipid, rich in linoleic acid), linseed oil (12% lipid, rich in linolenic acid), or low fat (4% lipid, low fat diet) for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined.ResultFA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level of fabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a given fabp gene. As such, up-regulation of the steady-state level of fabp mRNAs by FAs occurred at the level of initiation of transcription. None of the sister duplicates of these fabp genes exhibited an increase in their steady-state transcript levels in a specific tissue following feeding zebrafish any of the four experimental diets.ConclusionDifferential induction of only one of the sister pair of duplicated fabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.

Project description:Force, Lynch and Conery proposed the duplication-degeneration-complementation (DDC) model in which partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) were the two primary mechanisms for the retention of duplicated genes. The DDC model was tested by analyzing the transcriptional induction of the duplicated fatty acid-binding protein (fabp) genes by clofibrate in zebrafish. Clofibrate is a specific ligand of the peroxisome proliferator-activated receptor (PPAR); it activates PPAR which then binds to a peroxisome proliferator response element (PPRE) to induce the transcriptional initiation of genes primarily involved in lipid homeostasis. Zebrafish was chosen as our model organism as it has many duplicated genes owing to a whole genome duplication (WGD) event that occurred ~230-400 million years ago in the teleost fish lineage. We assayed the steady-state levels of fabp mRNA and heterogeneous nuclear RNA (hnRNA) transcripts in liver, intestine, muscle, brain and heart for four sets of duplicated fabp genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b in zebrafish fed different concentrations of clofibrate.Electron microscopy showed an increase in the number of peroxisomes and mitochondria in liver and heart, respectively, in zebrafish fed clofibrate. Clofibrate also increased the steady-state level of acox1 mRNA and hnRNA transcripts in different tissues, a gene with a functional PPRE. These results demonstrate that zebrafish is responsive to clofibrate, unlike some other fishes. The levels of fabp mRNA and hnRNA transcripts for the four sets of duplicated fabp genes was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). The level of hnRNA coded by a gene is an indirect estimate of the rate of transcriptional initiation of that gene. Clofibrate increased the steady-state level of fabp mRNAs and hnRNAs for both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b, but in different tissues. Clofibrate also increased the steady-state level of fabp10a and fabp11a mRNAs and hnRNAs in liver, but not for fabp10b and fabp11b.Some duplicated fabp genes have, most likely, retained PPREs, but induction by clofibrate is over-ridden by an, as yet, unknown tissue-specific mechanism(s). Regardless of the tissue-specific mechanism(s), transcriptional control of duplicated zebrafish fabp genes by clofibrate has markedly diverged since the WGD event.

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A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

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A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

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