Báo cáo khoa học: The fabp4 gene of zebrafish (Danio rerio) ) genomic homology with the mammalian FABP4 and divergence from the zebrafish fabp3 in developmental expression

The fabp4 gene of zebrafish (Danio rerio) ) genomic homology with the mammalian FABP4 and divergence from the zebrafish fabp3 in developmental expression Rong-Zong Liu1, Vishal Saxena1, Mukesh K. Sharma1, Christine Thisse2, Bernard Thisse2, Eileen M. Denovan-Wright3 and Jonathan M. Wright1

1 Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada 2 Institut de Ge´ ne´ tique et Biologie Mole´ culaire et Cellulaire, Department of Developmental Biology, CU de Strasbourg, Illkirch, France 3 Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Keywords brain vasculature; conserved synteny; gene phylogeny; linkage mapping; whole mount in situ hybridization

Correspondence J. M. Wright, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1 Fax: +1 902 494 3736 Tel: +1 902 494 6468 E-mail: jmwright@dal.ca Website: http://www.dal.ca/(cid:2)biology2/

(Received 1 December 2006, revised 15 January 2007, accepted 19 January 2007)

doi:10.1111/j.1742-4658.2007.05711.x

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

cise physiologic role(s) of each iLBP is far from being resolved. The proposed functions of iLBPs include cel- lular uptake and transport of long-chain fatty acids and retinoids, interaction with other transport and enzyme systems, regulation of gene transcription, and protection of cells against the detergent effects of excess fatty acids [4,5,9].

Phylogenetic analysis suggests that

Fatty acid-binding proteins (FABPs) are encoded by a multigene family termed the intracellular lipid-binding protein (iLBP) genes [1,2]. In vertebrates, at least 16 paralogous iLBPs, including 10 FABPs and six cellular retinoid-binding proteins, have been identified [3]. Each iLBP gene shows a specific pattern of expression during development and in adulthood of mammals [4,5]. Despite several iLBP gene-knockout experiments in mice that have attempted to provide direct evidence for the biological function(s) of FABPs [6–8], the pre-

the vertebrate iLBP multigene family may have arisen from a single encoding a universal hydrophobic ancestral gene

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Abbreviations dpf, days postfertilization; EST, expressed sequence tag; FABP, fatty acid-binding protein; hpf, hours postfertilization; iLBP, intracellular lipid- binding protein; LG, linkage group; 5¢-RLM-RACE, 5¢-RNA ligase-mediated RACE.

R.-Z. Liu et al. fabp4 gene in zebrafish

iLBP multigene

identity to Hh-FABP or H8-FABP from Antarctic fishes [17]. The differential patterns of distribution for the fabp3 and fabp4 transcripts in zebrafish embryos and larvae suggest distinct functions for these two paralogous genes during vertebrate development.

Results

cDNA sequence and gene structure of zebrafish fabp4

A blastn search using a previously cloned zebrafish fabp3 cDNA sequence [19] identified an expressed sequence tag (EST) from GenBank exhibiting sequence similarity to an fabp3 cDNA (accession number: CN511548). 3¢-RACE and 5¢-RNA ligase-mediated (RLM)-RACE generated a cDNA sequence (accession number: AY628221) with a complete coding capacity for an FABP, hereafter referred to as FABP4. The cDNA contained a 64-nucleotide 5¢-UTR, a 205-nuc- leotide 3¢-UTR, and a 405-nucleotide ORF that codes for a polypeptide of 134 amino acids. The deduced amino acid sequence had a theoretical molecular mass of 15.1 kDa and an isoelectric point of 7.8. A consen- sus polyadenylation signal (AATAAA) was located 19 nucleotides upstream of the poly(A) sequence.

sequence of

ligand-binding protein that underwent a series of gene duplications, starting some 900 million years ago [3]. family, FABP3 and Among the FABP4, together with FABP5, FABP7, FABP8 and FABP9, form the largest subfamily, subfamily IV [10]. Mammalian FABP4, also known as the adipocyte-type FABP gene (A-FABP), the adipocyte P2 gene (aP2) or the adipocyte lipid-binding protein gene (ALBP), was first described in mice almost two decades ago [11]. Mammalian FABP3 and FABP4 are both expressed in various tissues, with the transcripts and protein of FABP3 being most abundant in heart and skeletal muscle [12,13], and those of FABP4 being most abun- dant in adipose tissue [14]. A protein similar to FABP3 (H-FABP) was isolated from the bovine mammary gland, and initially termed mammary-derived growth inhibitor [15]; it was later shown to be a mixture of FABP3 and FABP4 (A-FABP) [16]. Two FABP cDNAs, named Hh-FABP and Had-FABP, have been isolated from mRNA extracted from the heart vent- ricle of four Antarctic teleost fishes [17]. Hh-FABP and Had-FABP cDNAs code for the proteins H8-FABP and H6-FABP, respectively. Whereas the Antarctic fish H6-FABP showed the highest sequence similarity to FABP4, fabp4 has not, thus far, been formally repor- ted in fishes, the largest and most diverse group of ver- tebrates, with distinct physiologic features in fat deposition and distribution [18]. Owing to these differ- ences in fat deposition and distribution, the role(s) of orthologous FABPs in fishes and mammals may differ markedly.

separated by three

is

The genomic sequence of zebrafish fabp4 was identi- fied in a zebrafish genomic DNA assembly sequence (accession number CR759777) in GenBank through a tblastn search. The fabp4 spanned 2459 bp and consisted of four exons (137 bp, 176 bp, 102 bp, and 258 bp) introns (217 bp, 101 bp, and 1468 bp) (Fig. 1), a gene struc- ture common to all FABP genes identified in verte- brates thus far (Fig. 2), with the exception of zebrafish fabp1a, which contains an additional intron in the 5¢- UTR [20]. The nucleotides at the splice site of each exon–intron junction of zebrafish fabp4 conform to the GT–AG rule [21]. Alignment of the cDNA sequence with the coding sequence of zebrafish fabp4 showed one nucleotide difference in the coding region of exon 2, resulting in an alteration of the ninth codon of this exon from GTT to GTC without changing the encoded amino acid. The genomic sequence and several EST sequences from GenBank (CN511548, CN168379, BC081489) had GTT at this position, whereas the ze- brafish fabp4 cDNA sequence reported here and another EST (CK355002) had GTC, suggesting that this T–C transition most likely represents an allelic variation.

Two important questions need to be answered. First, is there a functional fabp4 in teleost fish genomes orthologous to mammalian FABP4? Second, if there is a fish fabp4, how does the fish fabp4 differ functionally from the paralogous fabp3? To date, there has been no detailed comparative functional analysis of these two closely related paralogous genes in mammals, or any other species. Here we report the identification of a zebrafish gene transcript encoding a polypeptide with highest sequence identity to Had-FABP or H6-FABP from four Antarctic fishes [17]. On the basis of gene structure, phylogeny and conserved synteny, we deter- mined that this zebrafish fabp is the ortholog of the mammalian FABP4, and it therefore hereafter referred to as zebrafish fabp4. Taking advantage of the qualities of the zebrafish as a model system for study- ing gene expression during vertebrate development, we provide, for the first time in vertebrates, a detailed spa- tiotemporal expression profile for fabp4 during embry- onic and larval development, and compare this pattern of gene expression with that of zebrafish fabp3, a gene sequence encoding a polypeptide showing greatest

5¢-RLM-RACE generated a single product for the 5¢-end of the cDNA using zebrafish fabp4 cDNA-speci- fic nested antisense primers (data not shown). Align-

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Fig. 1. Nucleotide sequence of zebrafish fabp4 and its proximal 5¢-upstream region. Exons are shown in upper-case letters, with the coding sequences of each exon underlined and the deduced amino acid sequence indicated below. Numbers on the right indicate nucleotide posi- tions in the gene sequence. The initiation site for transcription is numbered + 1. A putative polyadenylation signal is highlighted in bold and underlined. PCR primers (s1, as1, as2) used in this study are double underlined and indicated. A putative TATA box, two 5¢-upstream AP1-binding sites and two CCAAT box sequences are boxed and indicated. A variation between the cDNA sequence and the genomic DNA sequence within exon 2 is highlighted in bold, with the variation indicated above. Zebrafish fabp4 and its 5¢-upstream sequence were identified from a Danio rerio DNA sequence assembly deposited in GenBank (accession number CR759777) by The Wellcome Trust Sanger Institute.

ment of the nucleotide sequences of the cloned 5¢- RLM-RACE product with the genomic sequence assigned the transcription start site to a position 64 bp

upstream of the initiation codon (Fig. 1). The length of the 5¢-UTR of zebrafish fabp4 is similar to that of mouse Fabp4 (previously termed aP2) (67 bp) [11]. A

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Exon 1 Intron 1 Exon 2 Intron2 Exon 3 Intron 3 Exon 4

24 aa 217 bp 59 aa 101 bp 34 aa 1468 bp 17 aa

Dr fabp4 24 aa 1241 bp 58 aa 224 bp 34 aa 1284 bp 16 aa Gg fabp4

24 aa 2496 bp 58 aa 968 bp 34 aa 500 bp 16 aa

Hs FABP4 24 aa 2316 bp 58 aa 607 bp 34 aa 670 bp 16 aa Mm Fabp4

Fig. 2. Comparison of the gene structure of zebrafish fabp4 and its chicken and mammalian orthologous genes. Exons are shown as solid boxes, introns as open boxes, and the UTRs of the first and fourth exon as dotted boxes. The number of amino acids encoded by each exon is shown above each box. The size of each intron is indicated in bp. The D. rerio (Dr), Gallus gallus (Gg), Homo sapiens (Hs) and Mus muscu- lus (Mm) FABP4 sequences were obtained from GenBank (accession numbers CR759777, NC_006089, NC_000008 and NC_000069, respectively).

clade

TATA box-like element (TTGAAAA) is located at nu- cleotides ) 26 to ) 32 (Fig. 1). The position of this putative TATA box of zebrafish fabp4 is identical to that of the TATA box (TTTAAAA) of mouse Fabp4 relative to their transcription start sites [11]. Inspection the upstream sequence of zebrafish fabp4 using of motif search (http://motif.genome.jp) identified two AP1-binding sites and two CCAAT box elements within 400 bp of the proximal promoter sequence (Fig. 1). These cis elements have been shown to be in controlling the expression of mouse important Fabp4 during adipocyte differentiation [22].

Zebrafish fabp4 is the ortholog of mammalian FABP4

The phylogenetic relationship of zebrafish fabp4 and the H6-FABP gene from Antarctic fishes was deter- mined using amino acid sequences of members of the FABP family from mammals and fishes (Fig. 4). A dis- tinct clade consisting of zebrafish FABP4, the four Antarctic fish H6-FABPs and putative FABP4 homo- logs from three other teleost fishes was evident. The teleost fish FABP4 (H6-FABP) clustered with mamma- lian FABP4s in an extended clade (Fig. 4). Zebrafish FABP3 and the four Antarctic fish H8-FABPs, along with other fish and mammalian FABP3s, formed a separate (Fig. 4). The phylogenetic analysis resolved fish FABP4 (and H6-FABPs) and FABP3 (and H8-FABPs) as proteins encoded by distinct genes, and suggested that these genes are orthologs of mam- malian FABP4 and FABP3, respectively.

the divergence of

(Fig. 3). These

The deduced amino acid sequence of zebrafish FABP4 showed highest sequence identity to Had-FABP (H6- FABP) from Antarctic teleost fishes (83–84%, Fig. 3) and to the putative orthologs of other teleost fishes, including Takifugu rubripes (85%, deduced from a cDNA sequence deposited in GenBank with accession number AL837220), Oryzias latipes (84%, BJ899828) and Cyprinus carpio (88%, CF661735). The second highest identity of zebrafish FABP4 was to mammalian FABP4 (51–53%), FABP3 (54–56%) and FABP7 (54– 55%). In contrast to the divergence in amino acid sequence between fish FABP4s (83–88%) and mamma- lian FABP4s (51–53%), zebrafish FABP3 exhibited similar amino acid sequence identities to other fish (71–72%) and mammalian (72–73%) FABP3s (Fig. 3). their primary protein Despite sequence, residues R107, R127 and Y129 of zebrafish FABP4 and Antarctic Had-FABPs (H6-FABPs) are conserved with residues R106, R126 and Y128 of residues are mammalian FABP4s believed to play a critical role in the specificity and affinity of ligand binding by mammalian FABP4 [23].

To further confirm that zebrafish fabp4 is indeed the orthologous gene of mammalian FABP4, we first mapped zebrafish fabp4 to a particular linkage group (LG) in the zebrafish genome using the radiation hybrid mapping panel LN54 [24], and then compared its syntenic relationships with the mammalian FABP4s (Table 1). Zebrafish fabp4 was assigned to LG 19 with a mapping distance of 14.73 centi-Rads (cR) to the genome framework marker fa04h09. Using a pair of the closest flanking markers (fd60g10 and Z22532), ze- brafish fabp4 was located between 50.8 and 53.1 cM on the merged ZMAP. The syntenic relationship of 19 gene loci mapped so far surrounding fabp4 on zebra- fish LG 19 was conserved with human FABP4 on chromosome 8 (Table 1). The majority of these con- served syntenies span a mapping distance of around 10 cM (46.86–57.80 cM) on zebrafish LG 19 and 8q21–8q24.3 on human chromosome 8 (Table 1). In addition to sequence identities (Fig. 3) and the phylo- genetic analysis (Fig. 4), the well-conserved synteny provides compelling evidence that the putative zebra- fish fabp4 and human FABP4 are orthologous genes.

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A

B

Fig. 3. Alignment of zebrafish FABP4 and FABP3 with the orthologous protein sequences from other teleost fishes and mammals. (A) D. re- rio (Dr) FABP4 (GenBank accession number AY628221) was aligned with Chaenocephalus aceratus (Cha, AAC60350), Cryodraco antarcticus (Ca, AAC60351), Gobionotothen gibberifrons (Gog, AAC60354), Notothenia coriiceps (Nc, AAC60352), H. sapiens (Hs, CAG33184) and M. musculus (Mm, AAH02148) FABP4s. (B) D. rerio FABP3 (Dr, AAL40832) was aligned with Ch. aceratus (AAC60356), Cr. antarcticus (AAC60357), Go. gibberifrons (AAC60359), N. coriiceps (AAC60358), H. sapiens (CAG33148) and M. musculus (AAH89542) FABP3s. Dots indicate amino acid identity, and dashes represent gaps. Positions of amino acids are marked and numbered. Three residues implicated in ligand-binding specificity and affinity and conserved among the zebrafish, Antarctic fish and mammalian FABP4s are boxed. Amino acid sequence identity values between the zebrafish FABP4 or FABP3 and the Antarctic fish, human and mouse FABP4s or FABP3s are indicated at the end of each alignment.

Differential distribution of the zebrafish fabp3 and fabp4 transcripts during embryonic and larval development

The spatiotemporal distribution of the fabp4 and fabp3 transcripts during zebrafish embryonic and larval in situ development was analyzed by whole mount hybridization [25] using probes generated from their specific cDNA sequences (Figs 5 and 6). At middle somitogenesis [17 h postfertilization (hpf)], fabp4 tran- scripts were detected only in the proximal region of

the retina, whereas fabp3 transcripts were distributed in several structures, including the diencephalon, hind- brain, spinal cord, and somites (Fig. 5A). At 24 hpf, the fabp4 transcripts were abundant in the lens and in the dorsal diencephalon, and visible in the choroid fis- sure. The pattern of expression of fabp3 in the devel- oping brain was similar at 17 hpf and 24 hpf. Levels of fabp3 transcripts, however, were higher at 24 hpf than at 17 hpf. In addition, at 24 hpf, fabp3 transcripts were detected in the retina, tectum, and posterior bran- chial arches (Fig. 5B). At the 36 hpf larval stage, the

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Fig. 4. Phylogenetic relationship of the zebrafish and other teleost fish FABP4s and FABP3s in the FABP family. The bootstrap neighbor-join- ing phylogenetic tree was constructed with CLUSTALX [43] using H. sapiens LCN1 (GenBank accession number NP_002288) as an outgroup. The bootstrap values (based on number per 1000 replicates) are indicated above or under each node. Amino acid sequences used in this analysis include: D. rerio (Dr) FABP4 (derived from AY628221), FABP3 (AAL40832), FABP2 (AAP93851), FABP7a (AAH55621), FABP7b (AAQ92970), and FABP10 (AAH76219); H. sapiens (Hs) FABP1 (CAG46887), FABP2 (AAH69617), FABP3 (CAG33148), FABP4 (CAG33184), FABP5 (AAH70303), FABP6 (AAH22489), FABP7 (CAG33338), and FABP8 (AAH34997); M. musculus (Mm) FABP1 (NP_059095), FABP2 (AAS00550), FABP3 (AAH89542), FABP4 (AAH02148), FABP5 (NP_034764), FABP6 (NP_032401), FABP7 (NP_067247), FABP8 (XP_485204), and FABP9 (NP_035728); Rattus norvegicus (Rn) FABP1 (NP_036688), FABP2 (NP_037200), FABP3 (NP_077076), FABP4 (NP_445817), FABP5 (NP_665885), FABP6 (NP_058794), FABP7 (NP_110459), and FABP9 (NP_074045); Sus scrofa (Ss) FABP4 (CAC95166); Ga. gallus (Gg) FABP4 (NP_989621); Ch. aceratus (Cha) FABP4 (H6-FABP, AAC60350) and FABP3 (H8-FABP, AAC60356); Cr. antarcticus (Ca) FABP4 (H6-FABP, AAC60351) and FABP3 (H8-FABP, AAC60357); Go. gibberifrons (Gog) FABP4 (H6-FABP, AAC60354) and FABP3 (H8-FABP, AAC60359); N. coriiceps (Nc) FABP4 (H6-FABP, AAC60352) and FABP3 (H8-FABP, AAC60358); Parachaenichthys charcoti (Pc) FABP4 (H6- FABP, AAC60355); Ta. rubripes (Tf) FABP4 (deduced from AL837220); Tetraodon nigroviridis (Tn) FABP4 (deduced from CR723700); Or. lati- pes (Ol) FABP4 (deduced from BJ899828); and Cy. carpio (Cc) FABP4 (deduced from CF661735). Scale bar ¼ 0.1 substitutions per site.

R.-Z. Liu et al. fabp4 gene in zebrafish

Table 1. Conserved syntenies of the zebrafish fabp4 with human FABP4. –, data not available.

Humanb Zebrafisha

Gene symbol Mapping panel Gene symbol Chromosomal location Linkage roup position (cM) Gene name

mftc 19, 46.86 LN54 MFTC 8q22.3

gdf6b Mitochondrial folate transporter ⁄ carrier Growth differentiation 19, 47.30 HS GDF6 8q22.1 factor 6 spag1 Sperm-associated 19, 49.00 T51 SPAG1 8q22.2 antigen 1 Zinc finger protein, zfpm2b 19, 49.00 ZFPM2 8q23 T51

ptp4a3 19, 50.25 PTP4A3 8q24.3 multitype 2 Protein tyrosine HS

RRM2B 8q23.1 rrm2b phosphatase type IVA, member 3 Ribonucleotide T51 19, 50.60

Hey1 LN54 19, 50.80 HEY1 8q21 reductase M2 b Hairy ⁄ enhancer-of-split related to YRPW motif 1

rpl30 laptm4b LN54 LN54 19, 50.80 19, 50.80 RPL30 LAPTM4B 8q22 8q22.1 Ribosomal protein L30 Lysosomal-associated

fabp4 protein transmembrane 4b Fatty acid-binding 19, 50.80–53.10c LN54 FABP4 8q21

angpt2 stk3 LN54 T51 19, 50.80–57.80c 19, 51.88 ANGPT2 STK3 8q23.1 8q22.2

lyricl protein 4, adipocyte Angiopoietin 2 Serine ⁄ threonine kinase 3 Metadherin T51 19, 51.95 8q22.1

azin1 ndrg1 T51 LN54 19, 53.30 19, 55.13 MTDH (LYRIC) AZIN1 NDRG1 8q22.2 8q24.3 Antizyme inhibitor 1 N-myc downstream regulated gene 1 trps1 Trichorhinophalangeal LN54 19, 78.10 TRPS1 8q24.12

angpt1 atp6v1c1l T51 – 19, 81.93 19 ANGPT1 ATP6V1C1 8q22.3–q23 8q22.3

a Mapping information derived from the merged genetic map at the ZFIN website: http://www.shigen.nig.ac.jp:6070. b Mapping information derived from NCBI at http://www.ncbi.nlm.nih.gov. c Location defined by flanking framework markers on zebrafish linkage group 19.

syndrome I Angiopoietin 1 ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C1 Brain and acute Baalc – 19 BAALC 8q22.3 leukemia, cytoplasmic PTK2 protein tyrosine ptk2.2 – 19 PTK2 8q24-qter kinase 2

hybridization signal for fabp4 transcripts was detected in the head vasculature system and remained in the lens and dorsal diencephalon (Fig. 6B). In comparison, the distribution of fabp3 transcripts was similar to that of the 24 hpf stage, with the exception of a slightly decreased intensity of hybridization signal in the spinal cord and the appearance of transcripts at the level of myosepta in muscle pioneers (Fig. 6A). At 48 hpf, in addition to their continued presence in the lens and

dorsal diencephalon, fabp4 transcripts were elevated in the head vasculature and detected at low levels in the intersegmental blood vessels and in the aorta wall (Fig. 6B). The relative levels of fabp3 transcripts, as indicated by the hybridization signal, were dramatic- ally reduced in most of the structures at 48 hpf as compared to those at 36 hpf, with the fabp3 transcripts being first detected in the liver, intestinal bulb, pan- creas and one cranial ganglion (posterior lateral line

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ganglion) at this stage (Fig. 6A). Strong hybridization signals for fabp3 transcripts remained in the liver, pan- creas and posterior optic tectum of 5-day-old larvae (Fig. 6C). The zebrafish fabp4 transcripts were unde- tectable at the 5 days postfertilization (dpf) larval stage (data not shown).

Adult tissue-specific distribution of fabp3 and fabp4 transcripts detected by RT-PCR

RT-PCR was employed to detect the presence of fabp3 and fabp4 transcripts in adult zebrafish tissues. Both fabp3 and fabp4 mRNA were detected by the highly sensitive technique of RT-PCR in all the adult tissues examined, which included the ovary, liver, skin, intes- tine, brain, heart, testis, and muscle (Fig. 7). It is of note that fabp3 transcripts had only been detected pre- viously in adult zebrafish ovary and liver by the less sensitive technique of tissue section in situ hybridiza- tion [19]. No hybridization signal for fabp4 mRNA was observed in any of the adult tissues using the method of tissue section in situ hybridization (data not shown), suggesting the presence of low levels of fabp4 transcripts in adult zebrafish tissues.

Discussion

In the vertebrate iLBP multigene family, fabp3 and fabp4 are grouped in the same subfamily, subfamily IV [10]. Their primary amino acid sequences share high identities (63–68% between mammalian FABP3s and FABP4s). Besides the common three-dimensional fold of the protein backbone, mammalian FABP3 and FABP4 show additional similarities in their tertiary structures, related to their ligand-binding specificity and affinity [10]. In addition to the similar three- dimensional structure and ligand-binding specificity and affinity of FABP3 and FABP4, the transcripts and proteins of the two paralogous genes exist in multiple tissues and are colocalized in several mammalian tis- sues, including mammary gland [16], heart [17], skel- etal muscle [26], and adipose tissue [27]. In earlier studies, the gene products from these two paralogs were not readily resolved. For example, bovine FABP3 and FABP4 were once regarded as a single protein, termed mammary-derived growth inhibitor when it was first isolated from the bovine mammary gland [15]. Mammary-derived growth inhibitor was later shown to be a mixture of FABP3 and FABP4 [16].

Fig. 5. Spatiotemporal distribution of zebrafish fabp3 and fabp4 transcripts during development at 17 and 24 hpf. (A) Distribution of fabp3 transcripts (A1, A2) in the diencephalon (Di), hindbrain (Hb), spinal cord (Sc), and somites (So), and fabp4 transcripts (A3, A4) in the retina (Re) of 17 hpf embryos. (A1, A3) Lateral view, head to the left. (A2, A4) Dorsal view, head to the left. (B) Comparison of the distribution of fabp3 and fabp4 transcripts at 24 hpf. Abundant fabp3 mRNA was present in the diencephalon (Di), retina (Re), tec- tum (Te), hindbrain (Hb), spinal cord (Sc), and myotomes. fabp4 mRNA was restricted to the dorsal diencephalon, lens (Le) and choroid fissure (Cf) of 24 hpf embryos. (B1, B4) Lateral view, head to the left. (B3, B6) Dorsal view, head to the left. (B2) Magnified lateral view of the tail. (B5) Magnified lateral view of the head.

Vayda et al. [17] isolated two fabp gene transcripts, termed Hh-FABP, coding for H8-FABP, and Had- FABP, coding for H6-FABP, from the heart ventricle of four Antarctic fishes. On the basis of sequence

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Fig. 6. Comparison of the distribution of fabp3 and fabp4 transcripts at 36 hpf, 48 hpf and 5 dpf. (A) fabp3 mRNA was detected in the tec- tum (Te, 36–48 hpf), retina (Re, 36–48 hpf), hindbrain (Hb, 36 hpf), spinal cord (Sc, 36–48 hpf), branchial arches (Ba, 36 hpf), muscle pioneers at the level of the longitudinal myosepta (Mys, 36 hpf), cranial ganglion (Cg, 48 hpf), liver (Li, 48 hpf), intestine (In, 48 hpf), and intestinal bulb (Inb, 48 hpf). The relative positions of the notochord (No) and otic vesicle (Ov) are indicated in red (A5, A10). (B) fabp4 mRNA was dis- tributed in the diencephalon (Di, 36–48 hpf), lens (Le, 36–48 hpf), head vasculature (B1–B5, arrowheads, 36–48 hpf) intersegmental blood vessels (B6, arrows, 48 hpf), and aorta wall. (C) Detection of fabp3 mRNA in the posterior tectum (Te), liver (Li) and pancreas (Pa) of 5 dpf larvae. (A1, A6, A8, B1, B3, B4, C1) Lateral view, head to the left. (A2, A7, B2, B5) Dorsal view, head to the left. (A3, A4, A9, B6) Magnified lateral view of the tail. (A5) Magnified view of the tail, cross-section. (C2, C3) Magnified lateral view of the liver and pancreas.

comparison and phylogenetic analysis, these two fabp cDNAs, present as mRNA transcripts in cardiac tissue of Antarctic fishes, were proposed to be products of distinct genes, and their encoded proteins were thought

to be homologous to mammalian adipose FABP and heart FABP [17]. However, Vayda et al. [17] did not explore further the genomic relationship of these Ant- arctic fish FABPs with their mammalian orthologs. In

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Fig. 7. Zebrafish fabp3 and fabp4 transcripts detected by RT-PCR in RNA extracted from adult tissues. RT-PCR products were gener- ated from total RNA extracted from various adult zebrafish tissues (indicated below the panel showing stained agarose gels), using both fabp3 and fabp4 cDNA-specific primers. As a positive control, rack1 transcripts were detected by RT-PCR in RNA extracted from all adult tissues. A negative control (–) lacking RNA template gener- ated no RT-PCR products.

in developing embryonic

ing that Antarctic teleost fishes and the tropical zebrafish live in environments with extremely different temperatures, the differences in the tissue expression patterns of these two paralogous genes may reflect var- iations in fatty acid metabolism, energy utilization and storage between these two fish lineages. The expression of fabp3 and fabp4 may be regulated by environmental factors [27] and training [26]. We detected zebrafish fabp4 transcripts in the early head vascular system at 36 hpf, and the intensity of the hybridization signal in this structure had greatly increased at 48 hpf (Fig. 6). The distribution pattern of fabp4 transcripts in the developing brain vasculature system parallels the devel- opment dynamic of this tissue in zebrafish [28]. Blood circulation in zebrafish embryos starts at about 24–26 hpf through a single circulatory loop. Connec- tion of simple blood vessel branches can be seen in the head of the zebrafish embryos at 36 hpf (1.5 dpf), and a rather complex head vascular system is formed at 48 hpf [28]. Our detection of the specific and dynamic distribution of fabp4 transcripts in the early developing vasculature of the zebrafish embryonic brain suggests a function for this gene in brain angiogenesis. Our obser- vation of the abundant distribution of the zebrafish fabp4 transcripts tissues and low levels in adult tissues indicates a developmen- tal role for this gene in zebrafish that has not been well documented in the embryogenesis of mammalian species.

the present study, we have determined the cDNA sequence and the structure of a gene coding for a pro- tein similar to the Antarctic teleost Had-FABPs (or H6-FABPs). In addition to the evidence from sequence identity and gene phylogeny, the conserved syntenic relationship of this zebrafish fabp with human FABP4 strongly suggests that we have identified a gene ortho- logous with mammalian FABP4 and distinct from zebrafish fabp3. We concluded that zebrafish fabp4, along with the Antarctic teleost genes for Had-FABPs (or H6-FABPs), and zebrafish fabp3, along with the Antarctic teleost genes for Hh-FABP (or H8-FABP), are orthologous with mammalian FABP4 and FABP3, respectively.

[31,32] and rat

[36–39].

In this article, we provide for the first time the spa- tiotemporal distribution of fabp4 and fabp3 transcripts during development in vertebrates, using zebrafish as a model system. Although a similar distribution of the transcripts for these two genes has been observed in adult tissues from Antarctic fishes by northern blot analysis, we revealed strikingly different patterns of expression for the fabp4 and fabp3 transcripts during embryonic and larval development in zebrafish (Figs 5 and 6) that suggest distinct function(s) for these two genes during vertebrate development. Although the fabp4 and fabp3 cDNAs from four Antarctic fishes were isolated from the heart, and both gene transcripts were abundant in this tissue as determined by northern blot analysis [17], a previous study by us did not detect fabp3 transcripts in the adult zebrafish heart using tis- sue section in situ hybridization [19]. In the present study, we again did not observe hybridization signals for fabp3 and fabp4 transcripts in the embryonic or larval heart tissue. In contrast, zebrafish fabp3 mRNA was abundant in the adult [19], embryonic and larval liver, but the Antarctic fish fabp3 (Had-FABP or H8- FABP) is not present in the adult liver [17]. Consider-

The genes for FABP3 and FABP4 reside on different chromosomes in human (chromosomes 1 and 8, respect- ively) [29,30], mouse (chromosomes 4 and 3, respect- ively) (chromosomes 5 and 2, respectively) [33,34]. We assigned both fabp3 [19] and fabp4, however, to a single LG, LG 19, of the zebrafish genome using the same radiation hybrid mapping panel, LN54 [24]. Different radiation hybrid scoring vectors (Table 2) generated by gene-specific primers assigned zebrafish fabp3 and fabp4 to different loci in the same LG and confirmed that they are indeed dis- tinct genes with divergent linkage relationships with the genome markers. Interestingly, zebrafish LG 19 con- tained both conserved syntenies surrounding the fabp3 [19] and fabp4 loci (Table 1) with the chromosomal seg- ments harboring FABP3 and FABP4 on human chro- mosomes 1 and 8, respectively. The conserved synteny of zebrafish LG 19 to both human chromosomes 1 and 8 suggests chromosomal rearrangement after the diver- gence of fish and mammals, which is estimated to have occurred approximately 450 million years ago [35], and which is an event revealed by extensive zebrafish– human comparative genomic analyses In humans, FABP4 (8q21) [30], FABP5 (8q21.13) and

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R.-Z. Liu et al. fabp4 gene in zebrafish

Table 2. Comparison of the LN54 radiation hybrid mapping panel scoring data and gene location of the zebrafish fabp3 and fabp4.

Gene Location Scoring vectora

a1, positive hybrid; 0, negative hybrid; 2, missing or ambiguous hybrid data.

products were cloned, and three clones for each product were sequenced. The complete cDNA sequence coding for zebrafish FABP4 was determined by aligning and combi- ning all 3¢- and 5¢-cDNA end sequences.

fabp3 LG 19, 365.69 cR 000000011111100000210010000000000000000010000000000100010000000000000000100000100000000001000 fabp4 LG 19, 287.93 cR 000000000011000000000010100000011102000000000001010101010000001100100000100000100010011000010

FABP8 (8q21.13-q22.1) [40] exhibit closely linked synt- enies. The latter two genes, however, have not been described to date in fishes. It would be interesting to know whether functional fabp5 and fabp8 exist in the zebrafish genome, and whether their syntenic relation- ships with fabp4 are conserved in fishes.

Phylogenetic analysis

In summary, we have determined the

Phylogenetic analysis of zebrafish fabp4 and fabp3 and other fish and mammalian FABP genes was performed using clustalx [43]. The Antarctic fish H6-FABP and H8- FABP sequences were included in this analysis, and the putative FABP4 sequences from Ta. rubripes, Cy. carpio, Oncorhynchus mykiss and Or. latipes were deduced from the cDNA sequences deposited in GenBank (http:// www.ncbi.nlm.nih.gov/), which are similar to the zebrafish fabp4 cDNA. A bootstrap neighbor-joining phylogenetic tree was constructed using the Homo sapiens lipocalin 1 precursor (accession number NP_002288) as an outgroup.

LG assignment of zebrafish fabp4 using the radiation hybrid mapping panel LN54

cDNA sequence, defined the gene structure and mapped the ge- nomic locus of a paralogous member of the iLBP multi- gene family in zebrafish. Analysis of amino acid sequence similarity, gene phylogeny, conserved synteny and developmental expression of gene transcription revealed that this newly identified fabp4, along with the H6-FABP genes described earlier in Antarctic fishes [17], is the ortholog of mammalian FABP4. We also show that zebrafish fapb4 is paralogous to zebrafish fabp3 [19], which is orthologous with the Antarctic fish H8-FABP and mammalian FABP3 genes. We have fur- ther demonstrated the different spatiotemporal distribu- tion of the zebrafish fabp3 and fabp4 transcripts during embryonic and larval development, which may provide further insights into the potential physiologic role(s) that these two genes play in vertebrate development.

Experimental procedures

Zebrafish culture and breeding

Genomic DNA from radiation hybrids of the LN54 panel [24] was kindly provided by M Ekker, University of Ottawa, and used to assign zebrafish fabp4 to a specific LG. The sequences of the primers used to amplify the genomic DNA from radiation hybrids of the LN54 panel are shown in Fig. 1 (s1, as2). The PCR conditions have been previously described [42].

Whole mount in situ hybridization to zebrafish embryos

Zebrafish were purchased from a local aquarium store and cultured in filtered, aerated water at 28.5 (cid:2)C in 35 L aqua- ria. Fish were maintained on a 24 h cycle of 14 h light and 10 h darkness. Fish were fed with a dry fish feed, TetraMin Flakes (TetraWerke, Melle, Germany), in the morning, and hatched brine shrimp (Artemia cysts from INVE, Grants- ville, UT, USA) in the afternoon. Fish breeding and embryo manipulation were conducted according to estab- lished protocols [41].

Cloning of the zebrafish fabp4 cDNA

Digoxigenin-labeled RNA probes were transcribed from the linearized fabp3 and fabp4 cDNAs generated by 3¢-RACE. Whole mount in situ hybridization to zebrafish embryos and larvae at different developmental stages (gastrula, early somitogenesis, middle somitogenesis, 24 hpf, 36 hpf, 48 hpf, and 5 dpf, respectively) was performed as previously described [25].

RT-PCR

zebrafish EST (GenBank

accession

a

RT-PCR was employed to determine the tissue-specific dis- tribution of fabp4 and fabp3 transcripts in adult zebrafish according to Liu et al. [18]. Primers used in RT-PCR for detection of fabp4 transcripts are shown in Fig. 1 (s1, as1).

To obtain the complete cDNA sequence encoded by zebra- fish fabp4, both 3¢-RACE and 5¢-RLM-RACE were employed as previously described [19,42]. The sense and antisense primers used for 3¢-RACE (s1, Fig. 1) and 5¢- RLM-RACE (as1, as2, Fig. 1) were designed on the basis of number CN511548). The 3¢-RACE cDNA and 5¢-RLM-RACE

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increases cellular fatty acid levels. J Lipid Res 40, 967– 972.

9 Stewart JM (2000) The cytoplasmic fatty-acid-binding proteins: thirty years and counting. Cell Mol Life Sci 57, 1345–1359.

The constitutively expressed gene for receptor for the activa- ted C kinase 1 gene, rack1, was used as positive control for all RT-PCRs. Conditions for detection of rack1 transcripts were the same as those used for detection of fabp3 and fabp4 transcripts. The primers used for RT-PCR amplification of fabp3 and rack1 mRNA are described in Liu et al. [19].

10 Lucke C, Gutierrez-Gonzalez LH & Hamilton JA

R.-Z. Liu et al. fabp4 gene in zebrafish

Acknowledgements

(2003) Intracellular lipid binding proteins: evolution, structure and ligand binding. In Cellular Proteins and Their Fatty Acids in Human Health and Disease (Duttaroy AK & Spener F, eds), pp. 95–114. Wiley-VCH GmbH KGaA, Weinheim, Germany.

11 Hunt CR, Ro JH, Dobson DE, Min HY & Spiegelman BM (1986) Adipocyte P2 gene: developmental expres- sion and homology of 5¢-flanking sequences among fat cell-specific genes. Proc Natl Acad Sci USA 83, 3786– 3790.

12 Zschiesche W, Kleine AH, Spitzer E, Veerkamp JH &

Glatz JF (1995) Histochemical localization of heart-type fatty acid binding protein in human and murine tissues. Histochem Cell Biol 103, 147–156.

13 Heuckeroth RO, Birkenmeier EM, Levin MS & Gordon

This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada (to J. M. Wright), the Canadian Institutes of Health Research (to E. Denovan-Wright), and the Institut National de la Sante´ et de la Recherche Me´ di- cale, Centre National de la Recherche Scientifique, Hoˆ pital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, National Institute of Health (to C. Thisse and B. Thisse), and Izaak Walton Killam Memorial Schol- arships (to M. K. Sharma and R.-Z. Liu). We thank Aline Lux and Vincent Heyer for help with whole mount in situ hybridization experiments.

JI (1987) Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein. J Biol Chem 262, 9709–9717.

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