With the advent of high-throughput DNA sequencing technology, the genomic sequence of many disparate species has led to the relatively new discipline of genomics, the study of genome structure, function and evolution. Much work has been focused on the role of whole genome duplications (WGD) in the architecture of extant vertebrate genomes, particularly those of teleost fishes which underwent a WGD early in the teleost radiation > 230 million years ago (mya). Our past work has focused on the fate of duplicated copies of a multigene family coding for the intracellular lipid-binding protein (iLBP) genes in the teleost fishes. To define the evolutionary processes that determined the fate of duplicated genes and generated the structure of extant fish genomes, however, requires comparative genomic analysis with a fish lineage that diverged before the teleost WGD, such as the spotted gar (Lepisosteus oculatus), an ancient, air-breathing, ray-finned fish. Here, we describe the genomic organization, chromosomal location and tissue-specific expression of a subfamily of the iLBP genes that code for fatty acid-binding proteins (Fabps) in spotted gar. Based on this work, we have defined the minimum suite of fabp genes prior to their duplication in the teleost lineages 230-400 mya. Spotted gar, therefore, serves as an appropriate outgroup, or ancestral/ancient fish, that did not undergo the teleost-specific WGD. As such, analyses of the spatio-temporal regulation of spotted gar genes provides a foundation to determine whether the duplicated fabp genes have been retained in teleost genomes owing to either sub- or neofunctionalization.

Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.

In the duplication-degeneration-complementation model, duplicated gene-pairs undergo nonfunctionalization (loss from the genome), subfunctionalization (the functions of the ancestral gene are sub-divided between duplicate genes), or neofunctionalization (one of the duplicate genes acquires a new function). These processes occur by loss or gain of regulatory elements in gene promoters. Fatty acid-binding proteins (Fabp) belong to a multigene family composed of orthologous proteins that are highly conserved in sequence and function, but differ in their gene regulation. We previously reported that the zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters underwent subfunctionalization of PPAR responsiveness. Here, we describe the regulation at the duplicated zebrafish fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b gene promoters. Differential control at the duplicated fabp promoters was assessed by DNA sequence analysis, responsiveness to PPAR-isoform specific agonists and NF-κB p50 antagonists in zebrafish liver and intestine explant tissue, and in HEK293A cells transfected with fabp promoter-reporter constructs. Each zebrafish fabp gene displayed unique transcriptional regulation compared to its paralogous duplicate. This work provides a framework to account for the evolutionary trajectories that led to the high retention (57%) of duplicated fabp genes in the zebrafish genome compared to only ~ 3% of all duplicated genes in the zebrafish genome.

BACKGROUND:In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter.RESULTS:In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR.CONCLUSIONS:The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication.

Gene duplication is thought to facilitate increasing complexity in the evolution of life. The fate of most duplicated genes is nonfunctionalization: functional decay resulting from the accumulation of mutations. According to the duplication-degeneration-complementation (DDC) model, duplicated genes are retained by subfunctionalization, where the functions of the ancestral gene are sub-divided between duplicate genes, or by neofunctionalization, where one of the duplicates acquires a new function. Here, we report the differential regulation of the zebrafish tandemly duplicated fatty acid-binding protein genes, fabp1b.1 and fabp1b.2, by peroxisome proliferator-activated receptors (PPAR). fabp1b.1 mRNA levels were induced in tissue explants of liver, but not intestine, by PPAR agonists. fabp1b.1 promoter activity was induced to a greater extent by rosiglitazone (PPARγ-selective agonist) compared to WY 14,643 (PPARα-selective agonist) in HEK293A cells. Mutation of a peroxisome proliferator response element (PPRE) at -1232 bp in the fabp1b.1 promoter reduced PPAR-dependent activation. fabp1b.2 promoter activity was not affected by PPAR agonists. Differential regulation of the duplicated fabp1b promoters may be the result of PPRE loss in fabp1b.2 during a meiotic crossing-over event. Retention of PPAR inducibility in fabp1b.1 and not fabp1b.2 suggests unique regulation and function of the fabp1b duplicates.

The increased use of plant oil as a dietary supplement with the resultant high dietary lipid loads challenges the lipid transport, metabolism and storage mechanisms in economically important aquaculture species, such as rainbow trout. Fatty acid-binding proteins (Fabp), ubiquitous in tissues highly active in fatty acid metabolism, participate in lipid uptake and transport, and overall lipid homeostasis. In the present study, searches of nucleotide sequence databases identified mRNA transcripts coded by 14 different fatty acid-binding protein (fabp) genes in rainbow trout (Oncorhynchus mykiss), which include the complete minimal suite of seven distinct fabp genes (fabp1, 2, 3, 6, 7, 10 and 11) discovered thus far in teleost fishes. Phylogenetic analyses suggest that many of these extant fabp genes in rainbow trout exist as duplicates, which putatively arose owing to the teleost-specific whole genome duplication (WGD); three pairs of duplicated fabp genes (fabp2a.1/fabp2a.2, fabp7b.1/fabp7b.2 and fabp10a.1/fabp10a.2) most likely were generated by the salmonid-specific WGD subsequent to the teleost-specific WGD; and fabp3 and fabp6 exist as single copy genes in the rainbow trout genome. Assay of the steady-state levels of fabp gene transcripts by RT-qPCR revealed: (1) steady-state transcript levels differ substantially between fabp genes and, in some instances, by as much as 30 × 104-fold; (2) some fabp transcripts are widely distributed in many tissues, whereas others are restricted to one or a few tissues; and (3) divergence of regulatory mechanisms that control spatial transcription of duplicated fabp genes in rainbow trout appears related to length of time since their duplication. The suite of fabp genes described here provides the foundation to investigate the role(s) of fatty acid-binding proteins in the uptake, mobilization and storage of fatty acids in cultured fish fed diets differing in lipid content, especially the use of plant oil as a dietary supplement. These nutritional dietary supplements may well lead to high lipid loads with the resultant challenges to lipid homeostasis and, thus, health of cultivated fish which may be mediated by appropriate transcriptional control of fabp genes.

The fatty acid-binding protein (fabp) genes belong to the multigene family of intracellular lipid-binding proteins. To date, 12 different FABPs have been identified in vertebrate genomes. Owing to the teleost-specific genome duplication event, many fishes have duplicated copies of the fabp genes. Here, we identified and characterized the fabp genes of spotted green pufferfish (Tetraodon nigroviridis). Seven fabp genes were identified, out of which, two were retained in the pufferfish genome as duplicated copies. Each putative pufferfish Fabp protein shares greatest sequence identity and similarity with their teleost and tetrapod orthologs, and clustered together as a distinct clade in phylogenetic analysis. Conserved gene synteny was evident between the pufferfish fabp genes and the orthologs of human, zebrafish, three-spined stickleback, and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the teleost-specific genome duplication event. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes.

All fabp genes, except fabp2, fabp3 and fabp6, exist as duplicates in the zebrafish genome owing to a whole genome duplication event ~230-400 million years ago. Transcription of some duplicated fabp genes is modulated by fatty acids (FAs) and/or clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. We had also shown previously that the steady-state level of acyl-CoA oxidase 1 (acox1) mRNA, a marker of PPARα activation, was elevated in liver, intestine, heart and muscle of fish fed clofibrate demonstrating that zebrafish, unlike some fishes, is responsive to this drug. acox1 transcripts were not induced in the brain of fish fed clofibrate, which suggests this drug may not cross the blood brain barrier. Here, we investigated the effect of dietary FAs and clofibrate on the transcription of single copy fabp genes, fabp2, fabp3 and fabp6, in five tissues of inbred zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of fish fed diets differing in FA content. In fish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, were elevated. Based on these findings, modulation of fabp2, fabp3 and fabp6 transcription by FAs and/or clofibrate in zebrafish implicates control of these genes by PPAR interaction with peroxisome proliferator response elements (PPRE) most likely in fabp promoters. Moreover, transcriptional induction of these fabp genes by dietary FAs and/or clofibrate is over-ridden by a tissue-specific mechanism(s), e.g., transcriptional activator or repressor proteins.

Here we report the genomic organization of duplicated cellular retinoic acid-binding protein genes, crabp1 and crabp2, in medaka (Japanese ricefish; Oryzias latipes), the phylogenetic relationship of medaka Crabp1a, Crabp1b, Crabp2a and Crabp2b with other Crabp/CRABP sequences from teleosts/tetrapods, and the tissue-specific distribution of crabp1a, crabp1b, crabp2a, and crabp2b transcripts in adult medaka. The duplicated medaka crabp1 and crabp2 genes contain four exons separated by three introns, which encode polypeptides of 137 and 142 amino acids, respectively. Sequence alignment revealed that medaka Crabp sequences share highest sequence identity and similarity with their orthologs from vertebrates. Phylogenetic analysis confirmed the orthology of the medaka Crabps as they form a distinct clade with their orthologous polypeptides from vertebrates. Conserved gene synteny was evident between the duplicated crabp1 and crabp2 genes from medaka, and CRABP1 and CRABP2 genes from human, which provides compelling evidence that the identified duplicated crabp1 and crabp2 genes from medaka most likely arose owing to teleost-specific whole-genome duplication. The tissue-specific distribution of zebrafish (Danio rerio) and medaka crabp1a, crabp1b, crabp2a, and crabp2b gene transcripts suggests acquisition of new function by these genes in medaka, which may explain potential evolutionary processes that led to the retention of sister duplicates of crabp1 and crabp2 genes in the medaka genome.

In this study, we examined the evolutionary trajectories and the common ancestor of medaka rbp genes by comparing them to the well-studied rbp/RBP genes from zebrafish and other vertebrates. We describe here gene structure, sequence identity, phylogenetic analysis and conserved gene synteny of medaka rbp genes and their putative proteins as well as the tissue-specific distribution of rbp transcripts in adult medaka and zebrafish. Medaka rbp genes consist of four exons separated by three introns that encode putative polypeptides of 134-138 amino acids, a genomic organization characteristic of rbp genes. Medaka Rbp sequences share highest sequence identity and similarity with their orthologs in vertebrates, and form a distinct clade with them in phylogenetic analysis. Conserved gene synteny was evident among medaka, zebrafish and human rbp/RBP genes, which provides compelling evidence that the medaka rbp1, rbp2a, rbp2b, rbp5, rbp7a and rbp7b genes arose from a common ancestor of vertebrates. Moreover, the duplicated rbp2 and rbp7 genes most likely exist owing to a whole-genome duplication (WGD) event specific to the teleost fish lineage. Selection pressure and the nonparametric relative rate test of the medaka and zebrafish duplicated rbp2 and rbp7 genes suggest that these duplicated genes are subjected to purifying selection and one paralog might have evolved at an accelerated rate compared to its sister duplicate since the WGD. The steady-state levels of medaka and zebrafish rbp1, rbp2a, rbp2b and rbp5 transcripts in various tissues suggest that medaka rbp1, rbp2a and rbp2b genes have retained the regulatory elements of an ancestral RBP1 and RBP2 genes, and the medaka rbp5 gene has acquired new function. Furthermore, the tissue-specific regulations of rbp7a and rbp7b genes have diverged markedly in medaka and zebrafish since the teleost-specific WGD.

Here we describe the evolutionary relationship of the duplicated intestinal fatty acid binding protein genes fabp2a and fabp2b from medaka and three-spined stickleback by comparing them to the well-studied fabp2 gene from zebrafish. The duplicated fabp2 genes from medaka and three-spined stickleback consist of four exons separated by three introns, which code for a polypeptide of 132 amino acids. Fabp2a and Fabp2b of medaka and three-spined stickleback share highest sequence identity with zebrafish Fabp2. All Fabp2/FABP2 sequences from vertebrates form a distinct clade in a neighbor-joining phylogenetic tree with a robust 100% bootstrap value, which indicates that the medaka and three-spined stickleback fabp2a and fabp2b are orthologs of zebrafish fabp2. The syntenic genes of fabp2a and fabp2b from medaka and three-spined stickleback were shown to be conserved with the syntenic genes of fabp2/FABP2 from zebrafish and human, evidence that the duplicated fabp2 genes from medaka and three-spined stickleback most likely arose from the teleost-specific whole-genome duplication. The tissue-specific distribution of medaka and three-spined stickleback fabp2a and fabp2b transcripts, and zebrafish fabp2 transcripts, assayed by RT-qPCR suggests the acquisition of new function(s) by the medaka fabp2a, and the distinct evolution of fabp2b compared with fabp2a in the medaka and three-spined stickleback genomes.

A whole-genome duplication (WGD) early in the teleost fish lineage makes fish ideal organisms to study the fate of duplicated genes and underlying evolutionary trajectories that have led to the retention of ohnologous gene duplicates in fish genomes. Here, we compare the genomic organization and tissue-specific transcription of the ohnologous fabp7 and fabp10 genes in medaka, three-spined stickleback, and spotted green pufferfish to the well-studied duplicated fabp7 and fabp10 genes of zebrafish. Teleost fabp7 and fabp10 genes contain four exons interrupted by three introns. Polypeptide sequences of Fabp7 and Fabp10 show the highest sequence identity and similarity with their orthologs from vertebrates. Orthology was evident as the ohnologous Fabp7 and Fabp10 polypeptides of teleost fishes each formed distinct clades and clustered together with their orthologs from other vertebrates in a phylogenetic tree. Furthermore, ohnologous teleost fabp7 and fabp10 genes exhibit conserved gene synteny with human FABP7 and chicken FABP10, respectively, which provides compelling evidence that the duplicated fabp7 and fabp10 genes of teleost fishes most likely arose from the well-documented WGD. The tissue-specific distribution of fabp7a, fabp7b, fabp10a, and fabp10b transcripts provides evidence of diverged spatial transcriptional regulation between ohnologous gene duplicates of fabp7 and fabp10 in teleost fishes.

Here we describe the evolutionary relationship of the duplicated intestinal fatty acid binding protein genes fabp2a and fabp2b from medaka and three-spined stickleback by comparing them to the well-studied fabp2 gene from zebrafish. The duplicated fabp2 genes from medaka and three-spined stickleback consist of four exons separated by three introns, which code for a polypeptide of 132 amino acids. Fabp2a and Fabp2b of medaka and three-spined stickleback share highest sequence identity with zebrafish Fabp2. All Fabp2/FABP2 sequences from vertebrates form a distinct clade in a neighbor-joining phylogenetic tree with a robust 100% bootstrap value, which indicates that the medaka and three-spined stickleback fabp2a and fabp2b are orthologs of zebrafish fabp2. The syntenic genes of fabp2a and fabp2b from medaka and three-spined stickleback were shown to be conserved with the syntenic genes of fabp2/FABP2 from zebrafish and human, evidence that the duplicated fabp2 genes from medaka and three-spined stickleback most likely arose from the teleost-specific whole-genome duplication. The tissue-specific distribution of medaka and three-spined stickleback fabp2a and fabp2b transcripts, and zebrafish fabp2 transcripts, assayed by RT-qPCR suggests the acquisition of new function(s) by the medaka fabp2a, and the distinct evolution of fabp2b compared with fabp2a in the medaka and three-spined stickleback genomes.

Here we describe the structure of the fatty acid‐binding protein 11a and 11b genes (fabp11a and fabp11b) in medaka, and their evolutionary relationship to fabp11 genes from other teleost fishes. Initial studies indicated that the medaka fabp11a gene is intronless, but the fabp11b gene consists of four exons separated by three introns, a genomic organization that is characteristic of most members of the intracellular lipid‐binding protein family. Based on genomic sequence, we conclude that the intronless fabp11a gene most likely arose as a result of reverse transcription of its mRNA transcript into cDNA followed by integration into chromosomal DNA. The ancestral intron‐containing fabp11a gene was presumably lost from the medaka genome. The duplicated fabp11 genes extant in medaka encode polypeptides of 134 amino acids, which share highest sequence identity and similarity, and cluster in a distinct phylogenetic clade, with their orthologs in other teleost fishes. The fabp11a and fabp11b genes in medaka are therefore orthologs of the fabp11a and fabp11b genes, respectively, of other teleost fishes. No conserved gene synteny was found between medaka fabp11a and fabp11a genes from other teleost fishes, supporting our suggestion as to how this intronless gene arose. However, conserved gene synteny was evident between medaka fabp11b and fabp11b genes from other teleost fishes. The tissue‐specific distribution of transcripts for medaka and zebrafish fabp11a and fabp11b genes revealed acquisition of a new function(s) in various tissues by the medaka fabp11b gene, which may explain the retention of sister duplicates of fabp11 in the medaka genome.

We describe the evolutionary diversification of the duplicated ileal fatty acid-binding protein genes (fabp6a and fabp6b) from Japanese ricefish (Oryzias latipes; medaka) and three-spined stickleback (Gasterosteus aculeatus). The fabp6a and fabp6b genes from medaka and three-spined stickleback encode polypeptides of 125–127 amino acids, which share highest sequence identity with their orthologs in teleost fishes and tetrapods. All Fabp6a and Fabp6b from different species cluster together in a distinct clade in phylogenetic analysis and the topology of the tree suggests that fabp6a and fabp6b from medaka and three-spined stickleback are most likely duplicated genes of an ancestral FABP6 owing to teleost-specific whole-genome duplication. However, the topology of an alternate phylogenetic tree revealed that the duplication of the ancestral FABP6 that gave rise to the extant fabp6a and fabp6b possibly occurred before the divergence of tetrapods and fishes. Conserved gene synteny was evident between the teleost fabp6a and fabp6b genes and the human FABP6 gene. The tissue-specific distribution of fabp6a transcripts suggests the retention of ancestral function(s) of the fabp6a gene in medaka and three-spined stickleback with acquisition of new function(s) in different tissues. However, the tissue-specific regulation of the fabp6b gene has diverged markedly in medaka and three-spined stickleback since the duplication of the fabp6 gene.