Differential Transcriptional Modulation of Duplicated Fatty Acid-Binding Protein Genes by Dietary Fatty Acids in Zebrafish {Danio reno)
by
Santhosh Karanth
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
at
Dalhousie University Halifax, Nova Scotia August 2010
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Mom, Tara and Vish Bhat
"Life is short, craft long to learn, opportunity fleeting, experiments deceptive and judgment difficult". -Hippocrates
IV Table of Contents
List of Tables ix
List of Figures ?
Abstract xii
List of Abbreviations Used xiii
Acknowledgements xiv
Chapter 1: Introduction 1
Nomenclature and Classification of iLBP Genes 1
Gene and Genome Duplications, and the Molecular Evolution of iLBP 2 Multigene Family in Zebrafish Structure and Functions of iLBPs 7 Spatio-Temporal Distribution of Zebrafish iLBP Transcripts in Developing „ Embryos and Larvae Tissue-Specific Distribution of iLBP Transcripts in Adult Zebrafish 10
Transcriptional Regulation of iLBP Genes 1 1
Lipid Homeostasis in Teleost Fishes 14
Objectives of This Study 16 Chapter 2: Tandem Duplication ofTh&fabplb Gene and Subsequent Divergence of the Tissue-Specific Distribution oifabplb.l andfabplb.2 Transcripts in Zebrafish 17 (Danio rerio) Abstract 18
Introduction 19
Materials and Methods 21
Husbandry of Zebrafish 2 1
Nucleotide Sequence of the Zebrafish/a¿>pi¿>. 2 cDNA and its Gene 21
Phylogenetic Analysis 22
? Radiation Hybrid Mapping of the Zebrafishfabplb.2 Gene 22 In Situ Hybridization to Whole-Mount Embryos and Larvae, and to Sections of Adult Zebrafish 22 RT-PCR Detection offabplb.2 Transcripts in Adult Zebrafish 2_ Tissues Results and Discussion 24
Identification of a Duplicatedfabpl b Gene in the Zebrafish Genome 24 Duplicate Copies offabplb in the Zebrafish Genome may have Arisen by a Tandem Duplication Event 29 Distribution offabplb.2 Transcripts in Developing Zebrafish Embryos and Larvae 3 1 Tissue-Specific Distribution offabplb.2 Transcripts in Adult Zebrafish 34
Divergence of the Tissue-Specific Distribution offabplb. 1 and fabplb.2 Transcripts 36 Chapter 3: The Evolutionary Relationship Between the Duplicated Copies of the Zebïanshfabpl 1 Gene and the Tetrapod FABP4, FABP5, FABP8 and FABP9 39 Genes Abstract 40
Introduction 41
Materials and Methods 43
Husbandry of Zebrafish 43 Nucleotide Sequence of the Zebrafishfabpl 1 b cDNA and Its Gene 43 Phylogenetic Analysis 44
Radiation Hybrid Mapping of the Zebrafishfabpllb Gene 44 In Situ Hybridization to Whole-Mount Embryos and Larvae, and to Sections of Adult Zebrafish 44
RT-PCR Detection offabpllb Transcripts in Adult Zebrafish Tissues 45
Vl Database Searches of Genome Sequences and Identification of . , Transcription Factor Binding Sites Results and Discussion 46
Identification of a Duplicatedfabpllb Gene from Zebrafish 46 Distribution offabpllb Transcripts in the Retina of Developing Zebrafish Embryo and Larvae 49
Tissue-specific Distribution offabpllb Transcripts in Adult Zebrafish 49
Duplicate Copies offabpll in Zebrafish may have Arisen by a Fish Specific Whole-Genome Duplication Event 5 1 fabpll, FABP4, FABP5, FABP8, and FABP9 Evolved from a Common Ancestral Gene on a Single Progenitor Chromosome 58 The Tetrapod FABP5, FABP8, FABP9 and FABP4 Genes are Tandernly Arrayed and Likely Arose by Unequal Crossing-Over. f. Which Gene was Duplicated First? Chapter 4: Differential Transcriptional Modulation of Duplicated Fatty Acid- Binding Protein Genes by Dietary Fatty Acids in Zebrafish {Danio rerio): Evidence 66 for Subfunctionalization or Neofunctionalization of Duplicated Genes Abstract 67
Introduction 69
Materials and Methods 74
Diets and Fish Husbandry 74
RNA Isolation, cDNA Synthesis and RT-qPCR 78
Lipid Extraction, FAME Preparation and Gas Chromatography 80
Statistical Analysis 8 1
Results 81 The Steady-State Level oifabp mRNAs Does not Differ Between „ - Sexes of Zebrafish Effect of Diet on the FA Composition in Different Tissues of „ - Zebrafish
VIl Effect of Diet on the Steady-State Level of fabpla/fabplb.l/fabplb.2 mRNAs in Different Tissues 82 Effect of Diet on the Steady-State Level of Fabp7alFabp7b mRNA in Different Tissues. 89
Effect of Diet on Steady-State Level of Fabp11 alFabpllb mRNA in Different Tissues 89
Modulation of the Steady-State Level oífabp mRNA is Due to Up- Regulation of Transcriptional Initiation 94
Discussion 97
Effect of Diet on FA Profiles in Tissues of Zebrafish 97
Dietary FAs Modulate the Steady-State Level offabp mRNAs 97 Differential Modulation oîfabplb.l, but notfabpla andfabplb.2 1 __ Transcription by Dietary FAs Tissue-Specific Transcriptional Modulation offabp7a andfabp7b mRNAs by Dietary FAs 1 02 Transcriptional Modulation offabplla, but notfabpllb, by Dietary 1 m FAs U Conclusion 103
Acknowledgements 104
Chapter 5: Conclusion 105 Role of Gene and Whole-Genome Duplications in the Evolution 1 _ _ of iLBP Multigene Family in Zebrafish Retention of Duplicatedfabp Genes in the Zebrafish Genome 106 Subfunctionalization/Neofunctionalization of Duplicatedfabp Genes in the Context of Tissue-Specific Distribution of Their Transcripts 107
Subfunctionalization/Neofunctionalization of Duplicated fabp Genes in the 109 Context of Their Transcriptional Induction by Dietary FAs Bibliography 115
Appendix A. Copyright Permission Letter from FEBS Journal 132
Appendix B. Author's Rights Policy of NRC Press 133
vni List of Tables
Table 1 . 1 Zebrafish iLBP genes assigned to different linkage groups based on 5 radiation hybrid mapping (LN54 panel) Table 1.2 Transcript distribution of iLBP genes in different tissues of adult 12 zebrafish Table 2. 1 Tissue-specific distribution oífabpl transcripts in embryos and adults 37 of mammals (fabpl) and zebrafish (fabpla,fabplb.l andfabplb.2) Table 3.1 Conserved gene synteny of the duplicated copies of the zebrafish 57 fabpl 1 gene with the humanfabp4,fabp5,fabp8 andfabp9 genes Table 4. 1 Composition of experimental diets 75
Table 4.2 Major fatty acids in the experimental diets 77
Table 4.3 Primers used for the quantification offabp mRNA and hnRNA 79
Table 4.4 Fatty acid composition in intestine of zebrafish fed experimental diets 84
Table 4.5 Fatty acid composition in liver of zebrafish fed experimental diets 85
Table 4.6 Fatty acid composition in muscle of zebrafish fed experimental diets 86
Table 4.7 Fatty acid composition in brain of zebrafish fed experimental diets 87
Table 4.8 Induction of the steady-state level of mRNA and hnRNA coded by 99 fabp genes in tissues of zebrafish fed one of the four diets differing in FA content
Table 5.1 Tissue-specific distribution offabpl (fabp1 a, fabplb. 1 andfabplb.2) 108 andfabpl 1 (fabpl la andfabpllb) transcripts in embryos and adults of zebrafish
IX List of Figures Figure 2.1 The genomic organization of the zebvañshfabplb.2 gene 25
Figure 2.2 Phylogenetic tree of selected vertebrate Fabps showing the 27 relationship between zebrafish Fabpla, Fabplb.l and Fabplb.2. Figure 2.3 Genomic organization of the duplicatedfabplb. 1 (left) andfabplb.2 30 (right) genes on zebrafish chromosome 8 Figure 2.4 Spatio-temporal distribution offabplb.2 transcripts in embryos and 32 larvae of zebrafish and the tissue-specific distribution offabplb.2 transcripts in sections of adult zebrafish Figure 2.5 Detection offabplb. 1 andfabplb.2 transcripts by RT-PCR in RNA 35 extracted from tissues of adult zebrafish Figure 3.1 The sequence of the zebrafishfabpll b gene and its 5 -upstream 47 promoter region Figure 3.2 Spatio-temporal distribution offabpllb transcripts during zebrafish 50 embryonic and larval development Figure 3.3 Tissue-specific distribution offabpllb mRNA in adult zebrafish 52 sections determined by in situ hybridization Figure 3.4 RT-PCR detection offabpllb transcripts in RNA extracted from 53 adult tissues of zebrafish using fabpllb cDNA-specific primers Figure 3.5 Multiple sequence alignment of Fabp lib with Fabp 11a and other 55 vertebrates Fabps. Figure 3.6 Phylogenetic tree of selected vertebrate Fabps showing the 59 relationship between zebrafish Fabpl la and Fabpl lb Figure 3.7 Evolutionary relationship between the duplicated copies of thefabpll 63 gene in fishes andfabp4,fabp5,fabp8 mdfabp9 (perflS) in frog, chicken, mouse, and human. Figure 4. 1 Fatty acid composition of the four major fatty acids in intestine, liver, 83 muscle, and brain of zebrafish fed experimental diets Figure 4.2 The steady-state level offabpla anafabplb.l mRNA in the intestine 88 of zebrafish fed diets differing in FA content Figure 4.3 The steady-state level offabp7a andfabp7b mRNA in intestine, liver, 90 muscle, and brain of zebrafish fed diets differing in FA content
? Figure 4.4 The steady-state level offabplla mRNA in intestine, liver, muscle 92 and brain of zebrafish fed diets differing in FA content Figure 4.5 The steady-state level of hnRNA forfabplb.l in intestine, fabp7a in 95 liver, fabpla in muscle, fabp7b in intestine, fabp7b in brain, and fabplla in muscle of zebrafish fed diets differing in FA content Figure 5.1 A model to account for the dietary FAs mediated differential 111 induction of duplicatedfabp genes in zebrafish Figure 5.2 Divergent evolution and coevolution of DNA binding domains of 112 duplicated nuclear receptors and PPRE in the promoters of duplicated fabp genes leading to either no transcriptional activation or transcriptional activation offabpb.
Xl Abstract
In the Duplication-Degeneration-Complementation (DDC) model, subfuctionalization and neofunctionalization have been proposed as major 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. Many duplicated genes exist in teleost fishes as a result of a whole-genome duplication event that occurred early in the ray-finned fish lineage 250-400 million years ago. To test the DDC model, I chose to determine whether duplicated fatty acid-binding protein (fabp) genes are retained in the zebrafish genome owing to either subfunctionalization or neofunctionalization. I first determined the spatio-temporal distribution of transcripts for thefabplb.2 andfabpllb genes by two methods: (i) in situ hybridization of riboprobes to embryos, larvae and sections of adult zebrafish, and (ii) reverse-transcription, polymerase chain reaction (RT- PCR) using RNA extracted from adult tissues. The results of these initial studies showed that the spatio-temporal distribution of duplicated copies for the fabpl andfabp11 genes have markedly diverged. Furthermore, comparison of the spatio-temporal distribution of the duplicated copies of the zebrafishfabpl and fabp11 genes with that of their single- copy mammalian orthologs indicated that the zebrafishfabp duplicates had been retained in the zebrafish genome owing to either subfunctionalization or neofuctionalization. In a second experimental approach to test the DDC model, transcriptional induction of duplicated zebrafishfabp genes by fatty acids (FAs), compounds known to induce the transcription of several mammalianfabp genes, was assayed by quantitative RT-PCR. Adult zebrafish fed four diets differing in FA content exhibited different FA profiles in intestine, brain, muscle and liver depending on diet. Following quantitative RT-PCR, steady-state levels offabp7b transcripts were induced in brain by a diet rich in linolate; fabplb.l andfabp7b transcripts in intestine were elevated by a diet rich in linolenate; fabpVa transcripts in liver were elevated in fish fed a low lipid diet; andfabp7 and fabplla transcripts were elevated in muscle by diets that were either enriched in linolate, or contained low lipid content. None of the sister duplicates of thesefabp genes exhibited an increase in the steady-state transcript levels in tissues of fish fed one of the four diets. Moreover, the level of heterogeneous nuclear RNA for a givenfabp gene correlated with the induction of the steady-state level of mRNA transcripts indicating that up-regulation offabp transcripts occurred at the site of transcriptional initiation. The differential transcriptional induction of duplicated zebrafishfabp genes by dietary FAs provides further evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.
XIl List of Abbreviations Used
CRABP Cellular retinoic acid-binding protein
CRBP Cellular retinol-binding protein DDC Duplication-degeneration-complementation
DHA Docosahexaenoic acid
EPA Eicosapentaenoic acid EST Expressed sequence tag
FA Fatty acid FABP Fatty acid-binding protein HD Highly unsaturated FA-rich diet hpf Hours postfertilization iLBP Intracellular lipid-binding protein
LD Linoleic acid-rich diet
LFD Low fat diet
LG Linkage group
LND Linolenic acid-rich diet mya Million years ago
PPAR Peroxisomes proliferator activated receptor
RXR Retinoid X receptor RT-PCR Reverse transcription polymerase chain reaction Reverse transcription, quantitative polymerase chain RT-qPCR reaction
SEM Standard error of means
WGD Whole-genome duplication
XlIl Acknowledgements I thank my supervisor Dr. Jonathan M Wright for his kind and supportive approach in mentoring me. I am also grateful to him for patiently improving my writing skills. My co-supervisor Dr. Eileen M Denovan-Wright has been a source of technical expertise and also a detailed critique of experimental design and manuscripts, for which I am indebted to her. I thank my co-supervisor Dr. Santosh P LaIl for his overall support in general and for help in nutritional studies and fatty-acid analysis. Whole-mount in situ hybridization of zebrafish embryos and larvae was performed in the laboratory of Drs. Christine Thisse and Bernard Thisse. Sean Tibbetts and Joyce Milley helped me in diet preparation and fatty acid analysis. I am indebted to my family and friends for their love and camaraderie.
XlV Chapter 1: Introduction The intracellular lipid-binding proteins (iLBPs) belong to the calcyn protein super family, which also consists of lipocalins and avidins (Rüssel and Sternberg, 1996). The iLBP multigene family consists of the fatty acid- (FABP), cellular retinol- (CRBP) and cellular retinoic acid-binding protein (CRABP) genes which are involved in the intracellular transport of fatty acids (FAs) and other hydrophobic ligands, such as retinol and retinoic acid (Bernlohr et al., 1997; Glatz et al., 2003). The first iLBP gene emerged
after the divergence of animals from plants and fungi -930-1000 million years ago (Schaap et al., 2002). The ancestral iLBP gene might have undergone a series of duplications followed by sequence divergence, giving rise to the diversity observed in the extant iLBP multigene family. Currently, 18 paralogous iLBP genes have been identified
in vertebrates.
Nomenclature and Classification of iLBP Genes
Originally, iLBP genes and their proteins were named according to the initial tissue of isolation, e.g., liver-type (L-FABP), intestinal-type FABP (I-FABP). Owing to some confusion with this earlier nomenclature, I use the nomenclature proposed by
Hertzel and Bernlohr (2000), in which numerals distinguish the different FABP proteins and their genes, e.g., FABPl, FABP7, etc. The iLBP multigene family has been divided into four subfamilies based on the phylogenetic analysis of the amino acid sequences of the proteins (Haunerland and Spener, 2004). The members of subfamily 1 function as ligand acceptors for vitamin A and its derivatives. They are the cellular retinoic acid- binding proteins (CRABPl and 2) and cellular retinol-binding proteins (CRBPl, 2, 5, and 7) (Haunerland and Spener, 2004). Subfamily 2 consists of FABPl, FABP6, and
1 FABPlO. Subfamily 3 contains only one member, i.e. FABP2 and subfamily 4 comprises of FABP3, FABP4, FABP5, FABP7, FABP8, FABP9, FABPl 1 and FABP12.
Gene and Genome Duplications, and the Molecular Evolution of LLBP Multigene Family in Zebrafish There are different ways by which genes are duplicated. First, small-scale gene duplications arise mainly because of tandem duplication or retrotransposition (Hurles, 2004). Unequal crossing over, between homologous chromosomes during meiosis, may lead to tandem duplication of a single gene or many genes on a segment of chromosomes (Hurles, 2004). Tandem duplications can also occur because of nonhomologous recombination by replication-dependent chromosome breakages (Fan et al., 2008).
Retrotransposition describes the action of retrotransposons that integrates the reverse transcribed mature RNAs at arbitrary sites in a genome (Hurles, 2004). Because they are derived from mRNA, duplicated genes resulting from retrotransposition, lack introns and have poly-A sequence. In most cases, duplicates resulting from retrotransposition do not produce a viable mRNA transcript because they lack regulatory elements that activate their transcription (Hurles, 2004). Second, large-scale gene duplications like whole- genome duplications (WGDs) may result from polyploidy (Prince and Pickett, 2002). Polyploid organism possess more than two sets of haploid chromosomes. Allopolyploidy results from the hybridization of two closely related species and is very common in plants (Klug et al., 2006) whereas autopolyploids are the result of sexual hybridization within the same species. In Susumu Ohno's widely cited book, 'Evolution by Gene Duplication' (1970), he argued that one of the mechanisms that assists the increasing complexity in evolution of life is the duplication of genes and whole genomes. While a number of researchers before
2 Ohno, discussed the role of gene duplicates in evolution of morphological and functional diversity (Bridges, 1918, reviewed in Taylor and Raes, 2004), it was Ohno who combined the evidence on gene and genome duplications and proposed two different evolutionary fates for the duplicated genes (Hahn, 2009). Ohno argued that most duplicated genes are lost from the genome owing to nonfunctionalization, a claim that has been supported by Lynch and Conery (2000). Nonfunctionalization is a process where deleterious mutations accumulate in the coding region of a gene giving rise to either a dysfunctional protein or no protein product. Duplicated genes might, however, be retained in the genome owing to mutations in the coding region that led to a novel function for the protein product of the gene, a process Ohno termed 'neofunctionalization'. According to Ohno the process of 'neofunctionalization5 allowed duplicated genes to diversify by gaining a new function(s), and facilitate the evolutionary changes. The ancestor of present day ray-finned fishes is thought to have undergone a
WGD event 230 - 400 million years ago (Amores et al., 1998; Jaillon et al., 2004; Woods et al., 2005; Kasahara et al., 2007). Empirical data from the analysis of HOX gene clusters in human, mouse and zebrafish has been used as evidence for a WGD early in the radiation of the ray-finned fish lineage (Amores et al., 1998). Invertebrates have a single cluster of HOX genes, whereas humans and mice have four clusters ?? HOX genes distributed on four different chromosomes. The presence of four clusters of HOX genes
on four different chromosomes in tetrapods is thought be the result of two rounds of
genome duplications in the ancestral vertebrate (Prince and Pickett, 2002). Interestingly, zebrafish have seven hox clusters, apparently originated from the fish-specific WGD.
3 The double recessive model based on the Ohno's ideas predicts that most gene duplicates should become nonfunctional within a short period of time (Force et al. 1999). However, data from the genome sequencing projects suggest that a much greater proportion of gene duplicates are preserved in the eukaryotic genomes than predicted by the double recessive model based on the Ohno's ideas (Force et al. 1999). For example,
72% of the duplicated genes from a recent polyploidy event have been retained in maize (White and Doebley, 1998), and about 50% of duplicated genes have avoided nonfunctionalization in Xenopus laevis (Hughes and Hughes, 1993). In 1999, Force et al. proposed the Duplication-Degeneration-Complementation (DDC) model in which
subfunctionalization and neofunctionalization serves as alternative mechanisms, for the preservation of duplicated genes. According to the DDC model, duplicated genes are retained in the genome either by subfunctionalization, where the functions of the
ancestral gene are sub-divided between sister duplicate genes, or by neofunctionalization, where one of the duplicates acquires a new function. In this model, both processes occur by either loss or gain of c/s-acting regulatory elements in the promoters of the duplicated
genes.
To date, 20 zebrafish iLBP genes have been characterized. Among which 16 (eight pairs) iLBP genes are sister duplicates of each other (Table 1 . 1 and references
therein). With the exception offabplO andfabpll, mammalian orthologs have been described for all the zebrafish iLBP genes (Table 1.1 and references therein). The
4 Table 1.1. Zebrafish iLBP genes assigned to different linkage groups based on radiation hybrid mapping (LN54 panel).
Zebrafish Vertebrate Zebrafish Reference gene orthologue/s Linkage group fabpla FABPl (Sharma et al., 2006) fabplb fahp2 FABP2 (Sharma et al., 2004) 19 fahp3 FABP3 (Liu et al, 2003a) 21 fabp6 FABP6 (Alves-Costa et al., 2008) 17 FABP7 (Liu et al., 2004a) fabp7b 20
(Sharma et al., 2006) Chicken FABP10 19 (Venkatachalam et al., 2009) FABP4, FABP5, 19 (Liu et al., 2007) lfabpll FABP8, FABP9 15 rbpla (Cameron et al. 2002) RBPl rbplb (Liu et al., 2005b) 16 rbpla (Liu et al., 2004b) RBP2 rbplb (Liu et al., 2005b) rbp7a RBP7 (Belliveau et al., 2010) rbp7b 23 crabpla 25 CRABPl (Liu et al, 2005a) crabplb crabpla 16 CRABP2 (Sharma et al, 2005) crabplb 19
5 FabplO gene is present in the genomes of birds, reptiles and fishes, but not mammals suggesting thatfabplO was lost from mammalian genomes after the divergence of this lineage (Sharma et al., 2006). In zebrafish some duplicate genes (including several fabp genes) are clustered on duplicated chromosomal segments on chromosomes 2/24, 3/12, 5/10, 7/25, 16/19, and 17/20 with each chromosome pair harboring a copy of the duplicated gene (Woods et al., 2005). LN54 radiation hybrid panel was used to assign iLBP genes to zebrafish chromosomes. Four of the eight iLBP duplicate pairs are located on the chromosomes 7/25, 16/19, and 17/20, with each chromosome containing a copy of its sister duplicate
(Table 1.1 and references therein). Conserved gene synteny has been used to establish those regions of ancestral chromosomes which have not changed over the course of evolution (Woods et al., 2005). Duplicated iLBP gene pairs found on the different zebrafish linkage groups, together share the same gene order found in mammalian chromosomes, as shown in the case of crbpls, crbp2s, crabpls,fabp7s etc. (Liu et al., 2004a; Liu et al., 2004b; Liu et al., 2005a; Liu et al., 2005b; Sharma et al., 2005; Sharma et al., 2006; Liu et al., 2007;
Venkatachalam et al., 2009; Belliveau et al., 2010). Other zebrafish iLBP genes also show conserved gene synteny with their mammalian orthologs (Liu et al., 2003a; Sharma et al., 2004; Alves-Costa et al., 2008) suggesting that the ancestral vertebrate chromosomes are conserved in some instances in both zebrafish and mammals since they diverged from each other 400 million years ago (Kumar and Hedges, 1998). Zebrafish FABP genes, fabp2, fabp3 andfabp6 exist as single copies, whereas fugu, medaka and stickleback have two copies oïfabpl (Lai et al., 2009). mRNA of two fabp3 isoforms
6 has been detected in In Antarctic teleost fishes (Vayda et al., 1998). Most probably following the WGD event, the sister duplicates of zebrafishfabp2, fabp3 mdfabpó have presumably been lost by the accumulation of mutations leading to functional decay. The occurrence of duplicated genes in the duplicated stretches of chromosomes and conserved gene synteny in the duplicated blocks of zebrafish chromosomes and mammalian chromosomes provides compelling evidence that the duplicated copies of zebrafish iLBP genes originated from the teleost-specific WGD.
Structure and Functions of iLBPs
The molecular mass of iLBPs is approximately 15 kDa. iLBPs have a hydrophobic core surrounded by a twisted barrel (Chmurzynska, 2006). The protein has 10 antiparallel ß-strands (Stewart, 2000). The strands are arranged orthogonally and have a helix-turn-helix motif at the N-terminal (Sacchettini et al., 1988). The binding pocket opening is framed by the N-terminal helix-turn-helix domain. iLBPs play an important role in lipid, retinol, and retinoic acid metabolism. Functions of FABP, CRBP and CRABP include the intracellular transport of FAs, retinol and retinoic acid between different organelles, sequestering of excess hydrophobic ligands and protection of cells from the detergent effects of FAs (Bernlohr et al., 1997). Some FABPs also bind lysophospholipids, prostaglandins, phytanic acid, eicosanoids, heme and acyl-CoAs (Coe and Bernlohr, 1998; Wolfrum et al., 1999; and Zimmerman and Veerkamp, 2002). Furthermore, several intracellular functions have been proposed for iLBPs that include regulation of gene expression, and the control of cell growth and differentiation (Veerkamp and Maatmann, 1995). Different FABP knockout mouse models have been generated in the last decade (Reviewed in Haunerland and Spener,
7 2004). However, the deletion of particular FABP gene has not resulted in drastic phenotypical changes. It is thought that the compensatory over-expression of other members of the iLBP multigene family or other proteins involved in the transport of FAs is responsible for the viable phenotypes of FABP knockout mice (Haunerland and
Spener, 2004). It was assumed that different iLBPs display selective affinity for different ligands; therefore, positive selection will favor as many iLBP genes in the genome as possible. However, studies have shown that many FABPs demonstrate affinity for an array of FAs (Richieri et al., 2000), which raises the question why are there so many iLBP genes in vertebrate genomes? The probable answer is that the structural characteristics, of the individual iLBPs are responsible for their unique functional properties rather than their ligand binding specificity (Storch and McDermott, 2009). Examples from zebrafish (Calderone et al., 2002, Capaldi et al., 2007, Capaldi et al. ,2009) provide more evidence for Storch and McDermott' s (2009) hypothesis that changes in the structural conformation of iLBPs upon ligand binding gives them unique functional properties, which are different from each other, and enables iLBPs to carry out
their distinctive functions inside the cell.
Spatio-Temporal Distribution of Zebrafish iLBP Transcripts in Developing Embryos and Larvae
In zebrafish, neurulation occurs during the segmentation stages (10-24 hpf). The brain rudiment appears along the anterior neural keel with 10 divisions called
neuromeres. The first three neuromeres form the diencephalon, telencephelon, and mesencephalon. Transcripts offabp7a, fabpl 1, fabp3, fabplb.2, crabpla, and crabp2a
8 are detected in the developing forebrain of zebrafish (Liu et al., 2004a; Liu et al., 2005a; Sharma et al., 2005). The last seven neuromeres develop into rhombomeres. Transcripts
of the zebrafish iLBP genes show a distinct pattern of distribution in rhombomeres. fabp7a transcripts are detected in rhombomere 4, crabplb and crabp2a transcripts are detected in rhombomere 6, and the transcripts of crabpla and crabp2a are detected in rhombomere 7 (Liu et al., 2004a; Liu et al., 2005a; Sharma et al., 2005). rbpla, crabp2a,
and crabplb transcripts are detected at the early segmentation stage, along the posterior neural keel, which eventually develops into the spinal cord (Liu et al., 2004b; Sharma et
al., 2005).
Many iLBP transcripts are detected in mesodermal derivatives. Blood circulation
in zebrafish embryos starts at about 24-26 hpf (Isogai et al., 2001). Connection of simple blood vessel branches can be seen in the head of the zebrafish embryos at the mid pharyngula period (36 hpf), and a complex head vascular system is formed at the early hatching period (48 hpf). fabpll transcripts are abundant based on hybridization signal in
the developing head vasculature and are detected at low levels in the intersegmental blood vessels and in the aorta wall at mid pharyngula period (36 hpf) (Liu et al., 2005;
Flynn et al., 2009). In addition, the transcripts offabpll become more abundant at early hatching period (48 hpf). In addition fabp3, crabplb, and crabp2a transcripts are detected in somites at mid pharyngula period (36 hpf). In zebrafish, during the late blastula stage (4 hpf) the endoderm arises from the
four most marginal blastomere tiers (Warga and Nusslein-Volhard, 1999). These
blastomeres involute early during gastrulation and directly spread over the yolk syncytial layer (YSL) (Warga and Kimmel, 1990). Transcripts offabp2, fabplb, rbpla, and rbp2b
9 are detected in the YSL starting from the end of gastrulation (10 hpf) (Andre et al., 2000; Liu et al., 2004b; Sharma et al., 2006). The development of the intestine in zebrafish starts around the pharyngula stage (26 hpf) and continues until the larval stage (126 hpf) (Ng et al., 2005). In zebrafish, alimentary canal formation occurs by a wave of cavitation that is initiated at the anterior intestine (near the oesophagus and intestinal bulb) and advances into the posterior intestine (Ng et al., 2005). fabp2 transcripts were detected in the intestinal bulb of zebrafish between early to late pharyngula stage (24-36 hpf) (Sharma et al., 2004). The detection offabp2 transcripts at the posterior intestine occurs by late hatching period (72 hpf ) (Mudumana et al., 2004). fabp2 mRNAs display a characteristic cephalocaudal expression pattern in the developing intestine (Andre et al., 2000). Mudumana et al. (2004) suggest thatfabp2 can be used as a differentiation marker for intestinal epithelium because of its unique pattern of expression in the developing intestine. In addition tofabp2, transcripts of rbp2a,fabp3, andfabplb are also detected in the intestinal bulb of developing zebrafish larvae (Liu et al., 2004b; Sharma et al., 2006;
Liu et al., 2007). Tissue-Specific Distribution of iLBP Transcripts in Adult Zebrafish Transcripts of each iLBP gene exhibit a distinct, but sometimes overlapping, pattern of tissue-specific distribution with that of other iLBP gene transcripts (Table 1.2). Transcripts of all zebrafishfabp genes are detected in the intestine, and transcripts of most zebrafishfabp genes are detected in the liver (Table 1.2). This may be due to the ancestral gene from which all FABP genes have descended (Schaap et al., 2002). As such, they may have been expressed in the midgut of the common ancestor of present day vertebrates and invertebrates. As noted by Arrese et al. (2001), in insects digestion and
10 absorption of lipids occurs in the midgut. Two FABPs described in the Tobacco horn worm Manduca sexta, MFBl and MFB2, are detected in the midgut (Smith et al., 1992). In mammals transcripts of FABPl, FABP2, FABP4, and FABP6 genes are detected in intestine and transcripts of FABPl and FABP7 genes are detected in the liver (Yamamoto et al, 2009). Mammals, which evolved much later than fishes on an evolutionary time scale, might have lost the regulatory elements in some FABP genes, which drive the intestine and liver specific expression. In addition to the liver and intestine, transcripts of zebrafish/a?? genes are predominantly detected in ovaries, testis, muscle and heart, which are the sites of intense lipid metabolism in fish (Table 1.2 and the references therein). In contrast to the tissue-specific distribution of transcripts for mammalian CRABP and CRBP genes (Haunerland and Spener, 2004), transcripts of zebrafish crbp and crabp genes are not detected in all tissues with the exception of rbp7a and rbp7b (Table 1.2 and the references therein). Rbp7a and rbp7b are detected in all the tissues of zebrafish (Belliveau et al., 2010). Transcripts of crabp2a are not detected in any tissues in adult zebrafish (Sharma et al., 2005).
Transcriptional Regulation offabp Genes Our knowledge about the c/s-regulatory elements involved in the regulation of zebrafish iLBP genes is limited (Her et al., 2003a; Her et al., 2003b; Her et al., 2004a; Her et al., 2004b). Two distinct regulatory elements A (-1944 to -1623) and B (-1622 to - 1510) in a 435-bp distal region (-1944 to -1510) of thefabplOa promoter are regulating
11 O (N e O m O o cu O o O O (N O o CS CS o (N x> X) CS o CJ (N (N ? S (N (N e m m ? (U O cS O o O o o CU ·- O O o O o o CS (N cS (N (N (N CN (N ? C/2 (? -C ?? CU O cu 3 cS U ce CS O CS CS — (U O CO "ce (U (U oo (U M (U (U ? ? C CS co > O Ö O a 3 3 3 3 CS -tí _3 (U O CS (U X! t/0 < (N > (N J U 3 &0
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C ? O ce + + + CU + + + "o 3 B (U U ? cS CJ + + a e CS (? g H Is -ö •ft o (U 03 12 the expression of the gene in the liver of larvae and juveniles. Her et al. (2003b) have characterized two consensus HFH and a HNF-I alpha site in element A and a HNF-3 beta site in element B. Mutations in these sites will result in loss or decrease offabpl Oa expression in liver of zebrafish. However,fabpl Oa transcripts are also detected in intestine and testis in addition to liver of adults (Venkatachalam et. al., 2009) and Her et al. (2003b) fail to mention whether mutations in any of these sites have a bearing on the trascription of thefabplOa gene in intestine and testis of adult zebrafish. Her et al. (2004b) have also analyzed the promoter of zebrafishfabp2 gene, and reported that the proximal 192-bp region of the zebrafishfabpl promoter is sufficient to direct intestine- specific transcription of this gene during larval development. Several studies report the induction of some FABP genes in mammals by FA and molecular mechanisms for this induction have been proposed (Bass et al., 1985; Drozdowski et al., 2004; Mochizuki et al., 2007; Ockner and Manning, 1974; Schroeder et al., 2008). For example, some research groups (Huang et al., 2004; Schroeder et al., 2001; Schroeder et al, 2008) have suggested that FABPs transport long-chain FAs to the nucleus from the cytoplasm. Once inside the nucleus, FABPs interact with and transfer their long-chain FA ligands to nuclear receptors, such as PPARa and PPARy (Budhu and Noy, 2002; Delva et al., 1999; Tan et al., 2002). Dietary long-chain FAs are known to activate these nuclear receptors (Escher and Wahli, 2000; Gottlicher et al., 1992; Keller et al., 1993; Lemberger et al., 1996; Wolfrum et al., 2001). Once activated, these nuclear receptors form heterodimers with retinoic acid receptors (RAR) or retinoid X receptors (RXR) {e.g., PPAR-RXR or PPAR-RAR), which in turn bind to response elements in FABP genes and, thereby, stimulate initiation of transcription (Desvergne and Wahli, 13 1999). However, Qu et al. (2007) have shown that transcriptional induction by FAs of the Fabp3 gene from rat and desert locust is not due to the direct interaction of PPARs with the FABP promoter. Although many reports describing the effect of feeding and starvation, and the effect of different iLBP ligands, on the regulation of iLBP genes in mammalian model organisms have been published, there are only two reports concerning zebrafish (Jury et al., 2008; Flynn et al., 2009). Flynn et al. (2009) recently published their studies on the ontogeny of adipocytes and investigated the regulation oifabplla andfabpllb genes under the physiological conditions of feeding and starvation in zebrafish larvae. The authors observe that feeding stimulates the formation of adipocytes expressing the fabplla transcripts in 8 day old larvae. Jury et al. (2008) fed zebrafish either high (3 rations/day) or low (1 ration/7 days) calorie diets for 5 weeks and observed that protein levels of FABP7 are induced in high calorie diet compare to low calorie diet. To summarize, fabp genes are transcriptionally regulated not only by the quantity of lipid flux into the cell, but also by the composition of lipids. Interestingly no study has been reported yet on the effect of vitamin A or retinoids on transcript and protein levels of zebrafish Crbps and Crabps. Lipid Homeostasis in Teleost Fishes Lipids play an essential role in the life history and physiology of fishes (Tocher, 2003). Lipids are the main source of energy and are major organic components in fish. FAs in particular are a major source of ATP via ß-oxidation. They are also the constituents of biological membranes, and act as signalling molecules (Hotamisligil et al., 1996). 14 De novo FA biosynthesis and mitochondrial ß-oxidation of FAs are the major anabolic and catabolic pathways of lipid metabolism in fishes and other vertebrates (Tocher, 2003). De novo FA biosynthesis is a set of biochemical synthetic reactions that uses acetyi-CoA as building block to synthesize endogenous complex lipids. Saturated FAs, palmitic acid (C16:0) or stearic acid (18:0) are the products of de novo FA biosynthetic pathway (Tocher, 2003). High dietary lipid fluxes are known to down regulate the transcription of genes involved in the biosynthetic pathway and reverse effects have been observed when the animals are fed with low fat diets (Tocher, 2003). Similarly, mitochondrial ß-oxidation of FAs increases with high dietary lipid flux and decreases when animals are fed with low fat diets (Tocher, 2003). Saturated FAs can be further elongated and desaturated into polyunsaturated FAs. Stearic acid (18:0) can be desaturated into oleic acid (18:1 n-9). However, all vertebrates lack the enzymes to further desaturate and elongate oleic acid (18:1 n-9) to linoleic acid (18:2 n-6) and linolenic acid (18:3 n-3) (Tocher, 2003). Therefore, linoleic acid and linolenic acid are considered as the essential FAs and should be obtained from diet (Lands, 1992). Linoleic acid is biosynthesized to arachidonic acid and linolenic acid is biosynthesized to eicosapentaenoic acid and docosahexaenoic acid. These processes are catalyzed by the d6 and 85 desaturases and elongases. The synthesis of highly unsaturated FAs (HUFA) from their precursor FAs is also influenced by the FA profile of the diet. A diet rich in HUFA will down regulate the transcription of desaturase and elongase genes as a feedback mechanism, whereas a diet containing low quantities of HUFA will not down regulate the genes involved in the HUFA biosynthetic pathway, as shown in zebrafish and other teleost fishes (Tocher et al., 2002; Tocher, 2003). 15 Objectives of This Study Since FABPs are involved in the intracellular transport of FAs between different cellular organelles associated with the lipid metabolism, I proposed that a diet-mediated change in the metabolism of FAs would influence the requirement of FABPs for cellular processes and thus affect the transcription of their genes. Previous studies in mammals have shown that dietary FAs affects the transcription of FABPs (Bass et al., 1985; Lin et al., 1994; Mochizuki et al., 2007; Ockner et al., 1974; Poirier et al., 2001). The current study was undertaken to test the hypotheses that (i) zebrafish/a?t? genes might be regulated by dietary FAs, (ii) sister duplicatedfabp genes might be differentially modulated by dietary FAs and (iii) also to test the DDC model explaining the retention of duplicate genes in the genome by either subfunctionalization or neofunctionalization. 16 Chapter 2: Tandem Duplication of thefabplb Gene and Subsequent Divergence of the Tissue-Specific Distribution offabplb. 1 Andfabplb.2 Transcripts in Zebrafish {Danio rerio) The manuscript based on this study is presented below. Co-authors for this manuscript are Eileen M. Denovan-Wright, Christine Thisse, Bernard Thisse and Jonathan M. Wright. Originally published as: Karanth S, Denovan-Wright EM, Thisse C, Thisse B, Wright JM. 2009. Tandem duplication of thefabplb gene and subsequent divergence of the tissue-specific distribution ??fabplb. 1 andfabplb.2 transcripts in zebrafish. Genome 52: 985- 992. Authors' contributions: SK and JMW conceived and designed the research. SK conducted all the experimental work except for whole mount in situ hybridization to embryos and larvae (CT and BT) and in situ hybridizations to adult tissue sections (ED-W). SK and JMW drafted the manuscript with subsequent editorial comments from ED-W, BT and CT. All authors read and approved the final version of the manuscript. 17 Abstract We describe a fatty acid-binding protein 1 (fabplb.2) gene and its tissue-specific expression in zebrafish embryos and adults. The 3.5 kb zebrafish /a¿>/?i¿>.2 gene is the paralog of the previously described zebrafishfabpla andfabplb genes. Using the LN54 radiation hybrid mapping panel, we assigned the zebrafishfabplb.2 gene to linkage group 8, the same linkage group to whichfabplb.l was mapped, fabplb. 1 andfabplb.2 appear to have arisen by a tandem duplication event. Whole-mount in situ hybridization of a riboprobe to embryos and larvae detectedfabplb.2 transcripts in the diencephalon and as spots in the periphery of the yolk sac. In adult zebrafish, in situ hybridization revealedfabp 1 b. 2 transcripts in the anterior intestine and skin, and reverse transcription, polymerase chain reaction (RT-PCR) detectedfabplb. 2 transcripts in the intestine, brain, heart, ovary, skin and eye. By contrast, fabplb. 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 duplicatedfabpl b. 1 andfabplb.2 genes in adult tissues, and during development suggests that the duplicatedfabplb genes of zebrafish have acquired additional functions compared to the ancestral fabpl gene i.e., by neofunctionalization. Furthermore, these functions were subsequently divided between thefabplb. 1 and fabplb.2 owing to subfunctionalization. 18 Introduction Fatty acid-binding proteins (FABPs), members of the multigene family of intracellular lipid-binding proteins (iLBPs), are involved in the intracellular trafficking and sequestering of FAs (Bernlohr et al., 1997). A single ancestral iLBP gene is thought to have emerged some 1000 million years ago in animals after they diverged from plants and fungi (Chan et al, 1985; Schleicher et al., 1995; and Schaap et al., 2002). Schleicher et al. (1995) have convincingly argued that the multiplicity and diversity of the iLBP multigene family arose through a series of gene duplication events followed by sequence divergence of individual iLBP genes. To date, 12 paralogous genes coding for FABPs have been described in vertebrates (Agulleiro et al., 2007; Karanth et al., 2008; and Liu et al., 2008). Ockner et al. (1972) were the first to describe the properties of a FABP (FABPl, or the so-called liver-type FABP) from rat. Subsequently, FABPl has been identified in many animal species, including fishes (Sharma et al., 2006), birds (Sams et al., 1991) and amphibians (Di Pietro et al., 1999). An ortholog of FABPl has been reported in several invertebrate species (see e.g., Campbell et al., 2008). Several functions have been proposed for FABPl including FA uptake (Hertzel and Bernlohr, 2000), sequestering of FAs to protect cells against the detrimental effects of excess free FAs (Besnard et al., 2002), regulation of gene expression, and control of cell growth and differentiation (Veerkamp and Maatman, 1995). Ohno (1970) proposed that gene and entire genome duplications are major evolutionary forces driving the increase in complexity of life. Gene duplications can occur either by retrotransposition, or by tandem duplication (Hurles, 2004). WGDs result 19 from either allopolyploidy or allopolyploidy (Prince and Pickett, 2002). Force et al. (1999) argued that duplicated genes are retained in the genome, either by subfunctionalization, in which the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicates acquires a new function. In the Duplication-Degeneration-Complementation model proposed by Force et al. (1999), subfunctionalization and neofunctionalization occur by evolution of cis-regulatory elements in the promoters of the duplicated genes. In an earlier study (Sharma et al., 2006), we reported that the duplicated copies of the zebrafishfabpl gene, fabpla andfabplb, arose following the WGD event in the ray- finned fish lineage (Furlong and Holland, 2002). Furthermore, these duplicates were retained in the zebrafish genome owing to subfunctionalization (Sharma et al., 2006). In this communication, we describe another duplicated copy of thefabpl gene, fabplb. 2, recently identified in the zebrafish genome DNA sequence database (version Zv5, http://www.ensembl.org/Danio rerio/index.html) that most likely emerged in the zebrafish genome by tandem duplication during unequal crossing-over during meiosis, subsequent to the WGD in ray-finned fishes. The tissue-specific distribution of mRNA transcripts for the tandemly duplicatedfabplb.l andfabplb.2 genes in adult tissues, and during embryonic and larval development suggest that thesefabplb duplicates have been retained in the zebrafish genome owing to subfunctionalization. 20 Materials and Methods Husbandry of Zebrafish Adult zebrafish (AB strain) were maintained according to protocols described by Westerfield (2000). Animal care and usage followed the guidelines approved by the Canadian Council on Animal Care (http://www.ccac.ca). Experimental protocols were reviewed by the Animal Care Committee of Dalhousie University. Nucleotide Sequence of the Zebrafishfabplb.2 cDNA and its Gene Using thefabplb.l cDNA sequence as a query (Sharma et al., 2006), a previously unidentified gene (ENSDARG00000058536) and transcript (ENSDART0000081417), hereafter referred to as fabplb.l, were retrieved by a BLASTn search from the zebrafish genome sequence database at the Wellcome Trust Sanger Institute (version Zv5). The sequence of the transcript ENSDART0000081417 was used to retrieve ESTs and genomic DNA clones from NCBI Genbank. Based on the sequence of EST, EB880179.1 (GenBank accession number), primers were designed (Fig. 2.1: 5'- ATTCACCTTCATTTGTATAGCGC-3' as forward primer, cf, and 5'- GACTTTATCAGCATTCGTCTTCTC-3' as reverse primer, cr) to amplify a 485 bp fragment from cDNA prepared from total RNA extracted from a whole adult zebrafish by standard methods. PCR conditions were initial denaturation at 94.00C for two minutes, followed by 30 cycles at 94.00C for 30 seconds (denaturation), 60.30C for 30 seconds (primer annealing) and 72.00C for 1 minute (elongation) with a final elongation step at 72.O0C for 5 minutes. The resulting product was cloned into the pGEM-T vector (Promega, Madison, WI, USA) and five independent clones were sequenced. The sequence of eachfabplb.2 cDNA clone was identical to the coding sequence of the 21 genomic DNA clone, CU683892 (GenBank accession number). Upstream of the open reading frame of thefabplb.2 gene present in clone CU683892, we identified putative RNA polymerase and transcription factor-binding sites using the AliBaba2. 1 software (Grabe 2002). The World Wide Web version of the tandem repeat finder computer program (Benson, 1999) was used to find tandem repeats in clone CU683892. Phylogenese Analysis FABP sequences from zebrafish and other vertebrates were aligned using BLOSUM62 matrix and CLUSTALW (Thompson et al., 1997). The bootstrap neighbor- joining tree was constructed using the MEGA4 software (Tamura et al., 2007). Human LCNl (NP_002288) was used as the out-group. Radiation Hybrid Mapping of the Zebrafishfabplb.2 Gene Linkage group (chromosome) assignment oí the, fabplb.2 gene was performed using the LN54 radiation hybrid mapping panel of zebrafish (Hukriede et al., 1999). PCR amplification of thefabplb. 2-specific product employed the primers, rhmf (5'-CAAGAGCTGCTCAAGAAAGCCAA-3') and rhmr (5'-CTTGACTTTGTCTCCGCTCAGC-3'), as shown in figure 2.1. PCR conditions were initial denaturation at 94.0°C for two minutes, followed by 30 cycles at 94.0°C for 30 seconds (denaturation), 60.3 °C for 30 seconds (primer annealing) and 72.0°C for 1 minute (elongation) with a final elongation step at 72.O0C for 5 minutes. In Situ Hybridization to Whole-Mount Embryos and Larvae, and to Sections of Adult Zebrafish Whole-mount in situ hybridization to embryos and larvae of zebrafish used a riboprobe synthesized from one of thefabplb.2 cDNA clones isolated in this study using 22 the protocol described in Thisse and Thisse (2008). Detection oifabplb.2 transcripts in sections of adult zebrafish was performed by in situ hybridization of a synthetic oligonucleotide probe according to Denovan-Wright et al., (1998). Briefly, sagittal and transverse sections of adult zebrafish were hybridized to [a- P] dATP 3' end-labeled fabplh.2 antisense probe, isf (5'- CGTGTGTGTGGTCTCTCTTTTACTCTTCTGCTTACG-3'), shown in figure 2.1. Following hybridization and autoradiography, sections were stained with cresyl violet to identify specific tissues. RT-PCR Detection oifabplb.2 Transcripts in Adult Zebrafish Tissues Reverse transcription, polymerase chain reaction (RT-PCR) was used for the tissue-specific detection oïfabplb.2 transcripts in RNA extracted from tissues of adult zebrafish. Following synthesis of cDNA from RNA samples using the Omniscript RT kit (Qiagen, Mississauga, Ontario, Canada), fabplb.2 cDNA was PCR-amplified by the primers, rtf (5'-TGCCGTTCTCTGGGAAGTTTGAGTT-3') and rtr (5'- TGACTTTGTCTCCGCTCAGCATC-3'), as shown in figure 2.1. PCR conditions for the amplification of fabplb.2 transcripts were an initial denaturation step at 94.0°C for two minutes, followed by 30 cycles at 94.00C for 30 seconds (denaturation), 56.0°C for 30 seconds (primer annealing) and 72.O0C for 1 minute (elongation) with a final elongation step at 72.O0C for 5 minutes. As a positive control for the quality of cDNA prepared from RNA extracted from each tissue sample, transcripts for elongation factor la (efla) were assayed by RT-PCR using primers previously described by Pattyn et al. (2006). 23 Results and Discussion Identification of a Duplicatedfabplb Gene in the Zebrafish Genome A gene paralogous to the previously described zebrafishfabplb gene was identified from a BLAST search of the zebrafish genome sequence database at the Wellcome Trust Sanger Institute (version Zv5, http://www.ensembl.org/Danio rerio/index.htmQ using the GenBank sequence, DQ062096 (Sharma et al., 2006) as query. PCR Primers based on the sequence of EST EB880179.1 (GenBank accession number) were used to amplify a 485 bp fragment from template cDNA prepared from total RNA of an adult zebrafish. The resulting product was cloned and sequenced and the accuracy of the sequence was verified by alignment with sequences of other ESTs (EB882868.1, EB881605.1, and EB990446.1) and a finished DNA clone (CU683892) from the NCBI Genbank (see materials and methods). The 3.5 kb duplicated/a¿>pi¿> gene (hereafter referred to as fabplb.2) consists of four exons and three introns (Fig. 2.1), a gene organization common to all vertebrate iLBP genes (Bernlohr et al., 1997) with the exception of the zebrafishfabp 1 a gene, which contains an additional intron in the 5' untranslated region (Sharma et al., 2006). The nucleotides at the splice site of each exon-intron junction of the zebrafishfabplb. 2 gene conform to the GT-AG rule (Breathnach and Chambón, 1981). A putative CAAT box is located at position -86 to -82, and a putative TATA box is located at position -33 to -28 in the zebrafishfabplb. 2 gene. Multiple sequence alignments of selected zebrafish and human FABPs were performed using ClustalW (Thompson et al., 1997). Zebrafish Fabplb.2 showed highest sequence identity and similarity (48% and 69%, respectively) with zebrafish Fabplb. 1. 24 Figure 2.1. The genomic organization of the zebrafishfabplb.2 gene. Exons are shown in upper-case letters, with the coding sequences of each exon highlighted in bold and underlined and the deduced amino acid sequence indicated below. The solid square (¦) symbol indicates the stop codon. For introns 2 and 3, only the portions of sequences next to exon-intron splice junctions are shown. '+G indicates the transcription start site. A putative CAAT box, TATA box and polyadenylation signal (AATAAA), are also highlighted in bold and underlined. The in situ hybridization probe (isp) used for detection offabplb.2 transcripts in adult zebrafish tissues, and the PCR primers used for radiation hybrid mapping (rhmf, rhmr), cloning of cDNA from the RNA extracted from whole zebrafish (cf, cr), and RT-PCR detection of fabplb.2 transcripts in RNA extracted from adult zebrafish tissues (rtf, rtr) are indicated by dashed lines. 25 U ? U «e Cn cC (0 U U O ? PC 4 CC en en H O ? IO cC PC Cn « g Oi O 4-J U B ? E-· PC ß> Pt ? PC Di en pC U CC Cn 4-1 PC -P ? tO CS CH ¡? H Cn PC O ? ü U Cn .? Cn O PC en ? IO O (J Di ?ß IO H (0 to PC to ? ? tn en O (ß IO ?? U ? O Pt IO ? PC (O *>\ IO ? U s> ? IS < rt (O « U U ? ? O ? pC <ß (O O H ?«! a O ? ? ? « JQ pC (O O U ? Cn PC Q s I < Ol ? to pC to 13 ? O o PC U o U ? O ? U ? a U (O tO cC CJi O (O CC O +J TÍ H Cn ci (O 4-1 < Ü H Cn PC IO CC Cn PC ? Q ? pC Oi (J (O a (O en U m 0 U 4J J3 o E-i (O U (O H ? H H Eh (O cC ? n O U s H ?· Di s to fa O (O 0> U H ?? ? cC co pC pC ê> CJ ? H Z Eh ? U ? U ?? u H ?? ? o Z o (O ?? ? IO u ? ?a < ü to ?? H ? (0 ?? 4J Eh .0 Eh O cC s tO > O Cn H Cn Di H (O s? JJ IO U Cn (0 Oi ¡¿ -M O Cn ? U O 4-1 4J H O U 4-1 Cn pC ifl (0 U O cC U ¦? S io O ü ttì U U 51 (J 26 Figure 2.2. Phylogenetic tree of selected vertebrate FABPs showing the relationship between zebrafish Fabpla, Fabplb.l and Fabplb.2. The neighbor-joining tree was constructed using Homo sapiens lipocalin 1 precursor (LCNl, NP_002288) as outgroup. The bootstrap values (per 100 duplicates) are indicated above or below each node. Amino acid sequences used in this analysis include: Danio rerio, zebrafish (Dr) Fabpla (AAZ08575), Fabplb.l (ABF18598), and Fabplb.2 (derived from EB880179.1) FabplO (NP_694492), Fabp2 (NP_571506), Fabp3 (NP_694493), Fabp7a (NP_571680), Fabp7b (NP_999972), Fabpl la (NP_00 1004682), Fabpl lb (NP_001018394), and Fabp6 (NP_001002076); Takifugu rubripes, takifugu (Fr) Fabpl (AAC60290); Homo sapiens, human (Hs) FABPl (P07148), FABP2 (NP_000125), FABP3 (NP_004093), FABP4 (NP_001433), FABP5 (NP_001435), FABP6 (NP_001436), FABP7 (NP_001437) and FABP8 (AAH34997); Mus musculus, mouse (Mm) FABPl (Y 14660) and FABP9 (NPJB5728); Rattus norvégiens, rat (Rn) FABPl (P02692); Sus scrofa, pig (Ss) FABPl (P49924); Gallus gallas, chicken (Gg) FABPl (AAK58095); Xenopus tropicalis, African frog (Xt) FABPl (NPJ)Ol 116883); Pongo abelii, Sumatran orangutan (Pa) FABPl (derived from NPJ)0 1125017); Salmo salar, Atlantic salmon (Sas) Fabpl (ACI66705); Oncorhynchus mykiss, rainbow trout (Om) Fabpl (AAG30019); Epinephelus coloides, orange-spotted grouper (Epe) Fabpl (Q8JJ04); Equus caballus, horse (Ec) FABPl (derived from XPJXH497808); Ornithorhynchus anatinus, platypus (Oa) FABPl (derived from XPJ)015 10550); Canisfamiliaris, dog (Cf) FABPl (derived from XPJ532966); Bos taurus, cow (Bt) FABPl (X86904); Ambystoma mexicanum, axoltol (Ax) FABPl (P81399). Scale bar = 0.5 substitutions per site. 27 99r HsFABPl lìPaFABPl 16 rf- SsFABPl BtFABPl 29 CfFABPl MmFABPl 93 98 I-^ RnFABPl - EcFABPlOaFABPl AxFABPl XtFABPl —— DrFabpla ¦ OmFabpl - FrFabpl - EpcFabpl GgFABPl ¦ SasFabpl .DrFabplb.2 E DrFabplb.l ¦ DrFabplO ? HsFABPO 99 ¦ DrFabp6 1 oo ?— DrFabpl - HsFABP2 DrFabp3 98 MmFABP9 58 24 — HsFABP5 12 HsFABP8 33 M HsFABP4H 24 so ? DrFabplla CDrFabpllb HsFABP3 DrFabp7b 27 L-T Tolr- DrFabp7a 43 I— HsFABP7 HsLCNl 0.5 28 Zebrafish Fabplb.2 exhibits reduced sequence identity and similarity with zebrafish Fabpla, human Fabpl and other zebrafish Fabps. A neighbor-joining phylogenetic tree of selected vertebrate FABPs amino acid sequences revealed a common node for zebrafish Fabplb.2 and Fabplb.l, which are not linked by a common node on the tree to zebrafish Fabpla (Fig. 2.2). Fabpl sequences from other teleost fishes clustered together to form a clade separate from the mammalian FABPl clade. Based on sequence identity and similarity, and phylogenetic analysis, Fabplb.l and Fabplb.2 are more closely related to each other than to any of the other vertebrate Fabps, and presumably arose from a common ancestral gene by gene duplication. Duplicate Copies offabplb in the Zebrafish Genome may have Arisen by a Tandem Duplication Event Using the LN54 radiation hybrid mapping panel (Hukriede et al., 1999), we assigned the zebrafishfabplb.2 gene to linkage group (chromosome) 8 at a distance of 7.15 cR (centiRays) from the marker Z10731 with a logarithm (base 10) of odds (LOD) score of 14.7. Previously, the zebrafishfabplb.l gene had been mapped using the same LN54 panel to linkage group 8 at a distance of 4.81 cR from the same marker, Z10731, with a logarithm (base 10) of odds (LOD) score of 16.2. Based on the sequences of two finished clones from NCBI Genbank, CU683892 and CT027631, we deduced that fabplb.l andfabplb.2 are separated from each other by -3.8 kbp of DNA (Fig. 2.3). Downstream of each copy of the duplicatedfabplb genes is the triplet repeat, TAT, which suggests that this whole region was tandemly duplicated, most likely, owing to unequal crossing-over during meiosis. In addition to these TAT repeats, the intergenic region betweenfabplb.l anafabplb.2 as well as the 3' region of thefabplb.2 are AT rich 29 15 kb fabp1b.1 IGR fabp1b.2 1 2 3 4¿\ 1 234 /\ TAT (66) TAT (294) 30 kb Figure 2.3. Genomic organization of the duplicated/a¿>/?i&.i (left) andfabplb.2 (right) genes on zebrafish chromosome 8, as deduced from the sequences of clones CU683892 and CT027631 (GenBank accession numbers), fabplb. 1 anafabplb.2 are separated by -3.8 kbp of DNA in the intergenic region (IGR). The four exons offabplb. 1 and fabplb.2 are numbered as 1,2, 3, and 4. The locations of triplet repeats, TAT, are shown as arrows in the 3' downstream regions of thefabplb. 1 mafabplb.2 genes. The number of TAT repeats at each site is indicated in parentheses. 30 (data not shown), which might account for misalignment of the homologous chromosomes during recombination and the tandem duplication of thefabplb gene. Distribution offabplb.2 Transcripts in Developing Zebrafish Embryos and Larvae To determine the spatial and temporal distribution oífabplb.2 transcripts during zebrafish embryonic and larval development, we conducted whole-mount in situ hybridization to zebrafish embryos and larvae at different developmental stages (Fig. 2.4). fabplb.2 transcripts were first detected between 24-48 hours post-fertilization (hpf) in the periphery of the yolk sac. The specific cell type containing the, fabplb.2 transcripts could not be resolved, but the transcripts might be restricted to either macrophages or a subpopulation of mucous cells (Fig. 2.4A and 2.4D). In adult mice, Schachtrup et al. (2004) detected Fabpl mRNAs in the alveolar macrophages. However, Sharma et al. (2006) detectedfabplb. 1 transcripts in the yolk syncytial layer and in the intestinal bulb of developing zebrafish embryos at 48 hpf. In five day-old zebrafish larvae, the distribution offabplb. 1 transcripts was limited to the intestinal bulb and posterior intestine (Sharma et al., 2006). Whole-mount in situ hybridization of a riboprobe to zebrafish embryos, however, did not detectfabpla mRNA in embryos and larvae at any developmental stage (Sharma et al., 2006). Zebiañshfabplb.2 transcripts were also detected in embryos in the nucleus of the diencephalon between 24 and 48 hpf at the boundary between the hypothalamus and the ventral part of the posterior tuberculum (Figs. 2.4B and 2.4C). fabplb.2 transcripts were first detected on the left side of the diencephalon of embryos at 24 hpf and the distribution of these transcripts spread to the right of the diencephalon by 36 hpf (Figs. 2.4E and 2.4F). A weak hybridization signal was seen in the nucleus of the diencephalon 31 Figure 2.4. Spatio-temporal distribution oífabplb.2 transcripts in embryos and larvae of zebrafish was determined by whole-mount in situ hybridization and the tissue-specific distribution offabplb.2 transcripts in sections of adult zebrafish was determined by in situ hybridization. At 24 hours post fertilization (hpf), fabplb.2 transcripts were detected as distinct dots in the periphery of the yolk sac (A) and in the nucleus of the diencephalon (A-C). At 36 and 48 hpf, fabplb.2 transcripts were detected at 36 hpf and 48 hpf in the nucleus of the diencephalon (D-G) and in the periphery of yolk sac (Fig. 2.4D). Upper panels in H and I show the locations of sagittal and transverse sections from adult zebrafish, respectively, used for in situ hybridization assays. Middle panels show the distribution offabplb.2 transcripts in the anterior intestine (AI) and skin (S) detected by in situ hybridization of a riboprobe to sagittal (H) and transverse (I) sections of adult zebrafish. Lower panels (H and I) show the adjacent tissue sections stained with cresyl violet. 32 ïi » "5^ ''"""'"Vis ?. ,· '" J.M , ?^-Äf-i» fi*5#i' "4frJr ¦ ,ito* SC^S- i-*, jo/' U m 33 at 48 hpf (Fig. 2.4G). Zebmñshfabplla transcripts (Liu et al., 2007) and medakafabp7 transcripts (Maruyama et al., 2008) have been detected in the diencephalon at early stages of embryonic development which has led to the suggestion that Fabp/FABPs are involved in the neuronal growth and differentiation as transporters of docosohexanoic acid and other FAs during the early development (Sellner, 1994). Tissue-Specific Distribution offabplb.l Transcripts in Adult Zebrafish In situ hybridization was performed on sections of adult zebrafish to determine the tissue-specific distribution offabplb.2 transcripts (Fig. 2.4). We detectedfabplb.2 transcripts in the anterior intestine and skin (Fig. 2.4H, sagittal section; Fig. 2.41, transverse section). In mammals, the distribution and abundance of Fabpl transcripts shows a gradient along the intestine, with mRNAs being most abundant in the proximal jejunum and almost undetectable in the distal ileum, as assayed by in situ hybridization (Poirier et al., 1997). Sharma et al. (2006) employed whole-mount in situ hybridization assays to detectfabplb.l transcripts in the intestinal bulb and posterior intestine of five day-old larvae and found that the hybridization signal was more intense in the intestinal bulb (anterior intestine) than in the posterior intestine. Therefore distribution of transcripts oífabplb.2 in the adult zebrafish is distinct but overlaps with those of the zebrafishfabplb.l and its mammalian ortholog, Fabpl (Fig. 2.4H). Using the more sensitive assay of RT-PCR, fabplb.2 transcripts were detected in total RNA extracted from the intestine, brain, heart, eye, ovary and skin, but were not detected in total PvNA extracted from the liver, muscle, gills, testis, kidneys and swim bladder of adult zebrafish (Fig. 2.5). As a positive control for the quality of RNA in each tissue, transcripts for the constitutively expressed efla gene were assayed by RT-PCR and were detected in all 34 IMBHEGOTSK SB fabp1b.2 __ _ ß --1' **** **""* *^ 1^ fabp1b.1 \+F ¦ ?? m a vi ^i^^F "wPPHP ^BPWRT ^^^^flt «WP^Rr fl^^^B s ^J^^Pp ï^^mijp* l^p^MPj^4Vmpp< "^JpW * ^"Çi^pl Figure 2.5. Detection offabplb.l andfabplb.2 transcripts by RT-PCR in RNA extracted from tissues -of adult zebrafish. Top panel: fabplb.2 transcripts were detected in total RNA extracted from the intestine (I), brain (B), heart (H), eye (E), ovary (O) and skin (S). fabplb.2 transcripts were not detected in total RNA extracted from the liver (L), muscle (M), gill (G), testis (T), kidney (K) and swim bladder (SB). Middle panel: fabplb.l transcripts were detected in total RNA extracted from the liver (L), intestine (I), heart (H), gills (G), ovary (O) and testis (T). fabplb.l transcripts were not detected in total RNA extracted from the muscle (M), brain (B), eye (E), skin (S), kidney (K) and swim bladder (SB). Bottom panel: As a positive control for the quality of the extracted RNA, transcripts for the constitutively-expressed elongation factor la (efla) gene were detected by RT-PCR in RNA extracted from all adult tissues assayed. 35 tissues examined (Fig. 2.5). By contrast to the tissue-specific pattern of distribution of fabplb.2 transcripts, fabplb.l transcripts have been detected in the intestine (Sharma et al., 2006), liver, heart, gills, ovary and testis (Fig. 2.5). Transcripts of thefabpla gene have been detected only in the intestine of adult zebrafish (Sharma et al., 2006). Divergence of the Tissue-Specific Distribution offabplb.l anafabplb.2 Transcripts In adult mammals, Fabpl transcripts were detected in the intestine and liver but not in the heart, brain and testis using RNA dot-blot hybridization (Gordon et al., 1985) (Table 2.1). Fabpl transcripts were also detected in kidney (Maatman et al., 1992) and lung (Guthman et al, 1998) by RT-PCR (Table 2.1). Teratani et al. (2007), using RT- PCR, showed that Fabpl transcripts were not present in total RNA extracted from brain of mammals (Table 2.1). In adult zebrafish, fabpla transcripts were detected in the intestine (Sharma et al., 2006), and while zebrafishfabplb. 1 transcripts are abundant in the intestine (Sharma et al., 2006), they are also present at lower levels in the liver, heart, gills, ovary and testis (Fig. 2.5 and Table 2.1). In this study, we show that the tissue- specific distribution of zebrafishfabplb.2 transcripts is markedly different from that of zebrafishfabplb. 1 transcripts, as they were abundant in the intestine, ovary and skin, and detected at lower levels in the brain, heart and eye (Fig. 2.5 and Table 2.1). During embryogenesis, mouse Fabpl transcripts are first detected in the gut epithelium of the 17-19 day old foetus (Hauft et al., 1989). Although the tissue-specific distribution of zebrafishfabplb.l transcripts is similar to that of mammalian Fabpl transcripts during early development (Sharma et al., 2006), the tissue-specific pattern of expression for zebrafishfabplb. 2 transcripts is markedly different, fabplb. 2 transcripts were detected in the nucleus of the diencephalon between 24 and 48 hpf (Fig. 2.4). 36 (U oo r- Xi on ON o ? Qi oo ON ON o O O O U On (N O O O X) »-H S (N (N (N cd cd Ph "S "cd O s*-i (U (U S cd ? (U + X + -? (U + cd H ? -ö C O cd C O Dh (U C .S cd .& "cd o ? [? C "-3 cd + & (U 3 £ + e (U o X + + 3 ?"-1 -Scd ? <-> + + + 11 O cu cd "= 2 ? CSI «3 C/3 1? cu O O ? O .? s*. >> SÄ gf Lh S-H Lh Lh "S ci ö Z " 1/5 3 cd - a 1 S Lh . ^ W < W W -" ? ? C ? ? -*-* cd cd *—< CU .£ 'S = "ß £ — '—i cu £ £ •^ cd ·° ^ O £ r. -4—' es -g £ ^ ^ + "J 37 The different tissue-specific distributions of zebraûshfabplb.l andfabplb.2 transcripts compared to zebrañshfabpla and mammalian Fabpl transcripts suggests that the duplicatedfabplb genes of zebrafish acquired additional functions to the ancestral fabpl gene, i.e., neofunctionalizatiom. Furthermore, the function(s) of the duplicatedfabplb genes of zebrafish appear divided betweenfabplb.l andfabplb.2 owing to subfunctionalization. 38 Chapter 3: The Evolutionary Relationship Between the Duplicated Copies of the Zebrafish/a¿/7l7 Gene and the Tetrapod FABP4, FABP5, FABP8 and FABP9 Genes The manuscript based on this study is presented below. Co-authors for this manuscript are Eileen M, Denovan-Wright, Christine Thisse, Bernard Thisse and Jonathan M. Wright. Originally published as: Karanth S, Denovan-Wright EM, Thisse C, Thisse B, Wright JM. 2008. The evolutionary relationship between the duplicated copies of the zebrafish/aòpil gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes. FEBS Journal 275:3031- 3040. Authors' contributions: SK and JMW conceived and designed the research. SK conducted all the experimental work except for whole mount in situ hybridization to embryos and larvae (CT and BT) and in situ hybridizations to adult tissue sections (ED-W). SK and JMW drafted the manuscript with subsequent editorial comments from ED-W, BT and CT. AU authors read and approved the final version of the manuscript. 39 Abstract We describe the structure of a fatty acid-binding protein 1 1 (fabpllb) gene and its tissue-specific expression in zebrafish. The 3.4 kb zebrafishfabpllb gene is the paralog of the previously described zebrafish/a¿>/?iia gene with a deduced amino acid sequence for Fabpl IB exhibiting 65% identity with that of Fabpl IA. Whole-mount in situ hybridization of a riboprobe to embryos and larvae showed that the distribution of zebrafishfabpl lb transcripts were restricted solely to the retina and first detected at 24 hours postfertilization. In situ hybridization revealedfabpllb transcripts along the spinal cord in adult zebrafish. However, the highly sensitive assay of RT-PCR detectedfabpllb transcripts in the brain, heart, ovary, and eye in adult tissues. By contrast, fabplla 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 the zebrafishfabpllb gene to linkage group 16. Phylogenetic analysis and conserved gene senteny with tetrapod genes indicated that the emergence of two copies offabpl 1 in the zebrafish genome may have resulted from a fish-specific WGD event. Furthermore, we propose that the FABP4, FABP5, FABP8, FABP9 (PERF15) gene cluster on a single chromosome in the tetrapod genome and thefabp11 genes in the zebrafish genome originated from a common ancestral gene, which following their divergence gave rise to thefabp11 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. 40 Introduction The multigene family coding for vertebrate intracellular lipid binding proteins (iLBP) consists of fatty acid-binding protein (FABP), cellular retinoic acid-binding protein (CRABP) and cellular retinol-binding protein (CPvBP) genes. FABPs bind selectively to FAs, CRABPs bind to retinoic acid and CRBPs bind to retinol (Bernlohr et al., 1997). For many iLBP, their precise physiological function(s) is not completely understood or remains unknown. However, it is clear that iLBPs are involved in cellular uptake and intracellular transport of long-chain FAs, bile salts and retinoids, protection of cellular structures from the detergent effects of FAs by sequestering them until required in various metabolic processes, interaction with other enzymes and transport systems, and in the transcriptional regulation of specific genes (Corsico et al., 2004; Ho et al., 2002; Murata and Storch, 2005; Storch et al, 2002; Veerkamp and van Moerkerk, 1993). By functioning in the transport and metabolism of retinol and retinoic acid, CRBPs and CRABPs may play an important role in development, growth and reproduction, primarily by making retinoids available to receptors in the nucleus to regulated specific gene transcription (Ong et al., 1994). Originally, FABPs and their genes were named based on the tissue in which they were first isolated. Later, Hertzel and Bernlohr (2000) proposed a different nomenclature where FABPs are numbered according to the temporal order of their identification (e.g., fabpl, fabp2, etc.). We chose, for the sake of clarity, to use here the nomenclature adopted by Hertzel and Berhlohr (2000). Thus far, iLBPs have only been found in species from the animal kingdom suggesting that a single ancestral iLBP gene emerged in animals after their divergence from plants and fungi (Schaap et al., 2002). The diversity of the iLBP multigene family is 41 thought to have arisen through a series of gene duplication events followed by their sequence divergence (Schleicher et al., 1995). Estimates of the earliest iLBP gene duplication vary between 930 to 1000 million years ago (mya) (Chan et al., 1985; Schaap et al., 2002; Schleicheret al., 1995). Schaap et al. (2002) and Schleicher et al. (1995) have suggested that 600-700 mya, before the invertebrate and vertebrate lineages split, the gene(s) that would give rise to the FABP1/FABP2/FABP6 clade had already diverged from the gene(s) that would give rise to the FABP4/FABP8 clade. CRBPs and CRABPs, which are absent in invertebrates, might have diverged from the FABPl clade after the vertebrate and invertebrate split. To date, paralogs of 1 1 genes coding for FABPs have been described in vertebrates (Agulleiro et al., 2007). Zebrafish have attracted attention of evolutionary molecular biologists owing to the abundance of genetic and biological resources for this model organism for developmental studies, but particularly for the WGD event that occurred in ray-finned fishes some 250 to 400 mya (Furlong and Holland, 2002) that has led to investigations on the genesis and fate of duplicated genes. Gene duplication has been proposed by Ohno (1970) as a major evolutionary force in the increasing complexity of life. In addition to WGD, tandem duplication of individual genes by the process of unequal crossing-over during meiosis may also account for the increase in number of genes in eukaryotes (Vandepoele et al, 2004). Vayda et al. (1998) described afabp gene from four Antarctic fishes, termed Had-FABP, which they suggest is the ortholog of the mammalian FABP4 gene. In a previous communication (Liu et al., 2007), we described afabp4 gene from zebrafish showing greatest sequence identity to, and formed a clade in phylogenetic analyses with, 42 the Antarctic fish Ha(j-FABP. Recently, however, Agulleiro et al. (2007) recognized that the Fabp's reported by Vayda et al. (1998) and by us (Liu et al., 2007) constitute a new FABP clade likely restricted to fishes, and renamed the novel fish gene and protein, fabpll and Fabpl 1, respectively (Agulleiro et al., 2007). In this communication, we report the characterization of a duplicatedfabpl 1 gene (fabpl lb) from the zebrafish genome, the distribution offabpllb mRNA transcripts in adult tissues and during embryonic and larval development, and linkage group (chromosome) assignment of fabpllb by radiation hybrid mapping. Based on phylogenetic analysis and conserved gene syntenies of the zebrafishfabplla andfabpl lb genes with other vertebrate FABP genes, we propose that the duplicatedfabpll genes in fishes and the FABP4, FABP5, FABP8, and FABP9 gene cluster in tetrapods arose from a single progenitor gene. Materials and Methods Husbandry of Zebrafish Adult zebrafish were purchased from a local aquarium store and maintained according to established procedures (Westerfield et al., 2000). Experimental protocols were reviewed by the Animal Care Committee of Dalhousie University in accordance with the Canadian Committee on Animal Care. Nucleotide Sequence of Zebrafishfabpllb cDNA and Gene We retrieved a novel ensembl gene (ENSDARG0000000231 1) from a BLASTn search of the zebrafish genome sequence database at the Wellcome Trust Sanger Institute (version Zv6, http://www.ensembl.org/Danio_rerio/index.html) using thefabplla cDNA sequence as a query (Liu et al., 2007). The novel ensembl gene (ENSDARG0000000231 1) exhibited foremost sequence identity and similarity to the 43 zebrafish/a???a gene, hereafter referred to as the zebrafishfabpllb gene. We confirmed the sequence of the coding region for thefabpllb gene by comparison with fabpllb Expressed Sequence Tags (EST) obtained from the BLASTn search of GenBank at the National Center for Biotechnology Information. AU ESTs were identical to the coding region of the zebrafishfabpllb gene. Phyíogeneíic Analysis Blosum62 matrix and CLUSTALW (Thompson et al., 1997) were used to align FABP sequences from zebrafish and other vertebrates. The bootstrap neighbor-joining tree was constructed using the MEGA4 software (Tamura et al., 2007). Human LCNl (NP_002288) was used as the out-group. Radiation Hybrid Mapping of the Zebrafishfabpllb Gene A detailed protocol for radiation hybrid mapping of zebrafish genes is described by Hukriede et al. (1999). PCR reactions were carried out using the forward primer, rhf (5'-GTGTTGTGATTTTCGGTGG-3'; nucleotide [nt] positions 33 to 51), and the reverse primer, rhr (5'-TTCTGTCATCTGCTGTCGTC-3'; nt positions 396 to 423), as shown in Fig. 3.1. PCR conditions were initial denaturation at 940C for two minutes, followed by 30 cycles at 94°C for 30 seconds (denaturation), 54.50C for 30 seconds (primer annealing) and 72°C for 1 minute (elongation) with a final elongation step at 72°C for 5 minutes. In Situ Hybridization to Whole-mount Embryos and Larvae, and to Sections of Adult Zebrafish Whole-mount in situ'hybridization to zebrafish embryos and larvae was performed using a riboprobe synthesized fromfabpllb cDNA clone zgc: 1 10029 (GenBank accession number BC095142) as described by Thisse and Thisse (2008). In 44 situ hybridization of an oligonucleotide probe to sections of adult zebrafish followed the protocol of Denovan-Wright et al. (1998). Briefly, sagittal and transverse sections of adult zebrafish were hybridized to [a-33P]dATP 3' end-labeledfabpllb antisense probe, isf (5'-CACAACACAAGACGTTTGACAGATAATAGC-3'; nt positions 11 to 40), shown in Fig. 3 . 1 . Following hybridization and autoradiography, tissue sections were stained with cresyl violet to identify specific tissues. RT-PCR Detection offabpllb Transcripts in Aduit Zebrafish Tissues RT-PCR was employed for the tissue-specific detection offabpllb transcripts in RNA extracted from tissues of adult zebrafish. Following synthesis of cDNA from RNA samples using the Omniscript RT kit (Qiagen, Mississauga, Ontario, Canada), fabpllb cDNA was PCR-amplified by the forward primer, rtf (5'-GCTGTCACTACATTCAAGA CCCTGGA-3', nt positions 337 to 363) and the reverse primer, rtr (5'-ACCATCCGC AAGGCTCATAGTAGT-3'; nt positions 1369 to 1393), shown in Fig. 3.1. PCR conditions for the amplification of fabpllb transcripts employed an initial denaturation step at 940C for two minutes, followed by 30 cycles at 940C for 30 seconds (denaturation), 560C for 30 seconds (primer annealing) and 72°C for 1 minute (elongation) with a final elongation step at 720C for 5 minutes. PCR primers used for detection of elongation factor la (efla) transcripts in total zebrafish RNA are described in Pattyn et al. (2006). The PCR conditions were an initial denaturation step at 94°C for two minutes, followed by 30 cycles at 94°C for 30 seconds (denaturation) 620C for 30 seconds (primer annealing) and 720C for 1 minute (elongation) with a final elongation step of 72°C for 5 minutes. 45 Database Searches of Genome Sequences and Identification of Transcription Factor Binding Sites The genomic organization of the tetrapod FABP4, FABP5, FABP8 andFABP9 genes was derived from the Xenopus tropicalis, Gallus gallus, Mus musculus and Homo sapiens genome sequence databases at http://www.ensembl . org. Putative transcription factor-binding sites in the 5'-upsteam region of the zebrafishfabpllb gene were identified using the Alibaba 2.1 software (Grabe, 2002). Results and Discussion Identification ©f a Duplicatedfabpll Gene from Zebrafish A paralogous gene to the previously described zebrafishfabpl 1 (fabp4, but now referred to as fabplla) (Liu et al., 2007) was identified from a BLAST search of the zebrafish genome sequence database at the Wellcome Trust Sanger Institute (version Zv6, http://www.ensembl.org/Danio rerio/index.html) using as query the GenBank sequence, AY628221. The 3.4 kb duplicatedfabpl 1 (hereafter referred to as fabpl lb) consists of four exons and three introns (Fig. 3.1), a FABP gene organization seen for most vertebrate iLBP genes (Bernlohr et al., 1997). The four exons offabpllb code for 24, 59, 34 and 17 amino acids, respectively (Fig. 3.1), identical to that of the zebrafish fabplla gene (Liu et al., 2007). Forfabpllb, the splice junctions for intron one and intron two conform to the GT/AG intron/exon rule (Breathnach and Chambón, 1981), whereas the splice junction for intron three is TA/AG. A putative binding site for a GATA transcription factor was identified at position -471 to -483 in the 5' upstream region of thefabpllb gene (Fig. 3.1). Divine et al. (2006) reported that GATA-4, GATA- 5 and GATA-6 act cooperatively in activating the FABPl gene transcription in the murine small intestine. A putative TATA box is located at position -30 to -24. 46 Figure 3.1. The sequence of the zebrzñshfabpllb gene and its 5'-upstream promoter region. Exons are shown in upper-case letters, with the coding sequences of each exon underlined and the deduced amino acid sequence indicated below. The stop codon is indicated by the diamond symbol. Only partial nucleotide sequences were shown for introns two and three where the dotted lines indicate interruption in the sequence. +1 indicates the transcription start site. A putative polyadenylation signal, AATAAA, is highlighted in bold font and underlined. A putative TATA box and a site for GATA- binding factor are highlighted in bold font and underlined. The in situ hybridization probe (isp) used for detection oifabpllb transcripts in adult zebrafish tissues, PCR primers used for radiation hybrid mapping (rhf, rhr) and for detection of fabpllb transcripts in RNA extracted from adult zebrafish tissues (rtf, rtr) are either underlined or over-lined. 47 çh co in rí co co ai F ai ce CO if) G-* CtJ O 03 co o Oí ¿? ? ? « H t W co (M H s? Lf) rt N cO I I ? ? ? ? ? pH M co H CO CO (O U IO Ol U w ñ U U IB IB (? «. U U » 4-1 U ? Iti Ol O US M « U !O nj ?a ? io IO IO CU P ¦O (0 Oi Oi OI »J U öl id ? CU U Oi P U U Ol !0 o a ? Ü m ? P Ql id ? cn di ?a ?? b u IB O U IO (B P M P IO P IO ß U u m UTP P u rO LO jj Di JJ JJ o IO LC IO U 4-» 4J P P Ü Oi IO U !0 4J P P U U IO U IO 0» rd P io io O P Cn U 0 Di IO 4J Di IO •ß U U P ? « IO IO O Ü ?a id rd U U U U ID io ? DI P U P ? V u U U » "S US Oi Oi nS« ?-? P Oi ? u ? io P lfl Ol P "O ¡0 ? io ? S *j IO IO Di IO IO ?; JJ L) S i! U U id P" U io id Ol P !D IO Dl P U ID L-S Id io ?? Oi P 48 Distribution offabpllb Transcripts in the Retina of Developing Zebrafish Embryos and Larvae To determine the spatial and temporal distribution offabpllb transcripts during zebrafish embryonic and larval development, we conducted whole-mount in situ hybridization to zebrafish embryos and larvae at different developmental stages (Fig. 3.2). fabpl lb transcripts were first detected in the retina of developing embryos at 24 hours postfertilization (hpf) in the pigmented epithelium starting in the proximal part of the retina, fabpllb transcripts spread across this layer of the retina by 30 hpf (Fig. 3.2B). A homogeneous distribution offabpllb transcripts throughout the pigmented epithelium was observed at 48 hpf (Fig. 3.2C). In contrast tofabpllb, at 24 hpf and 36 hpf the fabplla transcripts were detected in the lens and diencephalon (Liu et al., 2007). In five day-old larvae, the hybridization signal forfabpllb transcripts was still observed in the same layer of the retina (Fig. 3.2D). Sellner (1994) reported a similar trend where retinal FABP appears to be maximally expressed in the chicken retina around the ninth day of the embryonic development and declines at later stages. Embryonic development in zebrafish spans three days, whereas embryonic development in chicken extends over 20- 21 days. Two other FABP transcripts, transcripts forfabp3 andfabp7b, have been detected in the developing retina of zebrafish embryos (Liu et al., 2004; Liu et al., 2007). It has been proposed that FABPs are involved in sequestering of FAs during retinal differentiation (Sellner, 1994). Tissue-Specific Distribution offabpllb Transcripts in Adult Zebrafish In situ hybridization was performed on sections of adult zebrafish to determine the tissue-specific distribution oífabpllb transcripts, fabpllb transcripts were detected along the vertebra in the spinal cord of adult zebrafish (Fig. 3.3A shows the hybridization 49 24h 30h Re i ¡fe* ? *,« m / Re a * Re Re « Jêsë. # S m m liei ma II Sd D Figure 3.2. Spatio-temporal distribution oîfabpllb transcripts during zebrafish embryonic and larval development was determined by whole mount in situ hybridization. fabpllb transcripts were first detected in the pigmented epithelium of the retina (Re) at 24 hour post fertilization (hpf) (Fig. 3.2A). The distribution oîfabpllb transcripts had spread across the retina by 30 hpf (Fig. 3.2B) and 48 hpf (Fig. 3.2C). In 5 day-old larvae, fabpllb transcripts were restricted to the circumference of the pigmented epithelium of the retina (Fig. 3.2D). 50 signal in a sagittal section and Fig. 3.3B a transverse section). While describing the distribution of FABP8 gene expression in the rabbit spinal cord, Narayanan et al. (1991) noted that the spinal cord has a high rate of FA biosynthesis. RT-PCR detectedfabpl 1 b transcripts in total RNA extracted from the brain, heart, ovary and eye of adult zebrafish (Fig. 3.4). fabpl lb transcripts were not detected in total RNA extracted from the liver, intestine, kidneys, gills or muscle of adult zebrafish. As a positive control for the quality of RNA in each tissue, transcripts for the constitutively expressed efla gene were assayed by RT-PCR and detected in all tissues examined (Fig. 3.4). The difference observed in the tissue-specific distribution offabpllb transcripts using in situ hybridization and RT- PCR is likely due to the sensitivity of the two assays; RT-PCR is far more sensitive than in situ hybridization. In contrast to zebrañshfabpl lb, fabplla transcripts were detected in liver, intestine, brain, heart, and muscle using RT-PCR (Liu et al., 2007). Abundant fabpl 1 transcripts were detected in liver, adipose tissue and the vitellogenic ovary, to a lesser extent in the previtellogenic ovary, heart, kidney, and muscle, and trace amounts were detected in testis by RT-PCR from tissues of adult Senegalese sole (Agulleiro et al., 2007). In adult Antarctic fishes (Vayda et al, 1998), fabpl 1 (Had-FABP) transcripts were detected in muscle, kidney, heart, and brain by the less sensitive assay of Northern blot and hybridization. Duplicate Copies oîfabpll in Zebrafish may have Arisen by a Fish Specific Whole- Genome Duplication Event Multiple sequence alignments of selected zebrafish and mouse FABP amino acid sequences and the prototypic Fabpl 1 from the Senegalese sole (Agulleiro et al., 2007) were performed using ClustalW (Thompson et al., 1997). Zebrafish Fabpl lb showed highest sequence identity and similarity (65% and 84%, respectively) with zebrafish 51 =Sc A Figure 3.3 Tissue-specific distribution oîfabpllb mRNA in adult zebrafish sections determined by in situ hybridization. Sagittal (A) and transverse (B) sections of adult zebrafish were hybridized to a [a-33P]dATP 3' end-labeledfabp 1 1 b antisense probe. The hybridization signal of the antisense probe was limited to the spinal cord (Sc) of adult zebrafish. 52 BLG H ! OEMK fcibDilb ' AÉHií 4ß?#?-: ••m**· ef1a Figure 3.4. RT-PCR detection oîfabpllb transcripts in RNA extracted from adult tissues of zebrafish using fabpllb cDNA-specific primers, fabpllb transcripts were detected by RT-PCR in RNA extracted from the brain (B), heart (H), ovary (O), and eye (E). Nofabpllb transcripts were detected in RNA extracted from the liver (L), gills (G), intestine (I), muscle (M), or kidney (K) of adult zebrafish (upper panel). As a positive control, constitutively expressed elongation factor la (efla) transcripts were detected by RT-PCR in RNA extracted from all adult tissues examined (lower panel). 53 Fabpl la, followed next by sequence identity and similarity with the Senegalese sole Fabpl 1 of 63% and 82%, respectively (Fig. 3.5). Sequence identity and similarity of zebrafish Fabpl 1 decreased with paralogous FABP/Fabps from zebrafish and mouse (Fig. 3.5). Radiation hybrid mapping using the LN54 panel (Hukriede et al., 1994) assigned zebrafishfabpllb to linkage group (LG) (chromosome) 16 at a distance of 26.59 cR from the marker fc09b04 with a logarithm of the Odds score of 10. Zdbr&ñshfabplla had previously been assigned to LG (chromosome) 19 by the same LN54 radiation hybrid panel (Liu et al., 2007). Conserved gene synteny on zebrafish LGs (chromosomes) 16 and 19 (Liu et al., 2007) with genes on human chromosome eight (Table 3.1) suggest that fabplla andfabpllb may have arisen by the teleost fish-specific WGD event that occurred approximately 250-400 mya (Furlong and Holland, 2002). Both fabplla (LG 19) andfabpllb (LG 16), and two other duplicated genes on these linkage groups, showed conserved gene synteny with the FABP4, FABP5, FABP8 and FABP9 gene cluster on human chromosome eight. For duplicated genes to be retained in the genome, Force et al. (1999) have proposed that both duplicated genes undergo either subfunctionalization, in which the functions of the ancestral gene are sub-divided between the sister duplicate genes, or one of the duplicates acquires a new function, called neofunctionaliszation. Force et al. (1999) further proposed that subfunctionalization of duplicated genes arises owing to the accumulation of mutations in the regulatory elements of duplicated genes which leads to divergence in their tissue-specific patterns of expression, fabplla mRNA transcripts were detected in ovary, liver, skin, intestine, brain, heart, testis and muscle in adult 54 Figure 3.5. Zebrafish (Danio rerio, Dr) Fabpl lb is aligned with zebrafish Fabpl la (deduced from AY628221), Fabp3 (AAL40832), Fabp7a (AAH55621) and Fabp7b (AAQ92970); Senegalese sole (Solea senegalensis, Sos) Fabpll (CAM58515); Mouse (Mus musculus, Mm) FABP4 (AAH02148), FABP5 (NP_034764), FABP8 (XP_485204), and FABP9 (NP_035728). Dashes specify gaps and dots indicate amino acid identity. The percentage of sequence identity and similarity of zebrafish, Senegalese sole and mouse FABP sequences with zebrafish Fabpl lb are shown at the end of each sequence. 55 - m w m h CQ CQ m CQ CQ CQ CQ CQ CQ < a ? m m o H : S > S É CQ CQ S j j · · · ? a • ? « · H H H OZOOOPOOO Q ..... O · · · Oi cqcq««cqq«J> H > > · . . K J J . Q ? ? ¦ · · · ·??^?£?^? o o O O O • J > H X H OS « -~ • J fe h dP Ui Ck HHMHCQHHoSiB U CQ CQ CQ CQ CQ CQ CQ W CQ · · HHHHMWWWa JJ ¦H Q Q M ^COOHCNCNOOOCn ß « OS H H Si ¦H S > g ¦H O O s W > > > CQ CQ O O K O w w dP J M CQ O O O O O O O O > > H > > > > 4J IflcyiHCNfiOOfnVOfn HJJH » J CQ CQ CQ CQ •H O CQ CQ · H ? JJ Ci (D ¡H ? ? an · a ? H a tu ? a « s ? ? œ w ? w • H ? ? . ? C» ? ? en ma · s · w · · IM *¡ < < H rt¡ > > > H «>>J>S>H • os = · oí S JJJ ? J J J J >¦> · · > . . „ . w ¡Z¡ . o . o d¡ . . is O O > > H H « H H H H ? H H H o • < . · . > H > J H H U U · · j à • CQ · · CQ b h h h It Cn tu W CQ CQ CQ CQ CQ • · e« W Ol W < < < w • H · · a a o Z · . . o · O O CO H H H H H O O W O Z Z • J h b « · · · > > H • J J J C^ CTt co co oo er* co o co oo aincoioooainocoœ HHHHH H H CN H H HHH inmininmminvoww HHHHH HHHHH ? ? H ? ?ß H ? n) H t-i T-i (ß ? H HH (ß ? H HH (ß ? H H H m > r- ft* in co ?? H H co c~ t- ft >*« in co en H H m c~ t- ft ·* m co s\ euaaftPi^ftciiCiiOi ftftftftftAPiaoiíi ? ? ? ? ? p) CQ CQ CQ CQ ? ? ? ? ? ?) CQ CQ CQ CQ ? ? ? ? ? ?ß CQ CQ CQ CQ fO(T>rOrOrOCnicC¡i=C;rt;i=q 56 Table 3.1. Conserved gene synteny of the duplicated copies of the zebrafish/aèpii gene with the human FABPA, FABP5, FABP8 and FABP9 genes. Gene name Zebrafísh Human Gene Linkage Mappi Gene Chromoso symbol group ng symbol mal position (cM) panel location ¦CI 7 7 Fatty acid-binding protei JU-Op 1 IU LN54 lib 53.10 Fatty acid-binding protein fabpllb 16,44.16- LN54 11a 45.60 Fatty acid-binding protein FABP4 4 8q21 Fatty acid-binding protein FABP5 5 8q21 Fatty acid-binding protein FABP8 3q21-8q22 Fatty acid-binding protein FABP9 8q21 der 1 -like domain family, derll 16,46.90 LN54 DERLl member 1 8q24.13 NADH dehydrogenase (ubiquinone)- 1 beta nduft>9 16, 24.20 T51 NDUFB9 8ql3.3 subcomplex 9 ATPase family, AAA atadl 16, 26.70 T51 ATAD2 8q24.13 containing 2 TAF2 RNA polymerase II, TATA box binding protein taß 16,31.25 LN54 TAF2 8q24.12 (TBP)-associated factor RAD21 homolog radii 16,61.07 LN54 RAD2 8q24 hyaluronan synthase 2 has2 16,31.67 T51 HAS2 8q24.12 Cadherin 17 cdhl7 16, 46.90 T51 CDH17 8q22.1 Mitochondrial folate 19,46.86 LN54 MFTC 8q22.3 transporter/carrier mftc Growth differentiation 19, 47.30 HS GDF6 factor 6 gdfSb 8q22.1 Sperm-associated antigen 1 spagl 19, 49.00 T51 SPAGl 8q22.2 Protein tyrosine phosphatase type IVA, zfpm2b 19,50.25 HS PTP4A3 8q24.3 member 3 Antizyme inhinbitorl azinl 19, 53.30 T51 AZINl 8q22.2 Angiopoietin 1 angptl 19,81.93 T51 ANGPTl 8q22.3 57 zebrafish (Liu et al., 2007). During the larval development, fabplla transcripts were detected in the lens and diencephalon (Liu et al., 2007). In contrastfabpllb transcripts were detected in brain, heart, ovary and eye by RT-PCR (Fig. 3.4) and in the spinal chord by in situ hybridization (Fig. 3.3) in adult zebrafish, and in the retina during the larval development (Fig. 3.2). Althoughfabpl 1 a andfabp 1 1 b transcripts exhibit strikingly different patterns of tissue distribution in developing embryos, larvae, and adult zebrafish, it is not possible to ascertain whether the duplicated copies offabpll have been retained in the zebrafish genome by either subfunctionalization or neofunctionalization, as there is no readily apparent ortholog of the fishfabp11 gene in tetrapods or other species studied thus far (Agulleiro et al., 2007). fabpll, FABP4, FABP5, FABP8, and FABP9 Evolved from a Common Ancestral Gene on a Single Progenitor Chromosome We constructed a neighbor-joining phylogenetic tree using the amino acid sequences of selected vertebrate FABPs (Fig. 3.6). Zebrafish Fabpl lb and zebrafish Fabpl la formed a clade with other teleost Fabpl Is (Fig. 3.6), a clade distinct from the frog, chicken and mammalian FABP4/FABP5/FABP8/FABP9 clade as previously shown by Agulleiro et al. (2007). The zebrafish Fabpl la and Fabpl lb amino acid sequences have sufficiently diverged from each other, such that they are not linked by a common node on the tree. Similarly, the putative Fugu and stickleback Fabpl la and Fabpl lb do not share a common node with their sister duplicates either, but are all clustered in the same clade with all other teleost fish Fabpl Is. The sequence identity of zebrafish Fabpl lb to mouse FABP4, FABP5, FABP8 and FABP9 (also known as PERF15) varied from 43% to 47% (Fig. 3.2). The sequence 58 Figure 3.6. Phylogenetic tree of selected vertebrate FABPs showing the relationship between zebrafish Fabpl la and Fabpl lb. The neighbor joining tree was constructed using Homo sapiens LCNl (NP_002288) as outgroup. The bootstrap values (per 100 duplicates) are indicated above or under each node. The teleost Fabpl Is formed a common clade which is indicated by a bracket. Amino acid sequences used in this analysis include: Danio rerio, zebrafish (Dr) Fabpl la (derived from AY628221), Fabpl lb (ENSDARP0000002311); Gobionotothen gibberifrons (Gog) Fabpl 1 (H6- Fabp, AAC60354); Notothenia coriiceps (Nc) Fabpl 1 (H6- Fabp, AAC60352); Parachaenichthys charcoti (Pc) Fabp 11 (H6- Fabp, AAC60355); Takifugu rubripes, takifugu (Fr) Fabpl la (deduced from AL837220), Fabpl lb (deduced from AL836636); Tetraodon nigroviridis (Tn) Fabpl 1 (deduced from CR723700); Oryzias latipes, rnedaka (OÏ) Fabpl 1 (deduced from BJ899828); and Cyprinus carpio, common carp (Cc) Fabpl 1 (deduced from CF661735); Solea senegalensis, Senegalese sole (Sos) Fabpl 1 (CAM58515); Gasterosteus aculeatus, stickleback (Ga) putative Fabpl la (ENSGACP00000004532), putative Fabpl lb (ENSGACP0000001 1538); Homo sapiens (Hs) FABP4 (CAG33184), FABP5 (AAH70303), and FABP8 (AAH34997); FABP9 (PERF15) (Uniprot ID Q0Z7S8); Mus musculus, mouse (Mm) FABP4 (AAH02148), FABP5 (NPJB4764), FABP8 (XP_485204), and FABP9 (NP_035728); Rattus norvégiens, rat (Rn) FABP4 (NP_445817), FABP5 (NP_665885) and FABP9 (NP_074045); Sus scrofa, pig (Ss) FABP4 (CAC95166); Gallus gallus, chicken (Gg) FABP4 (NP_989621), FABP5 (ENSGALP00000025375), FABP8 (ENSGALP00000025382); Xenopus tropicalis, African frog (Xt) Putative FABP4 (ENSXETP00000022878), Putative FABP4-like (NP_001096256.1), Putative FABP5 59 (ENSXETP00000Q22879); Pan troglodytes, chimpanzee (Pt) FABP9 (PERF15) (ENSPTRPOOOQ0053 1 26). 94rMmFABP9 RnFABP9 HSFABP9 78 L PtFABP9 I— GgFABP8 HSFABP8 P3 MmFABPS RnFABP8 GgFABP4 58 SsFABP4 r- HsFABP4 RnFABP4 90I LI MMmFABP4 — GgFABPS 89 I— HIsFABPS "¡Hj- RnFABPS 92L MmFABPS XtFABPS XtFABP4 85iL"I XtFABP4l FrFabpHb — GaFabpHb 58 57 — DrFabpHb 93)3J—1— DrFabpHa« 39 "L CcFae bp11 — SosFabpH 94|pOIFabp11 88 jTnFabpH 56 ft-FrFabpHa 3glialp— GaFabpHa 69 PcFa bp11 97 GogFabpH 91 NcFabpH HsLCNI identity of the Senegalese sole Fabpll with human FABP4, FABP5, FABP8 and FABP9 varied from 52,2% to 54.5% (Agulleiro et al., 2007). To date, we have no evidence for orthologs of the tetrapod FABP4, FABP5, FABP8 and FABP9 genes in teleost fishes. This is based on tBLASTn searches of complete or nearly complete sequences for several fish genomes {e.g., Danio rerio, Takifugu rubrifus and Tetradon nigroviridis) in ENSEMBL (http://www.ensembl.org) and in the extensive EST databases {e.g., salmonids) in GenBank (Benson et al., 2005). Agulleiro et al. (2007) were the first to suggest that fabpll formed a novel clade among vertebrate FABPs and that this gene is unique to teleost fishes. In addition io fabpll of the Senegalese sole, homologous fabps from the Antarctic fishes (Vayda et al., 1998), and zebmñshfabp genes reported here and previously (Liu et al, 2007), appear to belong to a sister group of FABP4, FABPS, FABP8 and FABP9 of mammals and that tetrapod FABP4, FABP5, FABP8, and FABP9 genes and the teleost fishfabpll genes might have arisen from a common ancestral gene (Agulleiro et al., 2007). Based on the evidence obtained from phylogenetic analysis, linkage mapping and conserved gene, we propose the following model for the evolution offabpll in fishes and the FABP4IFABP5IFABPSIFABP9 gene cluster in tetrapods (see Fig. 3.7): First, the fabp4, fabp5, fabp8 andfabp9 genes are absent in teleost fishes. Second, an ancestral gene diverged to give rise to the progenitors offabpll in fishes and FABP4, FABP5, FABP8, and FABP9 in tetrapods, probably, before the fish-tetrapod split some 450 mya (Kumar and Hedges, 1998). Third, the FABP4, FABP5, FABP8 and FABP9 gene cluster in tetrapods arose by successive tandem duplications and divergence of a single ancestral gene, as has been suggested for the evolution of some of the globin gene clusters 62 Ancestral gene Fishes Amphibians/Birds/Mammals Whole genome duplication/ wêêêêêêêêm\ci^^^EH^eeij frog fabp4 fabp4l fabp5 (scaffold 225) fabplla fabpllb ______In zebrafish FABP5 FABP8 FABP4 (chromosome 2) MOUSE Fabp5 Fabp8 Fabp9 Fabp4 (chromosome 3) FABP5 FABPB FABP9 FABP4 (chromosome 8) (PERFl 5) Figure 3.7. Evolutionary relationship between the duplicated copies of thefabp11 gene in fishes and FABP4, FABP5, FABP8 and FABP9 (PERFl5) in frog, chicken, mouse, and human. In zebrafish, fabpl 1 a is found on linkage group 19 andfabpllb is on linkage group 16. In frog (Xenopus tropicalis), fabp4, fabp4-like (fabp4l) and fabp5 are found on scaffold 225, wherefabp4l andfabp5 are immediately adjacent to each other. The chicken (Gallus gallus) FABP5, FABP8 and FABP4 genes are located next to each other on chromosome two. FABP5, FABP8, FABP9 (PERF15) and FABP4 are tandemly arrayed on human (Homo sapiens) chromosome eight and mouse (Mus musculus) chromosome three. Gaps in white indicate the presence of an additional gene between two FABP genes on a particular chromosome. 63 (Hardison, 1998). Last, the ancestral gene of thefabpll and FABP4, FABP5, FABP8 and FABP9 resided on a chromosome, or a part of it, that was the progenitor of zebrafish linkage groups (chromosomes) 16 and 19, chicken chromosome two, mouse chromosome three, human chromosome eight and frog scaffold 225 (see "Materials and Methods" for details of retrieval of genomic sequence data). The Tetrapod FABP5, FABP8, FABP9 and FABP4 Genes are Tandemly Arrayed and Likely Arose by Unequal Crossing-Over» Which Gene was Duplicated First? While speculative, it is possible to provide a parsimonious scheme for the tandem duplication events that gave rise to the cluster of four FABP on a single mammalian chromosome. In the frog genome, fabp4, fabp4-like andfabp5 are tandemly arrayed on scaffold 225 (Fig. 3.7). Owing to sequence identity and their relationship in the phylogenetic tree (Fig. 3.6), thefabp4 mdfabp4-like genes may have arisen in the frog genome as a result of tandem duplication of an ancestral gene on this chromosome. It is not readily apparent, however, if the tandem duplication that gave rise to the frogfabp5 gene occurred before or after the tandem duplication that produced thefabp4 andfabp4- like genes. The phylogenetic analysis would favour thefabp4/fabp4-like duplication occurred after the duplication event that generatedfabp5. Orthologs of mammalian FABP8 and FABP9 have not yet been identified, or more likely are not present, in the frog genome. The chicken FABP5, FABP8 and FABP4 genes are also tandemly-arrayed on chromosome two; based on database searches, it seems that FABP9 is absent from the chicken genome. Therefore, chicken FABP8 most probably arose from the tandem duplication of either FABP4 or FABP5. Again, the phylogenetic tree (Fig. 3.6) suggests that chicken FABP8 may have originated from the tandem duplication of FABP4 rather 64 than FABP5, as FABP8 and FABP4 form a common clade, whereas FABP5 was placed in a different clade (Fig. 3.6). Our model is consistent with the time-scale for these duplication events based on synonymous/nonsynonymous amino acid substitution rates in FABP4, FABP5, FABP8 and FABP9, and the topology of the phylogenetic tree reported by Schaap et al. (2002). Fabp5, FabpS, Fabp9, and Fabp4 are arranged in sequential order on the mouse chromosome three and the FABP5, FABP8, FABP9 and FABP4 gene cluster is present on the human chromosome eight (Fig. 3.7). Tandem duplication of FABP'8 may have resulted in the formation of FABP9, as FABP8 and FABP9 formed a common clade (Fig. 3.6). 65 Chapter 4: 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 The manuscript based on this study is presented below. Co-authors for this manuscript are Santosh P LaIl, Eileen M. Denovan-Wright and Jonathan M. Wright. Originally published as: Karanth S, LaIl SP, Denovan-Wright EM, Wright JM. 2009. 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. BMC Evol Biol 9:219. Authors' contributions: SK and JMW conceived and designed the research; SK conducted the experimental work and statistical analysis; SPL provided expertise in the design of diets and fatty acid analyses; ED-W assisted in design and interpretation of RT-qPCR analysis; SK and JMW drafted the manuscript with subsequent editorial comments from SPL and ED-W. All authors read and approved the final version of the manuscript. 66 Abstract 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 FAs in zebrafish. Adult zebrafish were fed diets containing either fish oil, sunflower oil, linseed oil, all contain 12% lipid, or low fat (4% lipid) diet for 10 weeks. FA profiles and the steady-state levels offabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. 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, fabpla/fabplb.l/fabplb.2,fabp7a/fabp7b, andfabpllalfabpllb, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level offabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level oifabplb.l andfabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level offabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level offabp7a and fabpl 1 a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level offabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a givenfabp gene. As such, up-regulation of the steady-state level offabp 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 67 following feeding zebrafish any of the four experimental diets. Differential induction of only one of the sister pair of duplicatedfabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization. 68 Introduction In his seminal book, 'Evolution by Gene Duplication', Ohno (1970) argued that one of the major mechanisms that facilitate the increasing complexity in the evolution of life is duplication of genes and whole genomes. Models to explain retention of duplicated genes in eukaryotic genomes have undergone a development of thought since Ohno proposed his model almost 40 years ago. Ohno (1970) argued that most duplicated genes are lost from the genome owing to nonfunctionalization, a claim which has been validated by empirical evidence from Lynch and Conery (2000). Nonfunctionalization is a process where deleterious mutations accumulate in the coding region of a gene giving rise to either a dysfunctional protein or no protein product. Duplicated genes might, however, be retained in the genome owing to mutations in the coding region that led to a novel function for the protein product of a gene, a process Ohno termed 'neofunctionalization'. This model by Ohno for the preservation of duplicated genes came to be known as the 'classical model'. Data primarily derived from genome sequencing projects over the past decade suggest that a much higher proportion of gene duplicates is preserved in the eukaryotic genome than predicted by Ohno' s "classical model". To explain this apparent "conundrum", Force et al. (1999) proposed the Duplication-Degeneration-Complementation (DDC) model in which subfunctionalization serves as an alternative mechanism, but not to the exclusion of neofunctionalization, for the preservation of duplicated genes. According to the DDC model, duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between sister duplicate genes, or by neofunctionalization, where one of the duplicates acquires a new function. In this model, both processes occur 69 by either loss or gain of cw-acting regulatory elements in the promoters of the duplicated genes. As with the "classical model" of Ohno (1970), the DDC model proposes that most duplicated genes are lost from the genome (i.e., nonfunctionalization) owing to an accumulation of deleterious mutations in coding or control regions leading to functional decay. We chose to test the DDC model of Force et al. (1999) that subfunctionalization or neofunctionalization results in the retention of duplicated genes in the genome by investigating the expression of duplicated copies of fatty acid-binding protein (Fabp) genes, members of the multigene family of intracellular lipid-binding protein (iLBP) genes, in zebrafish for two reasons. First, bioinformatic resources for zebrafish are readily available, including linkage maps, extensive expressed sequence tags (EST) [www.zfin.org] and an almost complete genome sequence database [www.ensembl.org/Danio_rerio]. Second, and most importantly, owing to a WGD event, an event that occurred early in the ray-finned fish radiation about 230-400 million years ago (Hurley et al, 2007; Mulley and Holland, 2004; Postlethwait et al., 2000; Woods et al., 2005), we predicted that many members of the iLBP multigene family might exist as duplicated copies. This prediction has proved to be correct (see below). The iLBPs are encoded by a highly conserved multigene family, which consists of the fatty acid- (FABP), cellular retinol- (CRBP) and cellular retinoic acid-binding protein (CRABP) genes (reviewed in Furushahi and Hotamisligil, 2008; Haunerland and Spener, 2004; Li and Norris, 1996; Schroeder et al., 2008; Storch and Corsico, 2008). Currently, 17 paralogous iLBP genes have been identified in animals, but no member of this multigene family has been identified in plants and fungi. Schaap et al. (2002) have 70 suggested, therefore, that the first iLBP gene emerged after the divergence of animals from plants -930-1000 million years ago. This ancestral iLBP gene presumably underwent a series of duplications followed by sequence divergence, giving rise to the diversity of the extant iLBP multigene family. This multigene family has been further augmented in ray-finned fishes by the WGD event mentioned above (Hurley et al., 2007; Mulley and Holland, 2004; Postlethwait et al, 2000; Woods et al., 2005). Originally, iLBP genes and their proteins were named according to the initial tissue of isolation, e.g., liver-type FABP (L-FABP), brain-type FABP (B-FABP), etc. Owing to some confusion with this earlier nomenclature, here we use the nomenclature proposed by Hertzel and Bernlohr (2000) in which numerals distinguish the different FABP proteins and their genes, [e.g., FABPl (liver-type), FABP7 (brain-type)]. FABPl, the first FABP isolated, was described almost 40 years ago (Ockner et al., 1972). Although extensive studies in mammals have focused on the tissue distribution and binding activities of FABPs, the regulation and evolution of their genes, and mice FABP gene knock-outs (Haunerland and Spener, 2004; Schroeder et al., 2008; Storch and Corsico, 2008), the precise physiological roles of FABPs remain unclear. However, accumulated data have provided evidence that FABPs play an important role in uptake, sequestering and transport of FA s and interaction with other transport and enzyme systems. Indirect evidence suggests other putative physiological functions for FABPs, such as: (i) transport of FAs to the nucleus to regulate gene transcription via activation of the nuclear receptors, the peroxisome proliferator-activated receptors (PPAR) [see e.g., (Furushahi and Hotamisligil, 2008; Leaver et al., 2005)]; (ii) essential functions in early development, especially neural growth and differentiation [see Liu et al. (2003) and Liu 71 et al. (2004) and references therein]; and (iii) a role in human diseases (Storch and Corsico, 2008; Furushahi and Hotamisligil, 2008). Although the coding sequence and structure of the FABP genes has been well conserved over millions of years, each FABP gene exhibits a distinct, yet sometimes overlapping pattern of tissue-specific expression with other FABP genes. If all FABP genes in this multigene family arose from a single ancestral gene as proposed (Schaap et al., 2002), the regulatory elements in the promoters must have evolved different functions in the subsequent duplicated genes giving rise to the complexity of the spatio-temporal expression of this multigene family. Regulatory elements in the promoters of some mammalian FABP genes and one insect have been identified (Haunerland and Spener, 2004; Wu et al., 2002). As such, cis-acting elements that determine the spatio-temporal expression of these genes, with a few exceptions, are not well defined. From sequence data, it appears that mammalian iLBP gene-promoters consist of a modular structure similar to many eukaryotic promoters, comprised commonly of a TATA box with proximal and distal regulatory elements (Carey and Smale, 1999). Currently, our understanding of the regulatory elements that control the expression of the FABP genes is modest and limited primarily to mammals [but see Her et al. (2003); Qu et al. (2007); Wu and Haunerland (2003)]. Based on the zebrafish/a¿piO sequence (Denovan-Wright et al., 2000), Her et al., (2003) cloned its promoter and by functional analysis, identified a 435 bp regulatory element that is sufficient to modulate the liver regional expression in transgenic zebrafish. How this cis-acting element functions is not known. In addition to functional promoter studies, FAs and peroxisome proliferators have been shown to induce the transcription of some FABP genes in mammals (Meunier-Durmort et al., 72 1996; Poirier et al., 2001) via activation of PPAR, or other unknown transcription factors, that bind to a FA response element (FARE) (Qu et al., 2007). To date, we have characterized 1 1 zebrafish FABP genes with respect to their cDNA sequence, gene structure, chromosome location, conserved gene synteny with their mammalian orthologs, and their spatio-temporal patterns of expression in embryos, larvae and adults (Alves-Costa et al, 2008; Denovan-Wright et al., 2000a; Denovan-Wright et al., 2000b; Raranth et al., 2008; Liu et al., 2003a; Liu et al., 2003b; Liu et al., 2004; Liu et al., 2007; Pierce et al., 2000; Sharma et al., 2004; Sharma et al., 2006). Based on phylogenetic analyses and conserved gene synteny with their single-copy, mammalian orthologs, eight (four pairs) out of 1 1 of the extant members of the zebrafish FABP genes arose as a result of the ray-finned fish-specific WGD event (Liu et al., 2004; Karanth et al, 2008; Karanth et al., 2009). One pair of genes, fabplb.l andfabplb.2, are tandemly- arrayed on chromosome 8 separated by ~4 kb of DNA (Karanth et al., 2009). This duplication, which was subsequent to the WGD but is not yet dated, is presumably the result of unequal crossing-over between homologous chromosomes during meiosis. The number of duplicated FABP genes (63%) retained in the zebrafish genome owing to the WGD event in the ray-finned fishes lineage is remarkable as Postlethwaith et al. (2000) estimate that only 20% of all the duplicated genes following WGD have been retained in the zebrafish genome. Other estimates for retention of duplicated genes in the zebrafish genome from the WGD are 14-30% (Woods et al., 2005). Three zebrafish FABP genes, fabp2, fabp3 andfabpó exist as single copies [a duplicate offabplO has recently been identified by us (Venkatachalam et al., 2009)]. Following the WGD event, the sister 73 duplicates of these genes have presumably been lost by accumulation of mutations leading to functional decay. In mammals, FAs up-regulate the expression of some FABP genes as evidenced by an increase in mRNA and protein levels (Meunier-Durmort et al., 1996; Poirier et al., 2001). We hypothesized that (i) zebrafishfabp genes might be up-regulated by dietary FAs, and (ii) sister duplicatedfabp genes might be differentially modulated by dietary FAs. We show by assaying steady-state mRNA and heterogeneous nuclear RNA (hnRNA) levels for three sets of duplicatedfapb genes, fabpla/fabpl b. 1/fabpl b.2, fabp7a/fabp7b andfabp11 a/fabpl lb, that dietary FAs modulate the transcriptional initiation of only one of the duplicatedfabp genes in specific tissues of zebrafish. This result provides compelling evidence that these duplicatedfabp genes were retained in the genome by either subfunctionalization or neofunctionalization owing to the divergence of c/s-acting regulatory elements in thefabp gene promoters. Materials and Methods Diets and Fish Husbandry Four isoproteic (41% crude protein) diets differing in FA composition and lipid content were formulated (Table 4.1). In addition to National Research Council recommendations on the nutritional requirements of warm-water fishes (NRC, 1993), we were guided by the results of previous dietary studies in zebrafish (Goolish et al., 1999; Mathew and Sherief, 1999; Meinelt et al., 1999; Meinelt et al., 2000; Tocher et al., 2001) in the formulation of the diets used in this study. All dry ingredients were mixed in a Hobart mixer. Butyl hydroxy anisóle was dissolved in the lipid source and choline chloride was dissolved in distilled water. Both of these solutions were then added to dry 74 Table 4.1. Composition of experimental diets (g/100 g of dry diet) Ingredient HD LD* LND LFD Vitamin free Casein 33 33 33 33 Wheat Gluten" 10 10 10 10 Gelatin Flax oil Omega Mix Sunflower oil Corn starch (pre-gelatinized) 29.1 29.1 29.1 37.1 CelufiT Vitamin Mix 1.3 1.3 1.3 1.3 t Mineral Mix Betaine 1.5 1.5 1.5 1.5 DL-Methionine ).2 0.2 0.2 ).2 Total 100 100 100 100 * HD - highly unsaturated FAs rich diet; LD - linoleic acid rich diet; LND - linolenic acid rich diet; LFD -low fat diet; BHA-butyl hydroxy anisóle. 1US Biochemical, Cleveland, OH, USA. 2Dover Mills Ltd. (Halifax, NS, Canada). 'Obtained from the local market. 4Jamieson omega-3 select capsules. 5National Starch and Chemical Co. (Bridgewater, NJ, USA). 6Vitamin added to supply the following (per kg diet): vitamin A, 8000 IU; vitamin D3, 4000 IU; vitamin E, 300 IU; vitamin K3, 40 mg; thiamine HCl, 50 mg; riboflavin, 70 mg; d-Ca pantothenate, 200 mg; biotin, 1.5 mg; folic acid, 20 mg; vitamin Bi2, 0.15 mg; niacin, 300 mg; pyridoxine HCl, 20 mg; ascorbic acid, 300 mg; inositol, 400 mg; choline chloride, 2000 mg; butylated hydroxy toluene, 15 mg; butylated hydroxy anisóle, 15 mg. 7Mineral added to supply the following (per kg diet): manganous sulphate (32.5% Mn), 40 mg; ferrous sulphate (20.1% Fe), 30 mg; copper sulphate (25.4% Cu), 5 mg; zinc sulphate (22.7% Zn), 75 mg; sodium selenite (45.6% Se), 1 mg; cobalt chloride (24.8% Co), 2.5 mg; sodium fluoride (42.5% F), 4 mg. 8Betaine anhydrous (96% feed grade), Finnfeeds, Finland. 75 ingredients to make a wet dough. The wet dough was scrubbed through an 800 µ?? screen and the resulting wet particles were freeze-dried and stored at -2O0C. The particle size was 600-800 µ?? in diameter, a size interval for feeding appropriate to the age and weight of the fish used in this study. Mean weight of fish at the start of the feeding trial was 0.26 g. Highly unsaturated FA-rich diet (HD), linoleic acid-rich diet (LD), linolenic acid-rich diet (LND) contained 12% lipid, whereas the low fat diet (LFD) was composed of 4% lipid (Table 4.1). The FA composition of the four diets is shown in Table 4.2. The concentration of linoleic acid (18:2 n-6) was highest in LD (44.82%), while the concentration of linolenic acid (18:3 n-3) was highest in LND (33.53%). Among the four diets, the concentration of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) was highest in HD (6.14% and 20.94%, respectively). One hundred and eighty wild-type inbred zebrafish siblings (mean wt. 0.26 g), bred from 2 males and 2 females (obtained from a local aquarium supply store) to control for genetic variance, were randomly assigned to 12 aquaria (35 1) with 15 fish per tank at approximately 20 weeks of age. Each dietary treatment had three tank replicates to control for environmental variance. Fishes were acclimatized for 15 days and fed Tetramin® (Tetrawerke, Melle, Germany) twice a day to satiation. The Tetramin diet was discontinued and then fish were fed one of the four diets twice a day at 0900 h and 1900 h to satiation for 10 weeks. A constant water temperature (28°C), a light/dark cycle (14/10 h) and other water parameters were maintained according to Westerfield (Westerfield, 2000). Feeding was stopped 24 h prior to sampling of tissue. Fish were individually euthanized by immersion in 0.2% MS-222, weighed and dissected to collect intestine, liver, muscle and brain tissues for RNA and FA analysis. Animal husbandry 76 Table 4.2. Major fatty acids in the experimental diets1 Fatty acid HD^ LD¿ LNDZ LFDZ 12:0 7.73 7.14 6.24 0.80 14:0 3.58 3.19 2.84 1.05 16:0 7.08 6.94 6.65 8.44 ?d.?1 g. A 3.36 3.94 Total Saturates 23.43 24.80 21.57 16.15 18:1 n-9 11.61 18.12 17.19 17.70 Total Monoenes 16.39 19.03 18.82 20.53 18:2 n-6 17.82 44.82 23.81 39.93 18:3 n-3 8.66 9.44 33.53 20.38 20:5 n-3 6.14 0.04 0.03 0.11 22:6 n-3 20.94 0.14 0.30 0.13 Total PUFAZ 60.14 56.12 )9.68 63.32 1DaIa expressed as area percentage of FA methyl esters. 2 HD - highly unsaturated FA-rich diet; LD - linoleic acid-rich diet; LND - linolenic acid- rich diet; LFD -low fat diet; PUFA - polyunsaturated FAs. 77 and protocols for this experiment were reviewed by the Animal Care Committee of Dalhousie University in accordance with the Canadian Council on Animal Care. RNA Isolation, cDNA Synthesis and RT-qPCR Total RNA was isolated from intestine, liver, muscle and brain of zebrafish using the standard TRIzol method (Invitrogen, Carlsbad, California, USA). The quality and quantity of extracted RNA was assessed by agarose gel-electrophoresis and spectrophotometry at 260 nm, respectively. Messenger RNA in 2 µg of total RNA was converted to cDNA using an oligo(dT) primer according to the manufacturer's instructions for the Omniscript RT kit (Qiagen, Mississauga, Ontario, Canada). cDNA was synthesized from heterogeneous nuclear RNA (hnRNA) using random hexamers. Primer pairs for quantification of mRNA and hnRNA encoded by differentfabp genes and the annealing temperature for primer pairs for eachfabp gene are shown in Table 4.3. For assay of gene-specific hnRNA, one primer was based on an intronic sequence, while the other was based on an exonic sequence (see Table 4.3 for details). Primers for the amplification of elongation factor 1 alpha (efla) mRNA by RT-qPCR are based on a previous study (Pattyn et al., 2003). The target sequence for each gene was quantified to generate a standard curve of known copy number. Amplification of cDNA samples and DNA standards was carried out using the SYBRGreen Quantitect PCR Kit (Qiagen, Mississauga, Ontario, Canada) following the manufacturer's instructions. For thermal cycling and fluorescence detection, a Rotor-Gene 3000 system (Corbett Research, Sydney, Australia) was used. PCR conditions were as follows: initial denaturation for 15 min at 95 0C followed by 40 cycles of 15 s denaturation at 94°C, 20 s annealing of primers at different temperatures 78 01 HH ¿ a; CJ; m s s W Sr, O in o Os IO Ko U H H < < < O CD O O E < "S O a. H a> H !ß < !- O H O H O S a U < ce U < S O e "S O ce ce 3 S s" CT O O < H ¦< U Z Z O PS a a: H s H a a a H H < O O •µe H ·- O < a U H Ü < H < H O a O fa < U < U O ? O U < < U U < O H U H H U -Si O JS 79 depending on the primer pairs (see Table 4.3), and 30 s of elongation at 72°C. Following the PCR cycles, the melting temperature of the PCR product was determined to assess its purity. Fluorescence was measured following each cycle. The copy numbers of mRNA and hnRNA for eachfabp gene were determined using the standard curves as explained by Bustin et al. (2005). As negative controls, the reverse transcriptase was omitted from cDNA synthesis reactions for each sample and these controls were subjected to RT- qPCR. To determine the relative steady-state level offabp mRNA and hnRNA in each tissue, the absolute copy number offabp mRNA and hnRNA transcripts was divided by the copy number of efla mRNA and hnRNA transcripts in each sample. Lipid Extraction, FAME Preparation and Gas Chromatography A modified Folch procedure (Folch-et al., 1957) described by Budge et al. (2001) was used to extract neutral lipid fractions from tissues. Briefly, the tissues were homogenized and sonicated for four minutes in 8:4:3 chloroform:methanol:water and the process was repeated four times. Following each extraction, the organic layer was removed, pooled and concentrated under a gentle stream of nitrogen. FA methyl esters (FAMEs) of tissue and dietary lipid were prepared with 7% boron trifluoride in methanol and heating to 100 0C for 60 minutes (Martins et al., 2007). FAMEs were separated by a gas Chromatograph equipped with a flame-ionization detector (Hewlett Packard 6890 GC system, Wilmington, Delaware, USA) on an Omegawax 320 capillary column (30 m ? 0.32 mm x 0.25 µp?; Supelco, Bellefonte, Pennsylvania, USA). FAMEs were identified by comparison of retention times with those of known standards (Supelco 37 and menhaden oil; Supelco, Bellefonte, Pennsylvania, USA). 80 Statistical Analysis Microsoft Excel® 2003 and SPSS® 14.0 (Chicago, USA) were used for statistical analysis. The relative abundance of mRNA and hnRNA encoded by eachfabp gene is presented as means ± S.E.M. The significance level was set at P<0.05. The effect of diet on FA composition and the relative abundance of mRNA and hnRNA encoded by each fabp gene in different tissues were analyzed by one-way ANOVA. Post hoc comparisons were conducted using the Tukey's HSD test. Results The Steady-State Level offabp mRNAs Does not Differ Between Sexes of Zebrafisfa Several authors have argued that lipid metabolism is influenced by the sex of an organism. For example, long-chain FAs are cleared from plasma by the liver more rapidly in female rats than male rats (Luxon and Weisiger, 1993). Bass et al. (1985) reported that female rats have a higher intracellular concentration of FABP3 than male rats. We, therefore, assayed by RT- the steady-state level of mRNA encoded by eachfabp gene in intestine, liver, muscle and brain of male and female zebrafish reared on Tetramin® (Tetrawerke, Melle, Germany) flake diet. No difference in the steady-state level offabp mRNAs in a specific tissue (i.e., intestine, liver, muscle or brain) was observed between male and female zebrafish (data not shown). Effect of Diet on the FA Composition in Different Tissues of Zebrafish Zebrafish readily ate all four diets and no differences were observed in their feed intake during the 10 week feeding period. Composition and abbreviated names for the four diets are shown in Table 4.1. The weight offish increased from 0.26 ± 0.02 g at the beginning of the feeding trial (-22 weeks of age) to 0.45 ±0.01 g at the end of the 81 feeding trial (-32 weeks of age) for all diets. At the end of 10 weeks, each diet resulted in a different FA profile for specific tissues. Figure 4. 1 shows the composition of four major FAs in intestine, liver, muscle and brain. For detailed composition of FAs in each tissue see: Tables 4.4 - 4.7. Intestine, liver, muscle and brain of fish fed HD and LND had higher proportions of n-3 FAs, due largely to elevated levels of linolenic acid (18:3 n-3), eicosapentaenoic acid (20:5 n-3), and docosahexaenoic acid (22:6 n-3), and reduced proportions of n-6 FAs. In contrast, intestine, muscle, and brain of fish fed LD and LFD exhibit higher percentages of n-6 FAs, linoleic acid (18:2 n-6) and arachidonic acid (20:4 n-6), and reduced proportions of n-3 FAs. The percentages of other FAs varied among tissues within each experimental group and also among the experimental groups (Tables 4.4-4.7). Effect of Diet on the Steady-State Level offabplalfabplb.ilfabplb.2 mRNAs in Different Tissues No modulation in the steady-state level oîfahpla mRNAs was observed in the intestine (Fig. 4.2A) regardless of diet fed to the fish. In the intestine of fish fed LND, the steady-state level offabplb.l mRNAs was higher than those in the intestine of fish fed the other three diets (Fig. 4.2B). The level oifabpla andfabplb.l mRNAs was below that quantifiable by reverse transcription, quantitative polymerase chain reaction (RT- qPCR) in liver, muscle and brain of zebmfish. fabplb. 2 mRNA was detectable in intestine and brain of fish fed the four diets, but the levels in liver and muscle were below the quantifiable range in RT-qPCR assays (data not shown). Furthermore, dietary FAs did not change the steady-state level offabplb. 2 transcripts in the intestine and brain. 82 A. Intestine B. Liver 35 30 H 2s a DHA 20 ss HEPA 15 10 BLNA 5 ¦ LA 0 ini HD LD LND LFD HD LD LND LFD C. Muscle D. Brain 25 ? 20 HDHA 15 H s? 20 4 HEPA 10 mm m- -m « i QLNA QLA HD LND LFD LND LFD Figure 4.1. Composition of the four major fatty acids, linoleic acid (18:2 n-6, LA), iinolenic acid (18:3 n-3, LNA), eicosapentaenoic acid (20:5 n-3, EPA), and docosahexaenoic acid (22:6 n-3, DHA) in intestine (A), liver (B), muscle (C), and brain (D) of zebrafish fed either the highly unsaturated FA-rich diet (HD), the linoleic acid-rich diet (LD), the Iinolenic acid-rich diet (LND), or the low fat diet (LFD) for 10 weeks. Data expressed as area percentage of fatty acid methyl esters (n = 3). 83 Table 4.4= Fatty acid composition in intestine of zebrafish fed experimental diets1 Fatty acid HD¿ LW LND" LFDZ 12:0 1.95 ±0.19 1.65 ±0.13 1.45 ±0.26 0.61 ±0.03 14:0 2.47 ±0.1 2.28 ±0.32 1.88 ±0.23 1.26 ±0.19 z¿.yq ± i. i y lö.^v ± i.öu ? /.zu ±?.3? ll.il ±U.38 18:0 8.43 ± 0.68 10.09 ±0.53 10.29 ±0.85 10.63 ±0.54 ÏÏF Total saturates 39.52 ±0.7G 34.70 ±1.37 25.73 ± 1.22° 40.39 ± 0.04" 16:1 ?-7 2.79 ±0.21 1.15 ±0.19 1.36 ±0.12 2.76 ±0.17 18:1 ?-9 18.35 ±1.24 21.21 ±1.72 25.36 ±0.87 20.84 ± 1.89 20:1 ?-9 0.98 ±0.13 1.61 ±0.11 0.97 ±0.16 1.54 ±0.15 Total monoenes 22.79 ±1.37 24.27 ± 1.98 28.09 ± 0.93 25.78 ± 0.93 18:2 ?-6 15.52 ±1.30 21.40 ±1.89 17.63 ±1.47 18.78 ±0.72 18:3 ?-3 3.63 ±0.61 2.74 ± 0.28 9.35 ±1.4 4.43 ±0.01 20:4 ?-6 1.08 ±0.12 3.18 ±0.23 2.28 ± 0.23 2.25 ± 0.03 20:5 ?-3 3.94 ± 0.23 1.50 ±0.21 3.99 ±0.11 0.98 ±0.12 22:6 ?-3 10.49 ± 0.56 8.32 ±0.54 11.30 ±0.42 3.45 ±0.10 Total PUFA 37.20 ±1.23 39.84 ±0.56 45.94 ±1.22 33.12 ±0.86 1DaIa expressed as area percentage of FA methyl esters. Results are mean ± S.E. Within a row, means with different superscript letters differ significantly (P < 0.05). 2 HD, highly unsaturated fatty acids rich diet; LD, linoleic acid rich diet; LND, linolenic acid rich diet; LFD, low fat diet; PUFA, polyunsaturated fatty acids. 84 Table 4.5. Fatty acid composition in liver of zebrafish fed experimental diets Fatty acid HDZ LOZ LND^ LFD2 12:0 0.66 ± 0.29 ).53 ± 0.07 1.14 ±0.08 0.95 ±0.13 14:0 1.14 ±0.08 0.95 ±0.13 0.66 ± 0.29 0.53 ± 0.07 1Ö.U1 f,r\ 1.55 ¿o. /5 ± ?.?d 18:0 7.87 ± 0.66 15.02 ± 2.57 16.85 ±2.86 10.62 ±1.08 Total saturates 37.30 ±0.12 46.69 ± 4.76 39.05 ± 4.39 43.76 ±5.29 16:1 n-7 2.85 ±1.01 2.25 ± 0.42 2.14 ±0.40 1.66 ±0.38 18:1 n-9 21.22 ±2.06 22.16 + 0.69 19.60 ±2.18 29.27 ± 3.02 20:1 n-9 0.98 ±0.13° 1.61±0.ir 0.97±0.16D 0.34±0.14L Total monoenes 26.45 ±1.69 24.42 ± 0.86 23.54 ±1.92 31.27 ±3.32 18:2 n-6 12.87 ±0.51 14.10 ±2.31 15.98 + 0.48 11.38 ±0.40 18:3 n-3 3.00 ±0.35° 1.12±0.25c 10.52 ± 0.22a 2.04 ± 0.33c w 20:4 n-6 0.52 ± 0.06D 3.40 ± 0.54a 1.19 ± 0.48a 1.50 ±0.39 20:5 n-3 5.50±1.2r 0.67 ± 0.06b 1.02 ±0.170° 1.08±0.24ü 22:6 n-3 8.28 ±1.02 4.48 ± 0.85 5.37 ±1.68 4.33 ±1.41 w w Total PUFAZ 35.61 ±1.81 27.72 ±3.91 37.48 ± 2.50a 24.67 ± 2.68D !Data expressed as area percentage of FA methyl esters. Results are mean ± S.E. Within a row, means with different superscript letters differ significantly (P < 0.05). 2 HD, highly unsaturated fatty acids rich diet; LD, linoleic acid rich diet; LND, linolenic acid rich diet; LFD, low fat diet; PUFA, polyunsaturated fatty acids. 85 Table 4.6. Fatty acid composition in muscle of zebrafish fed experimental diets Fatty acid HD¿ LD^ LND^ LFDZ 12:0 1.53 ± 0.30 1.49 ±0.19 1.53 ±0.23 0.63 ±0.11 14:0 1.86 ±0.14 1.54 ±0.13 1.39 ±0.14 1.01 ±0.12 w 16:0 22.31 ±0.08" 20.03 ±0.71 17.22 ± 0.44E 18.57 ±1.16D 18:0 5.23 ± 0.56 5.92 ±0.17 6.34 ± 0.74 7.48 ± 0.56 Total saturates 31.51 ±1.55 29.99 ±0.17 29.12 ±0.48 29.70 ±1.27 16:1 n-7 3.03 ±0.16 1.99 ±0.38 1.53 ±0.24 1.77 ±0.71 18:1 n-9 24.14 ±0.37 23.42 ± 0.25 27.15 ±1.2 29.28 ±1.72 20:1 n-9 1.18 + 0.21 1.15 ±0.17 1.22 ±0.22 0.57 ± 0.08 Total monoenes 29.11 ±0.33 27.12 ±0.41 29.35 ± 1.78 32.03 ±1.56 18:2 n-6 15.04 ± 0.72° 22.79 ± 0.63a 16.22 ±0.74D 16.73 ±0.68° 18:3 n-3 2.68 ± 0.34D 2.37±0.15D 9.45 ± 0.56a 4.46 ±0.3 Ie W 20:4 n-6 1.59 ±0.37° 4.94 ± 0.32a 1.21±0.29b 2.51 ±0.32 20:5 n-3 4.99 ± 0.3 G 1.11 ±0.2D 2.24 ± 0.36w 1.15 ±0.19° 22:6 n-3 11.45 ±0.95 7.64 ±0.86 8.69 ±0.78 8.76 ± 0.29 Total PUFA 38.97 ±1.94 42.57 ±0.61 41.18 ±1.26 37.58 ±1.51 Data expressed as area percentage of FA methyl esters. Results are mean ± S.E. Within a row, means with different superscript letters differ significantly (P < 0.05). 2 HD, highly unsaturated fatty acids rich diet; LD, linoleic acid rich diet; LND, linolenic acid rich diet; LFD, low fat diet; PUFA, polyunsaturated fatty acids. 86 Table 4.7. Fatty acid composition in brain of zebrafish fed experimental diets Fatty acid HD¿ LD* LND¿ LFD¿ 12:0 0.46 ± 0.03 0.38 ±0.11 0.33 ±0.11 0.34± 0.05 iE" 14:0 1.08 ± 0.1 2a 0.83 ±0.13iE" 0.59 ± 0.02° 0.76 ±0.11 16:0 21.64 ±1.14 20.45 ± 0.33 19.98 ±0.69 22.48 ± 0.94 iE" 18:0 3.46 ± 0.46 3.85 ±0.46" 1.29 ±0.67° 3.11 ±0.22" iE" Total saturates 29.74 ± 1.39a 28.50 ± 0.33 24.34 ± 0.96D 29.96 ± 1.57" 16:1 n-7 0.89 ± 0.06 0.97 ± 0.08 0.91 ±0.04 1.09 ±0.09 18:1 n-7 27.93 ±1.46 26.03 ±0.51 26.27 ± 0.55 27.87 ± 0.5 18:1 n-9 11.11 ±0.6 11.95 ±0.7 13.06 ±0.94 13.19 ±0.88 20:1 n-9 0.36 ±0.03° o.50±o.or 0.34±0.01c 0.26 ± 0.03" Total monoenes 41.63 ±1.59 41.93 ±0.23 43.01 ±1.28 43.60 ± 1.04 ir iE" 18:2 n-6 8.11 ±0.56 9.60 ±1.19" 4.86±0.65ü 7.10 ±1.09 18:3 n-3 1.35 ±0.11 1.01 ±0.21 1.76 ±0.31 1.39 ±0.11 20:4 n-6 1.36±0.08c 3.18±0.21£ 1.71 ±0.09'w 2.10±0.17D 20:5 n-3 1.78 ± 0.22a 0.50±0.0G 1.40 ±0.12" 0.49 ± 0.04° 22:6 n-3 11.28 ±0.5 11.65 ±0.56 15.58 ±0.96 11.17 ±1.07 Total PUFA 26.11 ±1.71 27.19 ±0.14 29.20 ± 0.84 25.12 ±1.07 ]Data expressed as area percentage of FA methyl esters. Results are mean ± S.E. Within a row, means with different superscript letters differ significantly (P < 0.05). 2 HD, highly unsaturated fatty acids rich diet; LD, linoleic acid rich diet; LND, linolenic acid rich diet; LFD, low fat diet; PUFA, polyunsaturated fatty acids. 87 A. Intestine -fabpla B. Intestine -fabplb.l ISO N 1*1 3DUiI I Í.50 W 1W ?« 2OtJt ? F 1.00 ™i W« & ?? Ê*aÛ eaIbes IMuftß W >NT ? j I ! ???3 ^tt 0.00 tJ·"-" I HD LD LND LFD HD LD LMDi LFD Figure 4.2. The steady-state level offabpla andfabplb.l mRNA in the intestine of zebrafish fed diets differing in FA content. The level offabpla (A) andfabplb.l (B) mRNA in the intestine of fish fed either diet HD, LD, LND or LFD was determined by RT-qPCR using gene-specific primers. The steady-state level offabp transcripts was normalized to the steady-state level of efla transcripts in the same samples. Data are presented as the mean ratio ± S.E.M. (x 10"3). Significant differences (P<0.05) in the relative steady-state level offabp mRNA between fish (n = 6) fed different diets are indicated by an asterisk. 88 Effect of Diet on the Steady-State Level offabp7a/fabp7b mRNAs in Different Tissues. The steady-state level offabp7a mRNAs was not modulated in intestine (Fig. 4.3A) or brain (Fig. 4.3G) of fish fed any of the experimental diets. In contrast, the steady-state level offabp7b mRNAs was higher in intestine of fish fed LND compared to fabp7b mRNAs in the intestine of fish fed LD and LFD, but not different in intestine of fish fed HD (Fig. 4.3B). Also, the steady-state level offabp7b mRNAs was elevated (10- fold) in the brain of fish fed LD compared to the level offabp7b transcripts in brain of fish fed LFD and LND, but was not different in brain of fish fed HD (Fig. 4.3H). The steady-state level offabp7a mRNAs was 4-fold higher in the liver of fish fed LFD compared to the transcript levels in the liver of fish fed one of the three other diets (Fig. 4.3C). The steady-state level offabp7a mRNAs was 3-fold higher in muscle offish fed LND compared to mRNAs levels for this gene in muscle of fish fed LD, but was not different in the muscle of fish fed HD and LFD (Fig. 4.3E). No difference was observed in the steady-state level offabp7b mRNAs in liver of fish fed any of the four experimental diets (Fig. 4.3D). Finally, fabp7b mRNAs were not quantifiable by RT- qPCR in muscle (Fig. 4.3F). Effect of Diet on Steady-State Level oîFabpllalFabpllb niRNA in Different Tissues No difference was observed in the steady-state level offabplla mRNAs in the intestine, liver and brain of fish fed any of the four diets (Fig. 4.4A, 4.4B, 4.4D). The steady-state level offabplla mRNAs was, however, 2-fold higher in muscle of fish fed LFD compared tofabplla transcript levels in muscle of fish fed the other three diets (Fig. 4.4C). The level offabpl lb mRNAs was not quantifiable by RT-qPCR in liver, 89 Figure 4.3. The steady-state level oïfabp7a andfabp7h mRNA in intestine (A, B), liver (C, D), muscle (E, F), and brain (G, H) of zebrafish fed diets differing in FA content. The level of\fabp7a andfabp7b mRNA in intestine, liver, muscle, and brain of fish fed either diet HD, LD, LND, or LFD was determined by RT-qPCR using gene-specific primers. The steady-state level offabp transcripts was normalized to the steady-state level of efla transcripts in the same samples. Data are presented as the mean ratio ± S.E.M. (x 10~3). Significant differences (P<0.05) in the relative steady-state level offabp mRNA between fish (n=6) fed different diets are indicated by an asterisk. fabp7b mRNA was not detected (ND) in muscle (F). 90 A. Intestine -fabp7a B. Intestine -fabpîb T T Il - ¡1 - £ ? T Iv m I » ( I mm: ?? ß il i ¿i^'-g -^jSjI -? DJQ' 1 ? ..·. f^tS_-_-ìil KD Lf) LIB LFD m LD LNO LFD C. Liver -fabp7a D. Liver -fabp7h 2J0- 2JO- T Ut- a. a OO * ?}. ¦ l· ri -sp-n rx"- -a:-· 1 r-L- M LO LMD LFt» HD LD LND LFD E. Muscle -fabp7a Fo Muscle -fabp7b ß,§? Mn ND ND ND HD LO LND LFD G. Brain -fabp7a H. Brain -fabp7b ¦s 250.ÛO- -= 200.00- = % 150.00- ? T S. 1100.00- X HD LD LND LFD LD LiD LFD 91 Figure 4.4. The steady-state level oîfabplla mRNA in intestine (A), liver (B), muscle (C) and brain (D) of zebrafish fed diets differing in FA content. The level oîfabplla mRNA in intestine, liver, muscle, and brain of fish fed either diet HD, LD, LND, or LFD was determined by RT-qPCR using gene-specific primers. The steady-state level offabp transcripts was normalized to the steady-state level of efla transcripts in the same samples. Data are presented as the mean ratio ± S.E.M. (x 10" ). Significant differences (P<0.05) in the relative steady-state level oífabp mRNA transcripts between fish (n=6) fed different diets are indicated by an asterisk. 92 A. Intestine —fabplla B. Liver -fabplla Co Muscle -fabplla ?· ft I f i HP LD LND LFD D. Brain -fabplla 93 muscle and intestine of zebrafish owing to their low abundance. The steady-state level of fabpllb mRNAs weas not changed in the brain of zebrafish fed any of the experimental diets (data not shown). Modulation of the Steady-State Level offabp mRNA is Due to Up-Reguîation of Transcriptional Initiation Heterogeneous RNA (hnRNA) for a given gene is rapidly processed to a mature mRNA transcript by removal of intervening sequences and the addition of a 5' cap and a poly(A) tail (Watson et al., 2008). As such, the level of hnRNA for a given gene indirectly correlates with the rate of initiation of transcription for that gene. To test whether the observed increase in the steady-state level oífabp mRNAs by dietary FAs was due to increased transcriptional initiation, the steady-state level of hnRNA for a specific fabp gene was assayed by RT-qPCR in different tissues of zebrafish fed the four experimental diets. In the intestine of fish fed LND, the level offabplb.l hnRNA was higher than in the intestine offish fed one of the other three diets (Fig. 4.5A). The steady-state level of fabp7a hnRNA was unchanged in the liver of fish fed any of the four experimental diets (Fig. 4.5B). The level offabp7a hnRNA was higher in muscle offish fed LND compared tofabp7a hnRNA in muscle of fish fed one of the other three diets (Fig. 4.5C). fabp7b hnRNA was elevated in the intestine of fish fed LND compared tofabp7b hnRNA in the intestine offish fed one of the other three diets (Fig. 4.5D). fabp7b hnRNA was 6-fold higher in the brain of fish fed LD compared to fish fed the rest of the treatment diets (Fig. 4.5E), while the steady-state level oifabplla hnRNA was 2-fold higher in muscle of fish fed LFD compared to hnRNA transcript levels in muscle of fish fed HD and LD, but was unchanged in muscle of fish fed LND (Fig. 4.5F). 94 Figure 4.5. The steady-state level of hnRNA forfabplb.l in intestine (A), fabp7a in liver (B), fabp7a in muscle (C), fabp7b in intestine (D),fabp7b in brain (E), and fabplla in muscle (F) of zebrafish fed diets differing in FA content. The levels of hnRNA forfabp genes in intestine, liver, muscle and brain of fish fed either diet HD, LD, LND or LFD was determined by RT-qPCR. The steady-state level oifabp hnRNA was normalized to the steady-state level of efla hnRNA transcripts in the same sample. Data are presented as the mean ratio ± S.E.M. (x 10"3). Significant differences (P<0.05) in the relative steady-state level oifabp hnRNA transcripts between fish (n = 5) fed different diets are indicated by an asterisk. 95 A. Intestine -fabplb.l B. Liver -fabp7a •s 20,0 " " 115.0- m « sa H ¡Í¡ Sì e F^ F^l HO LD LMO LFD C. Muscle -fabp7a D. Intestine -fabp7b "^r* fffjllllfilîi'*|j ¡ß 26.©- il 5® 1SJ-= 'hm & e fi & HJUfM1 (îuf- a s,M 5.Ö ? -?—?— m LD LND LFD HD LD LND LFD E. Brain -fabp7b F. Muscle -fabplla «?- «h¡ ? .Sg «?-~'" :| «" ÍJMj S: ¡ ± m ??=*! ri-3 -J^- —?— - ?^^??^?^????- (S#fea HD LD LND LFD HD LD LIID LfD 96 Discussion Effect of Diet on FA Profiles in Tissues of Zebrafish The FA composition in tissues of fish is mediated by various metabolic activities, such as FA desaturation, elongation (Tocher, 2003), and ß-oxidation (Henderson and Sargent, 1985). The composition of FAs in tissues is also influenced by dietary lipids (Bell et al., 2001; Bell et al., 2002; Torstensen et al., 2000). In the current study, the FA composition of the four diets fed zebrafish affected the FA profiles of intestine, liver, muscle, and brain (Fig. 4.1 and Tables 4.4-4.7). Our results are consistent with other studies in which dietary FAs modified the FA profile in zebrafish and other fishes (Jaya-Ram et al., 2008; Jordal et al., 2006; Meinelt et al., 1999; Meinelt et al., 2000; Tocher et al, 2001; Zheng et al, 2004). Dietary FAs Modulate the Steady-State Level offabp mRNAs Several studies reported the induction of some FABP genes in mammals by FAs and molecular mechanisms for this induction have been proposed (Bass et al., 1985; Drozdowski et al., 2004; Mochizuki et al., 2007; Ockner and Manning, 1974; Schroeder et al., 2008). For example, some research groups (Huang et al., 2004; Schroeder et al., 2001; Schroeder et al., 2008) have suggested that FABPs transport long-chain FAs to the nucleus from the cytoplasm. Once inside the nucleus, FABPs interact with and transfer their long-chain FA ligands to nuclear receptors, such as PPARa and PPARy (Budhu and Noy, 2002; Delva et al., 1999; Tan et al., 2002). Dietary long-chain FAs are known to activate these nuclear receptors (Escher and Wahli, 2000; Gottlicher et al., 1992; Keller et al., 1993; Lemberger et al., 1996; Wolfrum et al., 2001). Once activated, these nuclear receptors form heterodimers with retinoic acid receptors (RAR) or retinoid X receptors 97 (RXR) (e.g., PPAR-RXR or PPAR-RAR), which in turn bind to response elements in FABP genes and, thereby, stimulate initiation of transcription (Desvergne and Wahli, 1999). In part, our results showing the increased transcription offabp genes by dietary FAs are consistent with this mechanism of induction (Figs. 4.2-4.5). However, none of thefabp genes were up-regulated in any of the tissues assayed in zebrafish fed HD (Table 4.8). Indeed, the steady-state level offabpla, fabplb.1 mdfabplla mRNAs was lowest in all of the tissues assayed in fish fed HD (Figs. 4.2 and 4.4). Also, the steady-state level of Fabp7 mRNAs in rat brain (Puskas et al., 2004), Fabp5 mRNAs in mouse liver (Berger et al., 2006), and the steady-state level offabp3 andfabplO mRNAs in the muscle of Atlantic salmon (Jordal et al., 2006) were not elevated in animals fed diets rich in fish oil. Similarly, Liu et al. (2008) showed that PC 12 cells from rats exposed to eicosapentaenoic acid and docosahexaenoic acid, both abundant in the HD diet fed zebrafish, did not change the level of Fabp5 transcripts. Based on the results reported here and previous studies (Berger et al., 2006; Jordal et al., 2006; Liu et al., 2008; Puskas et al., 2004), we propose that, besides up-regulation offabp genes by FAs (Fontaine et al., 1996), there may be other mechanisms of transcriptional regulation of thefabp genes, such as repression of transcriptional initiation. Berger et al. (2006) suggested that the observed down-regulation of Fabp5 mRNA levels in the liver of mice fed a fish oil diet was mediated via transforming growth factor, beta 1 (TGFßl). Although a few reports (Belaguli et al., 2007; Fontaine et al., 1996; Nagasawa et al., 2006) emphasize the role of TGFßl in the transcriptional regulation of Fabp genes, the exact mechanism remains unknown. 98 a ? x¡ o (U C O ? C/3 iß ci IH X) ? N ? £ ö £ ? ^ «s, ? ? -s ? ? < 'S, a XS -d e £ ^ ^ -? ,3 ? I >, e ? ? -^ e ? O < ? -a M) QO .? C/3 es ? H '-3 99 Differential Modulation oîfabplb.l, but notfabpla anâfabplh.2 Transcription by Dietary FAs FAs are known to induce the expression of the Fabpl gene in the mammalian intestine (Bass et al., 1985; Lin et al., 1994; Mochizuki et al., 2007; Ockner et al., 1974; Poirier et al., 2001). Feeding of high-fat diets rich in vegetable oils resulted in a 30- 40%increase in the cytosolic content of FABPl in the rat intestine (Bass et al., 1985; Ockner et al., 1974). Rats weaned to a high-fat diet showed higher levels of Fabpl mRNAs in their intestine than those weaned to a low-fat diet (Lin et al., 1994; Mochizuki et al., 2007). Also, feeding rats a diet containing 6% sunflower oil (rich in linoleic acid) resulted in the greater induction of Fabpl in intestine compared to rats fed a diet containing 3% sunflower oil (Poirier et al., 2001). Since FAs, such as oleic acid, linoleic acid and linolenic acid, induce the expression of the mammalian FABPl gene (Lin et al., 1994; Meunier-Durmort et al., 1996; Mochizuki et al., 2007; Poirier et al., 2001), we anticipated that the steady-state level of mRNAs for all the duplicated copies of zebrafish/û/?/?./ (fabpla,fabplb.l, and fabplb.2), might be modulated by the different FAs in one or more of the experimental diets. However, in this study, we only observed up-regulation of the steady-state level of fabplb.l mRNAs in the intestine offish fed LND (Fig. 4.2) at the level of transcriptional initiation (Fig. 4.5). The steady-state level oífabpla (Fig. 4.2) andfabplb.2 mRNAs remained unchanged in the different tissues of zebrafish fed experimental diets. We conclude, therefore, that thefabpla anafabplb.2 genes, but not the. fabplb.l gene, no longer respond to dietary FAs owing to loss of c/s-regulatory elements in their respective promoters. In their study of cz's-regulatory elements after a WGD event in yeast, Papp et al. (2003) concluded that the total number of ds-regulatory elements remained 100 unchanged over time between duplicated genes. They further suggest that the number of shared cw-regulatory elements between duplicated genes decreased. The difference in the mode of gene regulation reported here for the duplicatedfabpl s may be due to loss of cis- regulatory elements in thefabpla andfabplb. 2 genes, a mechanism consistent with the process of subfunctionalization. In contrast, zebrafish/aèpls exhibit a divergent pattern in their tissue-specific distribution of transcripts when compared to their mammalian ortholog. Mammalian Fabpl transcripts were abundant in the intestine, liver and were detected at lower levels in kidney, ovary, and lung (Furuhashi and Hotamisligil, 2008; Thierry-Mieg and Thierry- Mieg, 2006). In adult zebrafish, fabpla transcripts were detected in the intestine (Sharma et al., 2006), whereasfabplb. 1 transcripts were abundant in the intestine (Sharma et al, 2006) but detected at lower levels in the liver, heart, gills, ovary and testes (Karanth et al., 2009). Zebrafishfabplb. 2 transcripts were abundant in the intestine, ovary and skin, and detected at lower levels in the brain, heart and eye (Karanth et al., 2009). This divergent tissue-specific distribution of transcripts for the duplicatefabp 1 genes differs from previously reported examples of subfunctionalization (Force et al., 1999), where the combined tissue-specific expression of duplicated zebrafish genes equals the tissue- specific expression of their mammalian ortholog, as described for the duplicated hoxbla and hoxblb (Prince and Pickett, 2002), mitfa and mitfb (Lister et al., 2001; Altschmied et al., 2002), and soxlla and soxllb (De Martino et al., 2000) genes. Comparative functional analysis of ds-regulatory elements of Fabpl genes will be necessary to determine if the duplicatedfabpl genes are retained in the zebrafish genome due to either subfunctionalization or neofunctionalization. 101 Tissue-Specific Transcriptional Modulation offabp7a andfabp7b mRNAs by Dietary FAs In this study, duplicated copies of zdbrañshfabp7, fahpVa andfabpVb, exhibited distinct patterns of up-regulation by dietary FAs in different tissues for steady-state levels of both mRNA and hnRNA (Figs. 4.3 and 4.5). The steady-state level offabp7a mRNAs in muscle and liver were elevated in zebrafish fed LND and LFD, whereas the steady- state level offabp7b mRNAs was modulated in brain and intestine of the fish fed LD and LND. Mammalian Fabp7 transcripts are detected in brain, retina, spinal cord and mammary gland (Haunerland and Spener, 2004; Furuhashi and Hotamisligil, 2008). Zebrafishfabp7a transcripts are detected in brain, spinal cord, retina, testis, liver, intestine, and muscle (Liu et al., 2004) and zebrafish/a£p7¿> transcripts are detected in brain, retina, testis, liver, intestine, skin, and swim bladder (Liu et al., 2004). In addition, the steady-state level of zebrafishfabp7b transcripts, but notfabp7a transcripts, was up- regulated in the brain of zebrafish fed LD (Fig. 4.3). Since the duplicated copies of the zebrafishfabp 7, fabp7a andfabp7b, show a different tissue-specific pattern of expression compared to their mammalian ortholog and exhibit a different mode of gene regulation by dietary FAs from each other, it is possible that either subfunctionalization or neofunctionalization may account for the retention of both duplicatedfabp7 genes in the zebrafish genome. Transcriptional Modulation offabplla, but notfabpllb, by Dietary FAs Previously, we reported that the duplicated copies of the zebrafishfabpl 1 gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes are derived from a common ancestral gene (Karanth et al., 2008). To date, thefabpl 1 gene has only been identified in 102 fishes (Karanth et al., 2008; Agulleiro et al., 2007). This study shows that both the steady-state level offabplla mRNAs (Fig. 4.4) and hnRNAs (Fig. 4.5) was only elevated in muscle of fish fed LFD, but not in muscle of fish fed one of the other three diets. Althoughfabplla andfabpl lb exhibit different tissue-specific patterns of expression in embryos, larvae and adult zebrafish (Liu et al., 2007; Karanth et al., 2008) and differential regulation by dietary FAs, we are unable at this time to resolve whether these duplicated genes were retained in the genome by either subfunctionalization or neofunctionalization as no ortholog of this gene has been identified thus far in other species, such as birds or mammals. Conclusion In this study, we show that dietary FAs change the FA profile in intestine, liver, muscle and brain of zebrafish. The tissue-specific changes in FA content modulated the steady-state level of mRNAs for only one sister duplicate of three pairs of duplicated genes of thefabp multigene family in zebrafish (Table 4.8). Furthermore, changes infabp hnRNA directly correlated with changes in the steady-state levels offabp mRNAs, suggesting that the affects of FAs on thesefabp genes occurred at the site of transcriptional initiation (Table 4.8). These findings indicate that the retention of duplicatedfabp genes in the zebrafish genome is most likely the result of either subfunctionalization or neofunctionalization. To distinguish between these processes as outlined in the DDC model (Force et al., 1999) will require functional analysis of the fabp promoters to identify cis-elements responsible for transcriptional induction of zebrafishfabp genes by FAs. 103 Acknowledgements The authors acknowledge Sean Tibbetts for advice during diet preparation, Joyce Milley for advice on lipid analysis, and the help of Daniel Sawler with RT-qPCR during this study. We gratefully appreciate the comments of Dr. Vanya Ewart on a draft of the manuscript. This work was supported by funds from the Natural Sciences and Engineering Research Council of Canada (to JMW), Canadian Institutes of Health Research (to ED-W), and National Research Council of Canada (to SPL). SK is recipient of a Faculty of Graduate Studies Scholarship from Dalhousie University. 104 Chapter 5: Conclusion Role of Gene and Whole-Genome Duplications in the Evolution of the iLBP Multigene Family in Zebrafish To date 22 iLBP genes, includingfabplb. 2 andfabpllb, have been described in the zebrafish genome (Table 1.1 and references therein). When compared with the iLBP repertoire in birds and mammals, it is reasonable to assume that detection of iLBP gene copies in zebrafish genome is now complete. Of the 22 zebrafish iLBP genes, 19 (nine pairs + one) of them are sister duplicates of each other (Chapters 2 and 3, Table 1.1 and references therein), fabplb. 1 a.ndfabplb.2, are tandemly-arrayed on chromosome 8 separated by ~4 kb of DNA and originated by a tandem duplication event (Chapter 2). Fish genomes have many duplicate genes compared to mammalian genomes. In the last decade, it has been widely debated whether a fish-specific WGD or many independent duplications in the teleost lineage may account for the numerous duplicate genes found in fish genomes (Amores et al., 1998; Woods et al., 2000; Taylor et al., 2003; Robinson- Rechavi et al., 2001). However, recent reports have convincingly demonstrated that the ancestor of present day teleost fishes underwent a WGD (Woods et al., 2005; Hurley et al., 2007). My analyses of phylogeny and syntenic relationship of zebrafishfabplla and fabpllb are in accordance with similar previous analyses of zebrafish duplicate iLBP pairs. With the exception of zebrafish fabplb. 1 a.ndfabplb.2, these analyses favor the teleost-specific WGD hypothesis as the reason for the origin of zebrafish iLBP duplicates pairs (Chapters 2 and 3 and Table 1.1 and references therein). The duplicated zebrafish fabplb. 1 a.ndfabplb.2 genes are thought to have arisen owing to a tandem duplication event subsequent to the WGD of the teleost fish (Chapters 2). Furthermore, I propose that 105 the orthologs of tetrapod FABF'4, FABP5, FABP8, FABP9 (PERF15) are not present in the zebrafish genome (Chapter 3). The tandemly arranged FABP4, FABP5, FABP8, FABP9 (PERFl5) gene cluster in the tetrapod genome and thefabp11 genes in the zebrafish genome likely originated from a common ancestral gene. Following the divergence of the fish and tetrapod lineages, this ancestral gene gave rise to thefabpll genes in zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9 genes in tetrapods (Chapter 3). Retention of Duplicatedfahp Genes in the Zebrafish Genome The estimates for retention of duplicate genes following the WGD in zebrafish varies from 14-30% (Postlethwaith et al., 2000; Woods et al., 2005). Interestingly -85% of iLBP genes have retained their duplicates in the zebrafish genome (Table 1.1, Chapter 2 and 3). Why have so many/a?»/? genes been retained in the zebrafish genome after the WGD? In Arabidopsis thaliana (Blanc and Wolfe, 2004) and Tetraodon nigroviridis (Brunei et al., 2006), a bias towards the retention of duplicate genes involved in the transcriptional regulation and signal transduction after genome duplications has been observed (van de Peer et al., 2009). In a recent study Sato et al. (2009) compared the duplicate genes involved in the signal transduction, transcription and metabolism which originated form the ray finned fish-specific WGD and found that no significant difference exist between the retention of duplicate genes involved in the different cellular processes. Similarly, iLBP are involved in cellular metabolism of FAs (Bernlohr et al., 1997), and the high retention of duplicated iLBP genes in the zebrafish genome cannot be readily explained by their function. I propose that higher retention of duplicated iLBP genes in the zebrafish genome may be explained by the mode of their transcriptional regulation. 106 The promoters of Fabp genes are known to harbour many regulatory elements (Haunerland and Spener, 2004). The DDC model proposes that the probability of subfunctionalization of a given pair of duplicates increases with the increasing independent mutable subfunctions in the regulatory region of those duplicate genes (Force et al, 1999). Over time, subfunctionalized duplicates may gain new functions (neofunctioanlization) and become fixed in the population. Subfunctionalization/Neofunctionalization of Duplicatedfabp Genes in the Context of Tissue-Specific Distribution of Their Transcripts To understand the tissue-specific pattern of transcript distribution forfabp genes in zebrafish adults and embryos, in situ hybridization of tissue sections, RT-PCR and whole mount in situ hybridization of embryos and larvae was used. The duplicate genes may diverge in their pattern of transcript distribution in adults and embryos and together they may complement the pattern of transcript distribution of the ancestral gene (Postlethwait et al., 2004). A single copy of a mammalian ortholog may be considered as the ancestral gene of the zebrafish duplicates, partly because mammals have not undergone a WGD after divergence from teleost fishes. In my studies, a complex pattern of subfunctionalization and neofunctionalization of duplicatefabpl andfabpll genes was found (Table 5.1). The different tissue-specific distributions of zebrafish fabplb. 1 and fabplb.2 transcripts compared to zebrafish/a¿>/?ia and mammalian Fabpl transcripts suggests that the duplicatedfabplb genes of zebrafish acquired additional functions to the ancestralfabpl gene, i.e., neofunctionalization. Furthermore, the function(s) of the duplicated/a¿pi¿> genes of zebrafish appear to be divided betweenfabplb.l andfabplb.2 owing to subfunctionalization. Moreover, fabpla is expressed only in the adult liver at 107 CU O U O s ? cy (D s. >> es s*- (N en I/) -t_j CS «J -^ ? B O cl C (33 cT .tí -ö co U U < Z < Z -ß < < Z Z ? ? ? ? ? Z -ö o s Z 'S.&1 Ü (/> ¦ + Z Lh 'S + + + + ? s + < Z ? -G + + + ? ¦ + + + + ? ? ? ? -? ¦ + ¦ (D + , —? ¦ja > + + + CS ce (D ? ca -O ss ? (D ce .tí U "»? t3 (? .ß aj cu eC a> ¿Si .JNl · — N ß JS 3 + 3 «. «> «. Oh 108 very low levels indicating it may eventually undergo nonfunctionalization. Similarly, fabplla andfabpllb transcripts exhibit strikingly different patterns of tissue distribution adult zebrafish (Chapter 3). Since there is no apparent ortholog of the fishfabp11 gene in other vertebrates (AguUeiro et al., 2007), it is not possible to conclude whether the duplicatedfabp11 genes are retained in the zebrafish genome by any mechanisms explained in the DDC model (Force et al., 1999). However, with the exception of liver, the tissue-specific expression pattern of zebrafishfabpl 1 s is similar to the collective tissue-specific expression pattern of tetrapod FABP4, FABP5, FABP8 and FABP9 genes (Yamamoto et al., 2009). Transcripts of tetrapod FABP4, FABP5, FABP8 and FABP9 genes are not detected in the liver of mammals (Yamamoto et al., 2009). Since the zebrafishfabpl 1 s and the tetrapod FABP4, FABP5, FABP8 and FABP9 originated from a single ancestral gene, I propose that the zebranshfabplla andfabpl lb might have undergone subfunctionalization. Subfunctionalization/Neofunctionalization of Duplicatedfabp Genes in the Context of Their Transcriptional Induction by Dietary FAs For the first time, this thesis describes the complementary pattern in the induction of duplicatedfabp genes in zebrafish fed with different dietary FAs (Chapter 4). The tissue-specific changes in FA content modulated the steady-state level of mRNAs for only one sister duplicate of three pairs of duplicated genes of thefabp multigene family in zebrafish (Table 4.8). Furthermore, changes in fabp hnRNA directly correlated with changes in the steady-state levels offabp mRNAs, suggesting that the affects of FAs on thesefabp genes occurred at the site of transcriptional initiation (Table 4.8). These findings indicate that the retention of duplicated/a¿>p genes in the zebrafish genome is most likely the result of either subfunctionalization or neofunctionalization. 109 One of the models for the regulation of iLBP expression by dietary FAs proposes that the FAs are transported to the nucleus by FABP, and inteeraction between peroxisome proliferator activated receptors (PPAR) and the FABP leads to the transfer of FAs to PPAR, which leads to the activation of PPAR (Schroeder et al., 2008). Further PPAR will dimerizes with other nuclear receptors, like retinoid X receptor (RXR) or retinoic acid receptor (RAR) and then binds to peroxisome proliferator response element (PPRE) in the promoters offabp genes and stimulate their transcription (Desvergne and Wahli, 1999). To explain the differential induction of duplicatedfabp genes by FAs in zebrafish I propose the following: (a) One of the sister duplicatefabp gene might have lost the regulatory elements responsible for the binding of nuclear receptors (Figure 5.1) leading to differential induction of sister duplicates. (b) Divergent evolution (Figure 5.2A) and coevolution (Figure 5.2B) of DNA binding domains of duplicated PPARs and PPREs in the promoters of duplicatedfabp genes lead to differential transcriptional activation of duplicatedfabp genes. A search in the zfm.org database shows that the many genes for duplicated nuclear receptors have been retained in the zebrafish genome. Corresponding to mammalian PPARa, PPARß, and PPARy, zebrafish genome has the duplicated pparaa, pparab, pparßa, pparßb genes, and a single copy of pparô gene. Similarly, functional rxraa, rxrab, rxrßa, rxrßb, rxiya, rxryb, raraa, rarab, rarya and raryb are extant in the zebrafish genome (Hale et al., 2006; Tallafuss et al., 2006; Waxman and Yelon, 2007). Studies in yeast suggest that the new transcription factors created by WGD are quickly 110 Whole genomeijdu,I duplication P ¡ fobpa 2 fabpb Figure 5.1 A model to account for the dietary FAs mediated differential induction of duplicatedfabp genes in zebrafish. In this model, owing to the mutations in nuclear receptor-biding site (PPRE), transcription of one of sisterfabp duplicates is not induced by the activated nuclear receptors (PPAR). Ill Figure 5.2 Divergent evolution and coevolution of DNA binding domains of duplicated nuclear receptors and PPRE in the promoters of duplicatedfabp genes leading to either no transcriptional activation or transcriptional activation offabpb. In model A, DNA binding domain for PPRAaa recognize the PPREa in the promoter oifabpa resulting in transcriptional activation oifabpa. Whereas because of divergent evolution of the DNA binding domain for PPRAab, the activated the nuclear receptors does not recognize the PPREb in the promoter ??fabpb and this does not results in transcriptional activation of fabpb. In model B, Coevolution of DNA binding domain of nuclear receptors and PPRE elements in the promoters of duplicatedfabp genes. DNA binding domain of PPRAaa recognize the PPREa in the promter oifabpa and PPRAab recognize the PPREb in the promoter oífabpb resulting in transcriptional activation of bothfabpa andfabpb. 112 Whole genomeïI duplication PPREa J fabpa C:T^ib fabpb A 1 fobp Whole genomei(I duplication 3 fabpa 2 fobpb B 113 incorporated into the regulatory network (Conant, 2010). Similarly, it is possible that the new nuclear receptors created by the fish-specific whole- genome duplication are incorporated into the regulatory network regulating the transcription oïfabp genes in zebrafish (Figure 5.2 A and B). I propose that duplicated nuclear receptors might have divided the ancestral regulatory functions between them through subfunctionalization in their coding region (Figure 5.2 A and B). Further, I propose that the subsequent divergent evolution or coevolution of DNA binding domains of duplicated nuclear receptors and the PPREs in the promoters of duplicatedfabp genes over 230-400 million years may explain the differential transcriptional activation of duplicatedfabp genes by dietary FAs (Figure 5.2). In model A (Figure 5.2A), DNA binding domain of PPRAaa recognize the PPREa in the promoter oîfabpa resulting in transcriptional activation oîfabpa. 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