Comparative analysis of teleost fish genomes reveals preservation of different ancient clock duplicates in different fishes

Clock (Circadian locomotor output cycle kaput) was the first vertebrate circadian clock gene identified in a mouse forward genetics mutagenesis screen. It encodes a bHLH-PAS protein that is highly conserved throughout evolution. Tetrapods also have the second Clock gene, Clock2 or Npas2 (Neuronal PAS domain protein 2). Conversely, the fruit fly, an invertebrate, has only one clock gene. Interrogation of the five teleost fish genome databases revealed that the zebrafish and the Japanese pufferfish (fugu) each have three clock genes, whereas the green spotted pufferfish (tetraodon), the Japanese medaka fish and the three-spine stickleback each have two clock genes. Phylogenetic and splice site analyses indicated that zebrafish and fugu each have two clock1 genes, clock1a and clock1b and one clock2; tetraodon also have clock1a and clock1b but do not have clock2; and medaka and stickleback each have clock1b and one clock2. Genome neighborhood analysis further showed that clock1a/clock1b in zebrafish, fugu and tetraodon is an ancient duplicate. While the dN/dS ratios of these three fish clock duplicates are all <1, indicating that purifying selection has acted upon them; the Tajima relative rate test showed that all three fish clock duplicates have asymmetric evolutionary rates, implicating that one of these duplicates have been under positive selection or relaxed functional constraint. These results support the view that teleost fish clock genes were generated from an ancient genome-wide duplication, and differential gene loss after the duplication resulted in retention of different ancient duplicates in different teleost fishes, which could have contributed to the evolution of the distinct fish circadian clock mechanisms.     

Comparative genomic analysis of teleost fish <Emphasis Type="Italic">bmal</Emphasis> genes

Bmal1 (Brain and muscle ARNT like 1) gene is a key circadian clock gene. Tetrapods also have the second Bmal gene, Bmal2. Fruit fly has only one bmal1/cycle gene. Interrogation of the five teleost fish genome sequences coupled with phylogenetic and splice site analyses found that zebrafish have two bmal1 genes, bmal1a and bmal1b, and bmal2a; Japanese pufferfish (fugu), green spotted pufferfish (tetraodon) and Japanese medaka fish each have two bmal2 genes, bmal2a and bmal2b, and bmal1a; and three-spine stickleback have bmal1a and bmal2b. Syntenic analysis further indicated that zebrafish bmal1a/bmal1b, and fugu, tetraodon and medaka bmal2a/bmal2b are ancient duplicates. Although the dN/dS ratios of these four fish bmal duplicates are all <1, implicating they have been under purifying selection, the Tajima relative rate test showed that fugu, tetraodon and medaka bmal2a/bmal2b have asymmetric evolutionary rates, suggesting that one of these duplicates have been subject to positive selection or relaxed functional constraint. These results support the notion that teleost fish bmal genes were derived from the fish-specific genome duplication (FSGD), divergent resolution following the duplication led to retaining different ancient bmal duplicates in different fishes, which could have shaped the evolution of the complex teleost fish timekeeping mechanisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative genomics of duplicate γ-glutamyl transferase genes in teleosts: medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), green spotted pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), and zebrafish (Danio rerio)

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we examine multiple paralogous genes of γ-glutamyl transferase (GGT) in several distantly related teleost species including medaka, stickleback, green spotted pufferfish, fugu, and zebrafish. Through mining genome databases, we have identified multiple GGT orthologs. Duplicate (paralogous) GGT sequences for GGT1 (GGT1 a and b), GGTL1 (GGTL1 a and b), and GGTL3 (GGTL3 a and b) were identified for each species. Phylogenetic analysis suggests that GGTs are ancient proteins conserved across most metazoan phyla and those paralogous GGTs in teleosts likely arose from the serial 3R genome duplication events. A third GGTL1 gene (GGTL1c) was found in green spotted pufferfish; however, this gene is not present in medaka, stickleback, or fugu. Similarly, one or both paralogs of GGTL3 appear to have been lost in green spotted pufferfish, fugu, and zebrafish. Syntenic relationships were highly maintained between duplicated teleost chromosomes, among teleosts and across ray-finned (Actinopterygii) and lobe-finned (Sarcopterygii) species. To assess subfunction partitioning, six medaka GGT genes were cloned and assessed for developmental and tissue-specific expression. On the basis of these data, we propose a modification of the "duplication-degeneration-complementation" model of subfunction partitioning where quantitative differences rather than absolute differences in gene expression are observed between gene paralogs. Our results demonstrate that multiple GGT genes have been retained within teleost genomes. Questions remain, however, regarding the functional roles of multiple GGTs in these species.     

Diverse forms of guanylyl cyclases in medaka fish -- their genomic structure and phylogenetic relationships to those in vertebrates and invertebrates

Fish species such as medaka fish, fugu, and zebrafish contain more guanylyl cyclases (GCs) than do mammals. These GCs can be divided into two types: soluble GCs and membrane GCs. The latter are further divided into four subfamilies: (i) natriuretic peptide receptors, (ii) STa/guanylin receptors, (iii) sensory-organ-specific membrane GCs, and (iv) orphan receptors. Phylogenetic analyses of medaka fish GCs, along with those of fugu and zebrafish, suggest that medaka fish is a much closer relative to fugu than to zebrafish. Analyses of nucleotide data available on a web site (http://www.ncbi. nlm.nih.gov/) of GCs from a range of organisms from bacteria to vertebrates suggest that gene duplication, and possibly chromosomal duplication, play important roles in the divergence of GCs. In particular, the membrane GC genes were generated by chromosomal duplication before the divergence of tetrapods and teleosts.     

Consequences of hoxb1 duplication in teleost fish

Vertebrate evolution is characterized by gene and genome duplication events. There is strong evidence that a whole-genome duplication occurred in the lineage leading to the teleost fishes. We have focused on the teleost hoxb1 duplicate genes as a paradigm to investigate the consequences of gene duplication. Previous analysis of the duplicated zebrafish hoxb1 genes suggested they have subfunctionalized. The combined expression pattern of the two zebrafish hoxb1 genes recapitulates the expression pattern of the single Hoxb1 gene of tetrapods, possibly due to degenerative changes in complementary cis-regulatory elements of the duplicates. Here we have tested the hypothesis that all teleost duplicates had a similar fate post duplication, by examining hoxb1 genes in medaka and striped bass. Consistent with this theory, we found that the ancestral Hoxb1 expression pattern is subdivided between duplicate genes in a largely similar fashion in zebrafish, medaka, and striped bass. Further, our analysis of hoxb1 genes reveals that sequence changes in cis-regulatory regions may underlie subfunctionalization in all teleosts, although the specific changes vary between species. It was previously shown that zebrafish hoxb1 duplicates have also evolved different functional capacities. We used misexpression to compare the functions of hoxb1 duplicates from zebrafish, medaka and striped bass. Unexpectedly, we found that some biochemical properties, which were paralog specific in zebrafish, are conserved in both duplicates of other species. This work suggests that the fate of duplicate genes varies across the teleost group.     

Comparative genomics provides evidence for an ancient genome duplication event in fish

There are approximately 25 000 species in the division Teleostei and most are believed to have arisen during a relatively short period of time ca. 200 Myr ago. The discovery of 'extra' Hox gene clusters in zebrafish (Danio rerio), medaka (Oryzias latipes), and pufferfish (Fugu rubripes), has led to the hypothesis that genome duplication provided the genetic raw material necessary for the teleost radiation. We identified 27 groups of orthologous genes which included one gene from man, mouse and chicken, one or two genes from tetraploid Xenopus and two genes from zebrafish. A genome duplication in the ancestor of teleost fishes is the most parsimonious explanation for the observations that for 15 of these genes, the two zebrafish orthologues are sister sequences in phylogenies that otherwise match the expected organismal tree, the zebrafish gene pairs appear to have been formed at approximately the same time, and are unlinked. Phylogenies of nine genes differ a little from the tree predicted by the fish-specific genome duplication hypothesis: one tree shows a sister sequence relationship for the zebrafish genes but differs slightly from the expected organismal tree and in eight trees, one zebrafish gene is the sister sequence to a clade which includes the second zebrafish gene and orthologues from Xenopus, chicken, mouse and man. For these nine gene trees, deviations from the predictions of the fish-specific genome duplication hypothesis are poorly supported. The two zebrafish orthologues for each of the three remaining genes are tightly linked and are, therefore, unlikely to have been formed during a genome duplication event. We estimated that the unlinked duplicated zebrafish genes are between 300 and 450 Myr. Thus, genome duplication could have provided the genetic raw material for teleost radiation. Alternatively, the loss of different duplicates in different populations (i.e. 'divergent resolution') may have promoted speciation in ancient teleost populations.     

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.     

The "fish-specific" Hox cluster duplication is coincident with the origin of teleosts

The Hox gene complement of zebrafish, medaka, and fugu differs from that of other gnathostome vertebrates. These fishes have seven to eight Hox clusters compared to the four Hox clusters described in sarcopterygians and shark. The clusters in different teleost lineages are orthologous, implying that a "fish-specific" Hox cluster duplication has occurred in the stem lineage leading to the most recent common ancestor of zebrafish and fugu. The timing of this event, however, is unknown. To address this question, we sequenced four Hox genes from taxa representing basal actinopterygian and teleost lineages and compared them to known sequences from shark, coelacanth, zebrafish, and other teleosts. The resulting gene genealogies suggest that the fish-specific Hox cluster duplication occurred coincident with the origin of crown group teleosts. In addition, we obtained evidence for an independent Hox cluster duplication in the sturgeon lineage (Acipenseriformes). Finally, results from HoxA11 suggest that duplicated Hox genes have experienced diversifying selection immediately after the duplication event. Taken together, these results support the notion that the duplicated Hox genes of teleosts were causally relevant to adaptive evolution during the initial teleost radiation.     

Are all fishes ancient polyploids?

Euteleost fishes seem to have more copies of many genes than their tetrapod relatives. Three different mechanisms could explain the origin of these 'extra' fish genes. The duplicates may have been produced during a fish-specific genome duplication event. A second explanation is an increased rate of independent gene duplications in fish. A third possibility is that after gene or genome duplication events in the common ancestor of fish and tetrapods, the latter lost more genes. These three hypotheses have been tested by phylogenetic tree reconstruction. Phylogenetic analyses of sequences from human, mouse, chicken, frog (Xenopus laevis), zebrafish (Danio rerio) and pufferfish (Takifugu rubripes) suggest that ray-finned fishes are likely to have undergone a whole genome duplication event between 200 and 450 million years ago. We also comment here on the evolutionary consequences of this ancient genome duplication.     

Phylogenetic and evolutionary relationships and developmental expression patterns of the zebrafish <Emphasis Type="Italic">twist</Emphasis> gene family

Four members of the twist gene family (twist1a, 1b, 2, and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split. Phylogenetic analysis groups most of the vertebrate Twist1 peptides into clade I, except for the Twist1b proteins of the acanthopterygian fish (medaka, pufferfish, stickleback), which clustered within clade III. Paralogies and orthologies among the zebrafish, medaka, and human twist genes were determined using comparative synteny analysis of the chromosomal regions flanking these genes. Comparative nucleotide substitution analyses also revealed a faster rate of nucleotide mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b, thus accounting for their anomalous phylogenetic clustering. We also observed minimal expression overlap among the four twist genes, suggesting that despite their significant peptide similarity, their regulatory controls have diverged considerably, with minimal functional redundancy between them. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.     

A Novel Glucagon-Related Peptide (GCRP) and Its Receptor GCRPR Account for Coevolution of Their Family Members in Vertebrates

The glucagon (GCG) peptide family consists of GCG, glucagon-like peptide 1 (GLP1), and GLP2, which are derived from a common GCG precursor, and the glucose-dependent insulinotropic polypeptide (GIP). These peptides interact with cognate receptors, GCGR, GLP1R, GLP2R, and GIPR, which belong to the secretin-like G protein-coupled receptor (GPCR) family. We used bioinformatics to identify genes encoding a novel GCG-related peptide (GCRP) and its cognate receptor, GCRPR. The GCRP and GCRPR genes were found in representative tetrapod taxa such as anole lizard, chicken, and Xenopus, and in teleosts including medaka, fugu, tetraodon, and stickleback. However, they were not present in mammals and zebrafish. Phylogenetic and genome synteny analyses showed that GCRP emerged through two rounds of whole genome duplication (2R) during early vertebrate evolution. GCRPR appears to have arisen by local tandem gene duplications from a common ancestor of GCRPR, GCGR, and GLP2R after 2R. Biochemical ligand-receptor interaction analyses revealed that GCRP had the highest affinity for GCRPR in comparison to other GCGR family members. Stimulation of chicken, Xenopus, and medaka GCRPRs activated Gα_s-mediated signaling. In contrast to chicken and Xenopus GCRPRs, medaka GCRPR also induced Gα_q/11-mediated signaling. Chimeric peptides and receptors showed that the K¹⁶M¹⁷K¹⁸ and G¹⁶Q¹⁷A¹⁸ motifs in GCRP and GLP1, respectively, may at least in part contribute to specific recognition of their cognate receptors through interaction with the receptor core domain. In conclusion, we present novel data demonstrating that GCRP and GCRPR evolved through gene/genome duplications followed by specific modifications that conferred selective recognition to this ligand-receptor pair.     

Five Nkx5 genes show differential expression patterns in anlagen of sensory organs in medaka: insight into the evolution of the gene family

We report the identification and characterisation of five different Nkx5-related genes in medaka fish (Oryzias latipes). They constitute homologues of genes previously isolated in higher vertebrates, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3 and SOHo-1, and were named accordingly: OlNkx5--1.1, OlNkx5--2, OlNkx5--3 and OlSOHo. For the Nkx5--1 gene a new, second homologue, OlNkx5--1.2, was isolated. In medaka, Nkx5 and SOHo genes are differentially expressed in three developing sensory organs: eye, ear and lateral line and later in defined brain regions. Phylogenetic analyses of the entire Nkx5 family revealed that four paralogous Nkx5 groups, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3/GH6 and SOHo, are present in vertebrates. Only some of the Nkx5 family members have been identified in singular vertebrate species so far. Here we present, for the first time, the isolation of representatives of each Nkx5 subgroup in one species, the medaka fish. Based on similarities in sequence and expression patterns, and genomic organisation we propose a model of the evolutionary history of the Nkx5 family. The model predicts that the four vertebrate Nkx5 genes arose by a tandem duplication, followed by chromosomal duplication. The two Nkx5--1 genes identified so far exclusively in medaka most probably result from an additional genome duplication in the fish lineage.     

Expansion of transducin subunit gene families in early vertebrate tetraploidizations

Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.     

Functional diversification of kir7.1 in cichlids accelerated by gene duplication

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.