Evolution of the duplicated intracellular lipid-binding protein genes of teleost fishes.

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.

Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish.

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.

Divergent evolution of cis-acting peroxisome proliferator-activated receptor elements that differentially control the tandemly duplicated fatty acid-binding protein genes, fabp1b.1 and fabp1b.2, in zebrafish.

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.

Fatty acid-binding protein (fabp) genes of spotted green pufferfish (Tetraodon nigroviridis): comparative genomics and spatial transcriptional regulation.

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.

Comparative evolutionary genomics of medaka and three-spined stickleback fabp2a and fabp2b genes with fabp2 of zebrafish.

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.

Comparative genomic organization and tissue-specific transcription of the duplicated fabp7 and fabp10 genes in teleost fishes.

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.

Tissue-specific differential induction of duplicated fatty acid-binding protein genes by the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio).

BACKGROUND: 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. RESULT: 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. CONCLUSION: 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.

Lexical Diversity, Lexical Sophistication, and Predictability for Speech in Multiple Listening Conditions.

When talkers anticipate that a listener may have difficulty understanding their speech, they adopt a speaking style typically described as "clear speech." This speaking style includes a variety of acoustic modifications and has perceptual benefits for listeners. In the present study, we examine whether clear speaking styles also include modulation of lexical items selected and produced during naturalistic conversations. Our results demonstrate that talkers do, indeed, modulate their lexical selection, as measured by a variety of lexical diversity and lexical sophistication indices. Further, the results demonstrate that clear speech is not a monolithic construct. Talkers modulate their speech differently depending on the communication situation. We suggest that clear speech should be conceptualized as a set of speaking styles, in which talkers take the listener and communication situation into consideration.

Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctionalization of duplicated genes.

BACKGROUND: In 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. RESULT: FA 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. CONCLUSION: Differential 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.

Tandem duplication of the fabp1b gene and subsequent divergence of the tissue-specific distribution of fabp1b.1 and fabp1b.2 transcripts in zebrafish (Danio rerio).

We describe a fatty acid-binding protein 1 (fabp1b.2) gene and its tissue-specific expression in zebrafish embryos and adults. The 3.5 kb zebrafish fabp1b.2 gene is the paralog of the previously described zebrafish fabp1a and fabp1b genes. Using the LN54 radiation hybrid mapping panel, we assigned the zebrafish fabp1b.2 gene to linkage group 8, the same linkage group to which fabp1b.1 was mapped. fabp1b.1 and fabp1b.2 appear to have arisen by a tandem duplication event. Whole-mount in situ hybridization of a riboprobe to embryos and larvae detected fabp1b.2 transcripts in the diencephalon and as spots in the periphery of the yolk sac. In adult zebrafish, in situ hybridization revealed fabp1b.2 transcripts in the anterior intestine and skin, and reverse transcription PCR (RT-PCR) detected fabp1b.2 transcripts in the intestine, brain, heart, ovary, skin, and eye. By contrast, fabp1b.1 transcripts were detected by RT-PCR in the liver, intestine, heart, testis, ovary, and gills. The tissue-specific distribution of transcripts for the tandemly duplicated fabp1b.1 and fabp1b.2 genes in adult tissues and during development suggests that the duplicated fabp1b genes of zebrafish have acquired additional functions compared with the ancestral fabp1 gene, i.e., by neofunctionalization. Furthermore, these functions were subsequently divided between fabp1b.1 and fabp1b.2 owing to subfunctionalization.

The evolutionary relationship between the duplicated copies of the zebrafish fabp11 gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes.

We describe the structure of a fatty acid-binding protein 11 (fabp11b) gene and its tissue-specific expression in zebrafish. The 3.4 kb zebrafish fabp11b is the paralog of the previously described zebrafish fabp11a, with a deduced amino acid sequence for Fabp11B exhibiting 65% identity with that of Fabp11A. Whole mount in situ hybridization of a riboprobe to embryos and larvae showed that zebrafish fabp11b transcripts were restricted solely to the retina and were first detected at 24 h postfertilization. In situ hybridization revealed fabp11b transcripts along the spinal cord in adult zebrafish. However, the highly sensitive RT-PCR assay detected fabp11b transcripts in the brain, heart, ovary and eye in adult tissues. By contrast, fabp11a transcripts had been previously detected in the liver, brain, heart, testis, muscle, ovary and skin of adult zebrafish. Using the LN54 radiation hybrid panel, we assigned zebrafish fabp11b to linkage group 16. Phylogenetic analysis and conserved gene synteny with tetrapod genes indicated that the emergence of two copies of fabp11 in the zebrafish genome may have resulted from a fish-specific whole genome duplication event. Furthermore, we propose that the FABP4-FABP5-FABP8-FABP9 (PERF15) gene cluster on a single chromosome in the tetrapod genome and the fabp11 genes in the zebrafish genome originated from a common ancestral gene, which, following their divergence, gave rise to the fabp11 genes of zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9 genes in tetrapods after the separation of the fish and tetrapod lineages.

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio).

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene.

Fatty acid-binding protein genes of the ancient, air-breathing, ray-finned fish, spotted gar (Lepisosteus oculatus).

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.

The fabp4 gene of zebrafish (Danio rerio)--genomic homology with the mammalian FABP4 and divergence from the zebrafish fabp3 in developmental expression.

Teleost fishes differ from mammals in their fat deposition and distribution. The gene for adipocyte-type fatty acid-binding protein (A-FABP or FABP4) has not been identified thus far in fishes. We have determined the cDNA sequence and defined the structure of a fatty acid-binding protein gene (designated fabp4) from the zebrafish genome. The polypeptide sequence encoded by zebrafish fabp4 showed highest identity to the H(ad)-FABP or H6-FABP from Antarctic fishes and the putative orthologs from other teleost fishes (83-88%). Phylogenetic analysis clustered the zebrafish FABP4 with all Antarctic fish H6-FABPs and putative FABP4s from other fishes in a single clade, and then with the mammalian FABP4s in an extended clade. Zebrafish fabp4 was assigned to linkage group 19 at a distinct locus from fabp3. A number of closely linked syntenic genes surrounding the zebrafish fabp4 locus were found to be conserved with human FABP4. The zebrafish fabp4 transcripts showed sequential distribution in the developing eye, diencephalon and brain vascular system, from the middle somitogenesis stage to 48 h postfertilization, whereas fabp3 mRNA was located widely in the embryonic and/or larval central nervous system, retina, myotomes, pancreas and liver from middle somitogenesis to 5 days postfertilization. Differentiation in developmental regulation of zebrafish fabp4 and fabp3 gene transcription suggests distinct functions for these two paralogous genes in vertebrate development.

Differential regulation of the duplicated fabp7, fabp10 and fabp11 genes of zebrafish by peroxisome proliferator activated receptors.

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.

Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome.

Fatty acid-binding protein type 1 (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is encoded by a single gene in mammals. We cloned and sequenced cDNAs for two distinct FABP1s in zebrafish coded by genes designated fabp1a and fabp1b. The zebrafish proteins, FABP1a and FABP1b, show highest sequence identity and similarity to the human protein FABP1. Zebrafish fabp1a and fabp1b genes were assigned to linkage groups 5 and 8, respectively. Both linkage groups show conserved syntenies to a segment of mouse chromosome 6, rat chromosome 4 and human chromosome 2 harboring the FABP1 locus. Phylogenetic analysis further suggests that zebrafish fabp1a and fabp1b genes are orthologs of mammalian FABP1 and most likely arose by a whole-genome duplication event in the ray-finned fish lineage, estimated to have occurred 200-450 million years ago. The paralogous fabp10 gene encoding basic L-FABP, found to date in only nonmammalian vertebrates, was assigned to zebrafish linkage group 16. RT-PCR amplification of mRNA in adults, and in situ hybridization to whole-mount embryos to fabp1a, fabp1b and fapb10 mRNAs, revealed a distinct and differential pattern of expression for the fabp1a, fabp1b and fabp10 genes in zebrafish, suggesting a division of function for these orthogolous and paralogous gene products following their duplication in the vertebrate genome. The differential and complementary expression patterns of the zebrafish fabp1a, fapb1b and fabp10 genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated fabp1a and fabp1b genes, and the fabp10 gene in the zebrafish genome.

Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development.

The cellular retinoic acid-binding protein 2 (CRABP2) is believed to be involved in regulating access of retinoic acid to nuclear retinoic acid receptors. We have determined the cDNA sequence and the genomic organization of the duplicated crabp2 gene (crabp2b) in zebrafish. The crabp2b cDNA was 522bp in length and encodes a polypeptide consisting of 146 amino acids. Radiation hybrid mapping assigned the crabp2b gene to zebrafish linkage group 19. The comparison of the mapped human CRABP2 gene, zebrafish crabp2a and zebrafish crabp2b genes revealed that human chromosome 1 has a syntenic relationship to zebrafish linkage groups 16 and 19. Reverse transcription-polymerase chain reaction (RT-PCR) detected crabp2b mRNA in total RNA extracted from whole adult zebrafish, but not in any of the adult zebrafish tissues examined. The crabp2a mRNA was detected in total RNA extracted from whole adult zebrafish, adult zebrafish muscle, testes, and skin and to a lesser extent in heart, ovary and brain. No crabp2a mRNA-specific product was detected in kidney, liver or intestine of the adult zebrafish. Whole mount in situ hybridization detected crabp2b and crabp2a mRNA in a number of structures known to require retinoic acid signaling during embryonic development. The crabp2b mRNA was detected in the central nervous system, branchial arches, pectoral fins, retina (dorsal to the lens), epidermis and otic vesicle of the developing zebrafish. The crabp2a transcripts were detected by whole mount in situ hybridization in the central nervous system, epidermis, proliferative zone of the retina, intestinal bulb, oesophagus, pectoral fins and branchial arches during zebrafish embryonic development.

Retention of the duplicated cellular retinoic acid-binding protein 1 genes (crabp1a and crabp1b) in the zebrafish genome by subfunctionalization of tissue-specific expression.

The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals. We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b. These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs. The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively. Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus. Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication. Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis. crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages. In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization. The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome.

Divergent spatial regulation of duplicated fatty acid-binding protein (fabp) genes in rainbow trout (Oncorhynchus mykiss).

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 30×10(4)-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.

A cellular retinoic acid-binding protein from zebrafish (Danio rerio): cDNA sequence, phylogenetic analysis, mRNA expression, and gene linkage mapping.

We report the sequence of a cDNA clone coding for a cellular retinoic acid-binding protein (CRABP) in zebrafish. The encoded polypeptide is 142 amino acids in length with an estimated molecular mass of 15.8 kDa and a calculated isoelectric point of 5.2. The zebrafish CRABP exhibits highest sequence identity to the pufferfish CRABPIIa (83%) and CRABPIIb (79%), and human CRABPII (74%) than to any other member of the intracellular lipid-binding protein (ILBP) family. A phylogenetic tree for different members of the ILBP multigene family including fatty acid-binding proteins (FABPs), cellular retinol-binding proteins (CRBPs) and CRABPs shows that the cloned zebrafish cDNA encodes a protein that clusters with CRABPs from other species and not with CRBPs and FABPs. Reverse-transcription polymerase chain reactions (RT-PCR), using oligonucleotide primers specific to the zebrafish CRABP cDNA made from total RNA of embryos collected at various developmental stages, did not detect the CRABP mRNA until 12 h post-fertilization. In adult zebrafish, CRABP mRNA was detected by RT-PCR in total RNA extracted from muscle, testes and skin, barely detectable in heart, ovary and brain and undetectable in liver, kidney and intestine. Quantitative RT-PCR (qRT-PCR) revealed a similar tissue-specific distribution for zebrafish CRABP mRNA with highest levels of CRABP mRNA in muscle followed by testes, skin, ovary and much lower levels in heart. Radiation hybrid mapping assigned the CRABP gene to linkage group 16 in the zebrafish genome. Comparison of the mapped zebrafish CRABP and human CRABPII genes revealed that zebrafish linkage group 16 has a syntenic relationship with human chromosome 1. Based on phylogenetic analysis and the syntenic relationship to the CRABPII gene in human, the zebrafish cDNA clone appears to code for a type II CRABP.