Retention of the duplicated cellular retinoic acid-binding protein genes (crabp1a and crabp1b) in the zebrafish genome by subfunctionalization of tissue-specific expression Rong-Zong Liu1, Mukesh K Sharma1, Qian Sun1, Christine Thisse3, Bernard Thisse3, Eileen M Denovan-Wright2 and Jonathan M Wright1 Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada ´ ´ ´ Institut de Genetique et Biologie Moleculaire et Cellulaire, Department of Developmental Biology, UMR 7104, CNRS ⁄ INSERM ⁄ ULP, CU de Strasbourg, Illkirch, France Keywords crabp1; embryonic development; gene duplication; gene expression; subfunctionalization Correspondence J M Wright, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1 Fax: +1 902 4943736 Tel: +1 902 4946468 E-mail: jmwright@dal.ca Website: http://www.dal.ca/biology2/ (Received March 2005, revised May 2005, accepted 16 May 2005) doi:10.1111/j.1742-4658.2005.04775.x The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome Cellular retinoid-binding proteins belong to the large family of low molecular mass ( 15 kDa) intracellular lipid-binding proteins (iLBP) that bind fatty acids, retinoids and steroids [1–3] In mammals, two different groups of cellular retinoid-binding proteins with distinctive binding properties have been identified, the cellular retinol-binding proteins (CRBPs) and the cellular retinoic acid-binding proteins (CRABPs) CRBPs, including CRBPI, CRBPII, CRBPIII and CRBPIV, show greatest binding affinity for retinol and retinal, but not bind retinoic acid or retinyl esters [1,4–6] In contrast, the mammalian CRABPs, CRABPI and CRABPII, bind retinoic acid but not retinol or retinal However, the affinity of the rat CRABPI for all-trans RA (Kd ¼ nm) is much greater than that of CRABPII (Kd ¼ 64 nm), and the same relative affinity for Abbreviations CRABPIa and CRABPIb, cellular retinoic acid-binding protein type I a and b; CRBP, cellular retinol-binding protein; hpf, hours post fertilization; iLBP, intracellular lipid-binding protein; LG, linkage group; ORF, open reading frame; RA, retinoic acid FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS 3561 Duplicated crabp1 genes in zebrafish all-trans retinoic acid (RA) is also true for human CRABPI (Kd ¼ 0.06 nm) and CRABPII (Kd ¼ 0.13 nm) [7] The function of mammalian CRABPI is not fully understood The highly conserved amino acid sequence of CRABPI among different species and its distinct tissue-specific expression patterns during development and in adulthood suggest that it has an essential role in retinoic acid targeting and metabolism Homozygous CRABPI knockout mice, however, exhibit no overt abnormalities and appear essentially normal in terms of viability and fertility [8–10] As such, CRABPI function may be dispensable or compensated by another member of the CRABPI ⁄ CRABPII multigene family in these knockout mice A recent investigation of CRABPI function using a murine embryonic stem cell line deleted for crabp1 showed that homozygous deletion of this gene results in decreased intracellular RA concentrations and increased CRABPII expression suggesting a role for CRABPI in the RA homeostasis in cells and regulation of CRABPII expression [11] Further evidence that CRABPI may modulate RA signaling is the localization of CRABPI in both the cytoplasm and nucleus [12–14] Specific expression of CRABPI in the mammalian olfactory bulb and spinal cord, which are recognized as prominent sites of ongoing plasticity and response to RA, implies that CRABPI is involved in neurogenesis [15–18] The mammalian CRABPI and CRABPII proteins are encoded by single genes which, like all other paralogous members of the vertebrate iLBP family, consist of four exons separated by three introns [1] Except for the gene structure of CRABPI in pufferfish (Fugu rubripes) [19], no detailed characterization of CRABPI genes has been reported in fishes, the largest and most diverse group of vertebrates Furthermore, only a single copy of the gene encoding CRABPI has been identified in vertebrates Here we report the finding of duplicated genes coding for CRABPI (designated crabp1a and crabp1b) in the zebrafish genome Gene structure, syntenic relationship and primary protein sequence of the zebrafish crabp1a and crabp1b are well conserved with their orthologous mammalian CRABP1s Comparative genomic analysis suggests that the zebrafish duplicated crabp1a and crabp1b genes may have arisen from a teleost fish-specific chromosomal or whole genome duplication The differential distribution of crabp1a and crabp1b transcripts during development and in adult zebrafish tissues implies subfunctionalization after their duplication, which might be a mechanism for preservation of the crabp1 duplicates in the zebrafish genome 3562 R.-Z Liu et al Results Determination of cDNA sequence and gene structure of the zebrafish crabp1a A zebrafish EST sequence (GenBank accession number BI533516) showing sequence similarity to the mammalian CRABPI was identified and retrieved from the GenBank database Complete and overlapping 3¢ and 5¢ cDNA ends of the putative zebrafish crabp1 (designated crabp1a) were PCR-amplified using primers based on the EST sequence, cloned and sequenced The zebrafish crabp1a cDNA sequence was 719 bp including a 56 bp 5¢-UTR and a 246 bp 3¢-UTR (GenBank accession number AY242125) A polyadenylation signal was located 14 bp upstream of the poly A tail in the cDNA clones A 417 bp open reading frame (ORF) for the zebrafish crabp1a cDNA was identified, which codes for a peptide of 138 amino acids with a theoretical molecular mass of 15.68 kDa and a predicted isoelectric point of 5.05 The deduced amino acid sequence of the zebrafish CRABPIa showed highest identity with the mammalian and pufferfish CRABPIs (84–86%) (Fig 1A), followed by mammalian and zebrafish CRABPIIs (58–69%), mammalian and zebrafish CRBPIs (28–34%), mammalian and zebrafish CRBPIIs (28–30%), human CRBPIII (29%) and CRBPIV (30%) (data not shown) Comparison of the amino acid sequence of the zebrafish CRABPIa with its mammalian and pufferfish orthologs showed that there is a proline insertion in the amino acid sequence immediately following the initiator methionine (Fig 1A) Sequenced genomic DNA segments with identity to the cloned cDNA of the zebrafish crabp1a were identified by a BlastN search and retrieved from the zebrafish genome database (Wellcome Trust Sanger Institute) An assembly (GenBank accession number BX296538) contained the sequence of exon (129 bp), intron (5828 bp), exon (179 bp), a part of intron and a 5¢ upstream sequence of crabp1a A scaffold sequence, Zv4_scaffold2030.1, harbored the sequence of both exon (114 bp), intron (3262 bp) and exon (298 bp), and part of the sequence for intron The cDNA and genomic DNA sequences showed differences between three nucleotides in exon 2, resulting in alteration of the 15th codon of exon from GCC to GCA, 44th codon CGT to CGC and 47th codon GAA to GAG In spite of these nucleotide differences, the amino acids encoded by these codons remained unchanged suggesting that these changes represent allelic variants The RNA donor and acceptor splice sites present in each intron of the zebrafish crabp1a FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS R.-Z Liu et al Duplicated crabp1 genes in zebrafish A B Exon Intron 24 aa Exon Intron 60 aa Exon 5828 bp 23 aa 60 aa 60 aa 38 aa 60 aa 16 aa 1222 bp 38 aa 2277 bp 60 aa 511 bp 16 aa 7470 bp 3453 bp 554 bp 23 aa 16 aa 38 aa 6815 bp 1959 bp 23 aa Exon 3262 bp 7629 bp 23 aa Intron 38 aa 16 aa 4313 bp 38 aa 2925 bp 16 aa 4328 bp Fig Alignment of the amino acid sequences of the fish and mammalian CRABPIs and comparison of their gene structures (A) Zebrafish (Zf) CRABPIa (GenBank accession number: AAP44333) and CRABPIb (AAT38218) were aligned with the pufferfish (Pf; O42386), human (Hm; P29762) and mouse (Ms; P02695) CRABPIs Dots indicate amino acid identity and dashes represent gaps Positions of amino acids are marked and numbered Amino acid sequence identity values between the zebrafish CRABPIs and the pufferfish, human and mouse CRABPIs are indicated at the end of each alignment A proline insertion in the zebrafish CRABPIa is indicated by a ‘.’ (B) Comparison of the exon ⁄ intron organization of the zebrafish (Zf) crabp1 genes with the orthologous genes from pufferfish (Pf), human (Hm) and mouse (Ms) Exons are shown as boxes and introns as lines The number of amino acids encoded by each exon is shown above the boxes The size of each complete intron is indicated The complete sequence of the second intron in crabp1a has not been determined (indicated by dashes) The human and mouse CRABP1 gene sequences were obtained from GenBank (accession numbers NT_086829 and NT_039474) The structure of the pufferfish crabp1 gene was defined based on reference [19] gene conformed to the GT-AG rule [20] The zebrafish crabp1a gene structure, consisting of four exons and three introns, is the same as the mammalian and pufferfish crabp1 genes (Fig 1B) Exon of the crabp1a genomic sequence, like the cDNA sequence, had an insertion of a proline codon (CCT) immediately following the initiator methionine relative to mammalian and pufferfish orthologs [1,19] 5¢-RLM-RACE generated a single product corresponding to the position of the 7-methyl G cap of the mature crabp1a mRNA (data not shown) Alignment of the nucleotide sequence of the cloned 5¢-RLMRACE product with the genomic sequence assigned FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS the transcription start site to the 56 bp upstream of the initiation codon (data not shown) Identification of a second crabp1 gene, crabp1b A tblastn search using the deduced amino acid sequence of the zebrafish CRABPIa as a query identified a second crabp1-like gene in the zebrafish genome (GenBank accession number BX663612) This gene codes for an amino acid sequence showing similarity to the zebrafish CRABPIa and the mammalian CRABPIs (Fig 1A) The structure and coding capacity of this gene was identical to that of the mammalian and 3563 Duplicated crabp1 genes in zebrafish R.-Z Liu et al pufferfish CRABP1 genes (Fig 1B) We designated this gene as crabp1b 5¢-RLM-RACE located a single transcription start site 109 bp upstream of the initiation codon (data not shown) The zebrafish crabp1b gene was approximately 23 kb in length (Fig 1B) The complete cDNA sequence of the zebrafish crabp1b (GenBank accession number AY616861) was determined by sequencing cloned 5¢-RLM-RACE and 3¢-RACE products The primers used for the cDNA cloning were designed based on the coding sequence of the zebrafish crabp1b gene The length of the complete cDNA sequence was 895 bp excluding the poly(A) tail A polyadenylation signal was identified in the 3¢-UTR 13 nucleotides upstream of the polyadenylation site The cDNA sequence contained an ORF of 414 bp coding for a polypeptide of 137 amino acids The deduced amino acid sequence of the zebrafish CRABPIb showed highest identity to the zebrafish CRABPIa (88%) and the mammalian and pufferfish CRABPIs (84–85%) (Fig 1A), lower sequence identity to the mammalian and fish CRABPIIs (58–71%) and lowest identity to the mammalian and fish CRBPs (25–35%) (data not shown) The crabp1a and crabp1b genes arose from a fish-specific chromosomal duplication Phylogenetic analysis of the mammalian and fish cellular retinoid-binding proteins revealed two distinct clades: CRBPs and CRABPs (Fig 2) The zebrafish CRABPIa and CRABPIb clustered with the mammalian and pufferfish CRABPIs in the same clade with a robust bootstrap value of 998 ⁄ 1000 The gene phylogeny suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian and pufferfish genes for CRABPI, which may have arisen from a fish-specific chromosomal or whole genome duplication after the divergence of the tetrapod and fish lineages some 300 million years ago [21] Linkage group (LG) assignment of the zebrafish crabp1 genes was determined by radiation hybrid mapping using the LN54 panel [22] The zebrafish crabp1a gene was assigned to LG 25 with a mapping distance of 26.53 cR to marker Z7306, while crabp1b was assigned to LG at a site 2.94 cR to marker fd15c06 (mapping data available on request) Both zebrafish crabp1 genes displayed conserved syntenies with the human CRABP1 gene on chromosome 15 (15q24) ([23]; http://www.ncbi.nlm.gov/locuslink) (Fig 3) The conserved syntenies on the human chromosome 15 with both zebrafish crabp1 genes were distributed within the same chromosomal region 15q15–15q26 (Fig 3) Another pair of duplicated zebrafish genes, 3564 Fig Phylogenetic analysis of cellular retinoid-binding proteins The bootstrap neighbor-joining phylogenetic tree was constructed using CLUSTALX [23] The zebrafish (Zf) intestinal type fatty acid-binding protein (I-FABP) amino acid sequence (GenBank accession number AAF00925) served as an outgroup The bootstrap values (based on number per 1000 replicates) are indicated to the right of each node Amino acid sequences used in this analysis include zebrafish CRBPIa (AAQ54326), zebrafish CRBPIb(AAR31829), human (Hm) CRBPI (P09455), mouse (Ms) CRBPI (Q00915), rat (Rt) CRBPI (P02696), zebrafish CRBPIIa (AAL38648), zebrafish CRBPIIb (AAT40241), human CRBPII (P50120), mouse CRBPII (Q08652), rat CRBPII (P06768), human CRBPIII (P82980), human CRBPIV (AAN61071), zebrafish CRABPIa (AAP44333) and CRABPIb (AAT38218), human CRABPI (P29762), mouse CRABPI (P02695), pufferfish (Pf) CRABPI (O42386), zebrafish CRABPIIa (AAQ85530), zebrafish CRABPIIb (AAW23987), human CRABPII (P29373), mouse CRABPII (P22935), and rat CRABPII (P51673) Scale bar ¼ 0.1 substitutions per site foxb1.1 and foxb1.2, were also found to be located on the zebrafish LG and LG 25, respectively The single human orthologous FOXB1 gene is located at position 15q21-q26 Differential distribution of the crabp1a and crabp1b transcripts during embryonic and larval development The spatio-temporal distribution of the zebrafish crabp1a and crabp1b transcripts during embryonic and FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS R.-Z Liu et al Duplicated crabp1 genes in zebrafish part of rhombomere in the posterior hindbrain, ventral part of the anterior somites, hypaxial muscles and the tail bud (Fig 4B4–6 and 4C1–4) A weak crabp1bspecific hybridization signal was present in rhombomere (Fig 4B6) and posterior yolk syncytial layer (Fig 4B4) At 36 hpf, hybridization signals for crabp1a mRNA were observed in the nucleus of the diencephalon, the ventricular zone of the developing optic tectum and the dorsal retina, the epidermis of the tail tip in addition to neurohypophysis (Fig 5A,C,E) crabp1b transcripts were abundantly distributed in the posterior hindbrain and anterior spinal cord in which the signal was restricted to the dorsal spinal cord and gradually diminished along the length of the spinal cord (Fig 5B,F) The hybridization signal in the ventral part of the anterior somites, hypaxial muscles and tail tip showed the presence of crabp1b mRNA in these tissues (Fig 5B,D,F) At 48 hpf, crabp1a transcripts were detected in the nucleus of the diencephalons, optice tectum and neurohypophysis (Fig 5G,I,K), while crabp1b mRNA was most prominent in the posterior hindbrain and, to a lesser extent, in the spinal cord and ventral part of anterior myotomes (Fig 5H,J,L) Tissue-specific distribution of crabp1a and crabp1b mRNA in adult zebrafish Fig Comparison of the syntenic relationship of the zebrafish crabp1 genes with their human ortholog The zebrafish duplicated crabp1a and crabp1b genes have conserved syntenies (left column) with the human CRABP1 on chromosome 15 (right column) Orthologous crabp1 gene symbols are in bold Another pair of duplicated genes (foxb1.1 and foxb1.2) on zebrafish LG and LG 25 and the human FOXB1 ortholog on chromosome 15 are underlined The order of the human syntenic genes on human chromosome 15 was determined based on the cytogenetic mapping data from LocusLink (http://www.ncbi.nlm.nih.gov/locuslink) and the gene loci on the zebrafish LGs are listed in the order appearing on the human chromosome 15 larval development was determined by whole mount in situ hybridization (Figs and 5) The two duplicate paralogous genes showed differential and mostly nonoverlapping mRNA distribution patterns in the developing CNS The zebrafish crabp1b transcripts were detected during the middle somitogenesis stage at 17 h post fertilization (hpf) in the primary neurons throughout the spinal cord and the trigeminal placode (Fig 4A1–3) At 24 hpf, crabp1a mRNA was first detected in the neurohypophysis and the epidermis of the tail bud (Fig 4B1–3) In comparison, crabp1b mRNA-specific hybridization signal was distributed in the anterior and posterior spinal cord, ventro-lateral FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS The distribution of crabp1a and crabp1b transcripts in adult zebrafish tissues was analyzed by RT-PCR (Fig 6A) The crabp1a-specific primers generated RTPCR products of the expected size only from RNA of the brain, but not from any other tissues examined including the liver, ovary, skin, intestine, heart, muscle and testis crabp1b mRNA was detected by RT-PCR in the skin, intestine, brain and muscle of adult zebrafish As a positive control for RT-PCR, receptor for activated C kinase gene (rack1)-specific products were generated from RNA of all the adult tissues examined No RT-PCR products were detected in the negative control reactions that lacked reverse-transcribed cDNA templates for each of the three genes analyzed In addition to RT-PCR, tissue section in situ hybridization was performed with crabp1a- and crabp1bspecific oligouncleotide probes to determine tissuespecific distribution of the crabp1 gene transcripts in adult zebrafish A hybridization signal was detected by the crabp1a-specific oligonucleotide probe in the optic tectum of the adult zebrafish brain (Fig 6B) We have previously shown that the specific distribution of the brain-type fatty acid-binding protein (fabp7a) mRNA is localized to the pariventricular grey zone of the optic tectum of the adult zebrafish brain [24] Similar patterns of mRNA distribution in the optic tectum were 3565 Duplicated crabp1 genes in zebrafish R.-Z Liu et al A B C Fig Spatio-temporal distribution of transcripts of the zebrafish crabp1a and crabp1b genes during development (17–24 hpf) (A) The presence of crabp1b mRNA in the primary neurons (arrow heads) of spinal cord (Sp) and the trigeminal placode (Tp) during middle somitogenesis (A1) Dorsal view, head to the left; (A2) Dorsal view, head to the left; (A3) lateral view, head to the left (B) Comparison of the mRNA distribution of crabp1a and crabp1b during developmental stages of 24 hpf crabp1a mRNA was detected in the neurohypophysis (Nh) and tail bud (Tb) (B1-B3), while crabp1b mRNA was distributed in the rhombomere (r7), spinal cord and hypaxial muscles (Hm) (B4-B6) Presence of crabp1b mRNA in the posterior yolk synctial layer (YLS) (B4) and in the neurons of r6 and anterior spinal cord is indicated by arrow heads (B6) (C) Sequential cross sections (rostral to caudal) around the boarder of hindbrain and spinal cord showing transversal distribution of the crabp1b mRNA in r7, neurons of spinal cord (N Sp), dorsal and ventral spinal cord (D Sp and V Sp) and the anterior somites (So) of the 24 hpf embryos observed for crabp1a and fabp7a (Fig 6B) No hybridization signal was observed in the optic tectum of tissue sections hybridized to a negative control, sense oligonucleotide probe (Fig 6B) Tissue sections hybridized to an antisense oligonucleotides corresponding to the zebrafish crabp1b mRNA did not exhibit hybridization signal even in tissues where crabp1b mRNA had been detected by RT-PCR (data not shown) The levels of crabp1b transcripts may be below the level of sensi3566 tivity of detection by the technique of tissue section in situ hybridization Discussion The differentiation and development of primary motor neurons in vertebrates is controlled by RA signaling [25] The developing posterior hindbrain [26,27], the anterior spinal cord [26–28] and the retina [26,29] are FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS R.-Z Liu et al Duplicated crabp1 genes in zebrafish A B G H C D I J E F K L Fig Comparison of the mRNA distribution of crabp1a and crabp1b during developmental stages of 36 (A–F) to 48 (G–L) hpf crabp1a mRNA was detected sequentially in the neurohypophysis (Nh), tail bud (Tb), diencephalons (Di), tectum (Te), retina (Re) and anterior spinal cord (Sp), while crabp1b mRNA was distributed in r7, spinal cord, hypaxial muscles (Hm), Anterior somites (So) and myotomes (My), posterior hind brain (Hb), the pectoral fin (Pf), tail bud and in the posterior yolk synctial layer (YLS) (A, B, G, H) Lateral view, head to the left; (E, F, K, L) Dorsal view, head to the left; (C, D) magnified lateral view of the tail; (I, J) magnified lateral view, head to the left prominent sites of RA distribution and action The localization of crabp1b mRNA in regions of active neurogenesis in the developing CNS of zebrafish embryos suggests that CRABP1b may well act as a mediator of RA action during embryonic neurogenesis Neurogenesis in the CNS of adult teleosts is restricted to specific proliferative zones The optic tectum in fishes, birds and amphibians exhibits continuous neurogenesis [30–32] This generation of new neurons in the adult CNS most assuredly requires the expression a specific subset of neuronal genes One of the genes associated with neurogenesis in both the developing and adult CNS is the brain-type fatty acid binding protein (B-FABP) gene [33–35] In adult canary, B-FABP mRNA is distributed widely in the brain and is abundant in cell types known or suspected to undergo neurogenesis in the adult brain [35] In zebrafish, the transcripts of crabp1a (this study) and fabp7a coding for B-FABP [24] were both localized to the optic tectum RA and docosahexanoic acid, the ligands for CRABPI and B-FABP, respectively, are necessary FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS for neurogenesis As such, crabp1a and fabp7a may be essential genes in the signaling pathways for neurogenesis in the optic tectum To date, only a single copy of the gene coding for CRABPI has been reported in the genomes of various vertebrate species including fishes [1,19] Based on phylogenetic analyses and conserved synteny with human chromosome 15, we have identified duplicated copies of the crabp1 genes, crabp1a and crabp1b, in the zebrafish genome Furthermore, the two zebrafish crabp1 paralogs showed high amino acid sequence identity to each other, but differential spatio-temporal expression patterns during development and in adulthood In previous studies, we have identified duplicated genes for several other paralogous members of the iLBP multigene family in the zebrafish genome, the fabp7a and fabp7b [36], rbp1a and rbp1b [37], rbp2a and rbp2b [37], crabp2a and crabp2b [38] genes It appears that duplication is common for the iLBP gene family members in zebrafish, which attests to the hypothesis of ‘large scale gene duplication in fishes’ [21,23,39–43] 3567 Duplicated crabp1 genes in zebrafish A B Fig Tissue-specific distribution of the zebrafish crabp1a and crabp1b transcripts in adult zebrafish (A) Tissue-specific RT-PCR products were generated using cDNA derived from total RNA extracted from various adult zebrafish tissues (indicated above each lane) using primers corresponding to the cDNA for crabp1a and crabp1b A RT-PCR product corresponding to the constitutively expressed rack1 mRNA coding for receptor for activated C kinase was generated from RNA in all samples A negative PCR control without cDNA template did not generate RT-PCR product (B) Sagittal tissue sections of adult zebrafish were hybridized to [32P]dATP[aP] labeled crabpIa and fabp7a gene specific oligonucleotide probes (B1) Both crabp1a and fabp7a transcripts were colocalized in the adult zebrafish optic tectum (arrows, B1) The control section hybridized with a sense probe produced no signal in the optic tectum The hybridized tissue sections were stained with cresyl violet and the region corresponding to the optic tectum region is shown in panel B2 Arrows indicate the periventricular gray zone (PGZ) of the optic tectum (OT) An intriguing question is how have both duplicated copies of members of the iLBP mutigene family been preserved in the zebrafish genome? The fate of duplicated genes, in general, and the mechanism for preservation of ancient duplicated genes remains unclear Ohno [44] was the first to suggest that the fate of duplicated genes depends on the occurrence of null 3568 R.-Z Liu et al mutations, which commonly lead to gene silencing (i.e nonfunctionalization) He also suggests that some mutations may give rise to a gene with a new function that is distinct from the ancestral or duplicated sister gene (i.e neofunctionalization) More recently, it has been argued in the ‘duplication–degeneration– complementation’ (DDC) model [45,46] that subfunctionalization of duplicated genes accounts for the preservation of many gene duplicates owing to accumulation of mutations in regulatory elements Consequently, each copy of the duplicated gene pair shares a subset of the ancestral functions, e.g partitioning of tissue-specific gene expression In mammals, CRABP1 is expressed in the developing CNS and retina from an early embryonic stage [26,47–49] and is widely distributed in adult tissues including the brain, spinal cord, liver, ovary, testis, uterus, adrenal gland and retina [1] In the present study, we observed differential patterns of crabp1a and crabp1b expression in the developing and adult zebrafish The zebrafish crabp1a transcripts were detected in regions of the developing fore- and midbrain whereas crabp1b mRNA was detected in the developing hindbrain and spinal cord crabp1a mRNA was observed by tissue section in situ hybridization (Fig 6B) in the optic tectum of the adult zebrafish brain crabp1b mRNA was detected in the adult brain, muscle, intestine and skin but only by the sensitive technique of RT-PCR (Fig 6A) and not by in situ hybridization (data not shown) In addition to this spatial segregation, we observed a temporal segregation of crabp1a and crabp1b expression crabp1b mRNA was detected during early embryonic and larval development while crabp1a transcripts were only detected at later stages of embryonic development (24 hpf and thereafter) Together, the expression patterns of the zebrafish crabp1a and crabp1b generally sum to the expression of their mammalian orthologs in the developing nervous system [26,47–49] The partitioning of the temporal and spatial distribution of the zebrafish crabp1a and crabp1b gene transcripts during development and in adulthood compared to their mammalian CRABP1 orthologs suggests that retention of the duplicated crabp1 genes in zebrafish is the result of subfunctionalization during evolution Experimental procedures Maintenance of fish Zebrafish were purchased from a local aquarium store Fish feeding, breeding and embryo manipulation was conducted according to established protocols [50] FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS R.-Z Liu et al Cloning of cDNAs for the zebrafish crabp1a and crabp1b To obtain the complete cDNA sequence encoded by the zebrafish crabp1a and crabp1b genes, both 3¢ rapid amplification of cDNA ends (3¢-RACE) and 5¢-RNA ligase mediatedRACE (5¢-RLM-RACE) were employed as previously described [51,52] The nested sense and antisense primers used for 3¢-RACE and 5¢-RLM-RACE were designed based on a zebrafish expressed sequence tag (EST, GenBank accession number BI533516), which had sequence similarity to the mammalian CRABPI cDNA (crabp1a; 3¢-RACE: 5¢-CCC AACTTCGCCGGCACCTGG-3¢, 5¢-TGAAAGCTCTCG GCGTAAAC-3¢; 5¢-RLM-RACE: 5¢-GAGCTTTCAGA AGTTCGTCG-3¢, 5¢-GAATCTCCACATGCGGTTTG-3¢) and a zebrafish genomic DNA sequence assembly (BX663612) which harbors the coding sequence of another gene similar to the mammalian CRABPI (crabp1b; 3¢-RACE: 5¢-GCTAACAGATCAATAGGCTTC-3¢, 5¢-GATTTGAA AGCAAGAGGGTC-3¢; 5¢-RLM-RACE: 5¢-TTAGACGC AGCCGCACAAG-3¢, 5¢-CGGCCATCGACGGTCTC-3¢) Three 3¢-RACE cDNA and 5¢-RLM-RACE clones of each gene were sequenced and the complete cDNA sequences of crabp1a and crabp1b genes were determined by aligning and combining the sequences Phylogenetic analysis Phylogenetic analysis of the zebrafish crabp1a, crabp1b and other fish and mammalian cellular retinoid-binding protein genes was performed using clustalx [53] A bootstrap neighbor-joining phylogenetic tree was constructed using the zebrafish intestinal-type fatty acid-binding protein sequence (I-FABP, GenBank accession number AY266452) as an outgroup Linkage mapping of crabp1a and crabp1b with radiation hybrid panel Radiation hybrids of the LN54 panel [22] were used to assign the crabp1a and crabp1b genes to a specific zebrafish linkage group The sequences of the primers used to amplify the genomic DNA from cell hybrids of the LN54 panel are: crabp1a: 5¢-TGGTTTGCACGCGGATTTAC-3¢, 5¢-GAAC GATGACTACAGCAATGG-3¢; crabp1b: 5¢-GCAAATGT GAGCACTAAGTG-3¢, 5¢-CGGCCATCGACGGTCTC-3¢ The PCR conditions have been described previously [51] Whole mount in situ hybridization to zebrafish embryos To reveal the spatio-temporal distribution of the zebrafish crabp1a and crabp1b transcripts in developing zebrafish, whole mount in situ hybridization was performed as described by Thisse & Thisse at the website: http://zfin.org/ FEBS Journal 272 (2005) 3561–3571 ª 2005 FEBS Duplicated crabp1 genes in zebrafish zf_info/zfbook/chapt9/9.82 RNA probes complementary to zebrafish crabp1a and crabp1b mRNAs were generated from sequenced 3¢ RACE clones Reverse transcription-polymerase chain reaction (RT-PCR) Conditions for RT-PCR used to determine the tissue-specific distribution of the crabp1a and crabp1b transcripts in adult zebrafish were according to those previously described in [51] Primers employed in RT-PCR were designed based on cDNA sequences of the zebrafish crabp1a (5¢-TGAAAG CTCTCGGCGTAAAC-3¢, 5¢-GAAC GATGACTACAGC AATGG-3¢) and crabp1b (5¢-GATTTGAAAGCAAGAGG GTC-3¢, 5¢-CTGCAAGTGCTGGAATATTC-3¢) The reaction products were size-fractionated by agarose gel electrophoresis Adult zebrafish tissue section in situ hybridization Zebrafish tissue section in situ hybridization was performed according to [52] The antisense oligonucleotide sequences complementary to the zebrafish crabp1a (5¢-CATGCAAT GAAGTTTTCTGGGTTTTCTAGACAG-3¢) and fabp7a (5¢-GTAATATCGTCAAGTTGCCAGGGTAATACTGA AACG-3¢) mRNA sequences [24] were used as in situ hybridization probes A previously synthesized sense oligonucleotide probe [52] was used as a negative control Following hybridization and autoradiography, the tissue sections were stained with cresyl violet Acknowledgements This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada (to JMW), the Canadian Institutes of Health ´ Research (to ED-W), the Institut National de la Sante ´ et de la Recherche Medicale, Centre National de la Recherche Scientifique, Hopital Universitaire de Strasˆ bourg, Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, National Institute of Health (to CT and BT), and Izaak Walton Killam Memorial Scholarships (to R-ZL and MKS) We thank Marc Ekker for the DNA from LN 54 radiation 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(19 90) Retinoic acid receptors and cellular retinoid binding proteins I A systematic study of their differential pattern of transcription during mouse organogenesis Development 11 0, 11 33? ?13 51. .. crabp1 gene symbols are in bold Another pair of duplicated genes (foxb1 .1 and foxb1.2) on zebrafish LG and LG 25 and the human FOXB1 ortholog on chromosome 15 are underlined The order of the human