The Cellular Retinol-Binding Protein Genes Are Duplicated and Differentially Transcribed in the Developing and Adult Zebrafish (Danio rerio)
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《分子生物学进展》
* Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada; Institut de Génétique et Biologie Moléculaire et Cellulaire, Department of Developmental Biology, CU de Strasbourg, France; and Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
Correspondence: E-mail: jmwright@dal.ca.
Abstract
There are single copies of the genes encoding the cellular retinol-binding protein type I and II (CRBPI and CRBPII) in the human and rodent genomes. We have identified duplicate genes for both CRBPI and CRBPII in the zebrafish (Danio rerio) genome (rbp1b and rbp2b). The zebrafish rbp1b and rbp2b have conserved gene structures, amino acid sequence similarities, gene phylogenies, and syntenic relationships with their mammalian orthologs and zebrafish paralogs, rbp1a and rbp2a. Like the mammalian genes for CRBPI and CRBPII, the zebrafish rbp1b and rbp2b genes are closely linked on a single linkage group. Comparative analysis suggests that the duplicate genes of rbp1 and rbp2 in the zebrafish genome may have arisen by chromosomal or whole-genome duplication. During embryonic development, rbp1b transcripts were detected in the gall bladder of 5-day postfertilization (5 dpf) larvae. The rbp2b mRNA was abundant in the developing liver through 48 hours postfertilization (48 hpf) to 5 dpf. Using reverse transcription-polymerase chain reaction (RT-PCR), rbp1b transcripts were detected in the ovary, and rbp2b mRNA was observed predominantly in the adult liver. Tissue section in situ hybridization and emulsion autoradiography localized rbp1b mRNA to primary oocytes within the zebrafish ovary. The differential mRNA distribution patterns of the rbp1a, rbp1b, rbp2a, and rbp2b genes in the developing and adult zebrafish suggest that shuffling of subfunctions among duplicate copies of paralogous genes may be a mechanism for the retention of duplicated genes in vertebrates.
Key Words: gene duplication ? gene structure ? phylogeny ? genome evolution ? gene expression ? subfunctionalization
Introduction
Retinoids play crucial roles in organogenesis during embryonic development and, thereafter, in the maintenance of a wide range of biological processes, including vision, reproduction, growth, and immunity (Napoli 1996; Ross 2000). It has long been recognized that retinoids are essential micronutrients for normal reproductive function of animals (Thompson, Howell, and Pit 1964). Recent experiments show that retinoids may serve as important regulators in oogenesis and oocytes maturation in vertebrates (Livera et al. 2000; Morita and Tilly 1999; Whaley et al. 2000).
Retinoids are hydrophobic molecules, and their transport in the cytoplasm is thought to be facilitated by a group of intracellular carrier proteins, which specifically bind different isomeric retinoids and target them to various intracellular enzymes or receptors for retinoid metabolism and eventual actualization of their physiological activities. During the past decade, increasing evidence has suggested the involvement of intracellular retinoid-binding proteins in retinoid biological action and metabolism (reviewed by Napoli [1999], Noy [2000] and Adida and Spener 2002). 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 (reviewed by Ong, Newcomer, and Chytil [1994]. Glatz and Vusse [1996], and Bernlohr et al. [1997]). In mammals, two different types of cellular retinoid-binding proteins with distinct ligand-binding properties have been identified, the cellular retinol-binding proteins (CRBPs) and the cellular retinoic acid–binding proteins (CRABPs). Four types of CRBP, including CRBPI, CRBPII, CRBPIII and CRBPIV, are encoded by different genes in mammals (Bashor, Toft, and Chytil 1973; Ong, Newcomer, and Chytil 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002). Although ligand-binding affinity (Kd) differs among different CRBP types, all CRBPs have affinity for retinol and retinal but do not bind retinoic acid or retinyl esters (Ong 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002).
To date, only a single copy of the gene coding for either CRBPI or CRBPII has been identified in mammalian genomes. In previous studies, the gene structure, cDNA sequence, linkage relationship, and expression of the genes coding for cellular retinol-binding protein type I (hereafter referred to as rbp1a) and type II (rbp2a) from zebrafish has been determined (Cameron et al. 2002; Liu et al. 2004). Here, we report the discovery of a pair of duplicate genes coding for CRBPI (rbp1b) and CRBPII (rbp2b) in the zebrafish genome. Phylogenetic analysis, conserved gene structure, and linkage relationship suggest that the zebrafish rbp1b and rbp2b are orthologs of the mammalian genes for CRBPI and CRBPII, respectively, and arose by genome-wide duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003). Comparative analysis of the mRNA distribution patterns for the gene duplicates of rbp1 and rbp2 in developing and adult zebrafish indicated that the closely related rbp paralogs have different subfunctions, and that "subfunction shuffling" among duplicates of paralogous genes might be an important mechanism for preservation of duplicated genes.
Materials and Methods
5' RNA Ligase Mediated–Rapid Amplification of cDNA Ends (5' RLM-RACE)
5' RLM-RACE was performed as previously described (Liu et al. 2003a) to amplify the 5' end of the zebrafish rbp1b and rbp2b transcripts and map their transcription start sites. Nested antisense primers used for the 5' RLM-RACE were designed within the coding sequence of the zebrafish rbp1b and rbp2b gene, respectively (as1 and as2 for rbp1b and as3 and as4 for rbp2b in figure 1). The 5' RACE products were cloned and sequenced.
FIG. 1.— Structures of the zebrafish rbp1b and rbp2b genes. Exons are shown in uppercase letters with the coding sequences of each exon underlined and the deduced amino acid sequence indicated below. Numbers on the right indicate nucleotide positions in the gene sequence. The initiation site for transcription is marked by a star and numbered as +1, and a putative polyadenylation signal is double underlined. A potential TATA box in the 5' upstream region of rbp2b is in bold and boxed. Nucleotide sequences corresponding to sense (s) and antisense (as) PCR primers and the tissue section in situ hybridization probe (p) used in this study are boxed and indicated. The position of a potential GAG insertion variant in exon 1 of rbp1b gene is marked by "". The zebrafish rbp1b and rbp2b gene sequences were identified from a zebrafish genomic DNA sequence assembly of clone CH211 to 11908 (GenBank accession number AL953896).
3' RACE
To obtain the complete cDNA sequence encoded by the zebrafish rbp1b and rbp2b genes, 3' RACE was employed (Liu et al. 2003b). The nested sense primer sequences used for 3' RACE are shown in figure 1 (s1 and s2 for rbp1b and s4 for rbp2b). Three clones of each gene were sequenced and the complete cDNA sequences of rbp1b and rbp2b genes were determined by aligning the sequences of the 5' RLM-RACE and 3' RACE cDNA clones.
Phylogenetic Analysis
Phylogenetic analysis of the zebrafish rbp1b, rbp2b, and the other fish and mammalian cellular retinoid-binding protein genes was performed with ClustalX (Thompson et al. 1997), and the bootstrap neighbor-joining phylogenetic tree was constructed using the zebrafish intestinal-type fatty acid–binding protein sequence (I-FABP) as an outgroup.
Radiation Hybrid Mapping
Radiation hybrids of the LN54 panel (Hukriede et al. 1999) were used to assign the rbp1b and rbp2b genes to a specific zebrafish linkage group. The sequences of the primers employed to amplify the genomic DNA from cell hybrids of LN54 panel are shown in figure 1 (s3 and as2 for rbp1b and s4 and as3 for rbp2b), and the PCR conditions were the same as previously described (Liu et al. 2003a).
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Conditions for RT-PCR used to determine the tissue-specific distribution of the rbp1b and rbp2b transcripts in adult zebrafish were the same as those previously described (Liu et al. 2003a). Gene-specific primers used for RT-PCR are shown in figure1 (s3 and as1 for rbp1b and s4 and as4 for rbp2b), and reaction products were size-fractionated by agarose gel electrophoresis. The tissue-specific mRNA distribution patterns of rbp1b and rbp2b were compared with that of rbp1a and rbp2a (Liu et al. 2004).
Tissue Section in situ Hybridization and Emulsion Autoradiography
Zebrafish tissue section in situ hybridization and emulsion autoradiography were performed as previously described (Liu et al. 2003b). The antisense oligonucleotide was complementary to the zebrafish rbp1b cDNA sequence (p in figure 1).
Whole-Mount in situ Hybridization to Embryos
To reveal the spatio-temporal distribution of the zebrafish rbp1b and rbp2b transcripts in developing zebrafish, whole-mount in situ hybridization was performed as described by C. Thisse and B. Thisse at the Web site: http://zfin.org/zf_info/zfbook/chapt9/9.82. Antisense RNA probes were prepared from the zebrafish rbp1b and rbp2b cDNA generated by 3' RACE.
Results
Structure of the Zebrafish rbp1b and rbp2b Genes
A zebrafish genomic DNA sequence record (GenBank accession number AL953896) was identified in GenBank by a tBlastN search for sequences corresponding to the published CRBPI and CRBPII amino acid sequence (Calderone et al. 2002; Cameron et al. 2002; Liu et al. 2004). The identified genomic DNA sequence contained two segments corresponding to the potential duplicated copies of the zebrafish rbp1 gene (rbp1b) and rbp2 gene (rbp2b), respectively. The distance between them is approximately 10 kb. The gene size and structure (fig. 1) of rbp1b and rbp2b were further defined after aligning their corresponding cDNA sequences (see below) with the genomic DNA sequences. The zebrafish rbp1b gene spanned 8,267 bp from the transcription start site (see below) to the polyadenylation site and consisted of four exons separated by three introns. The four exons of rbp1b were 159 bp, 179 bp, 102 bp, and 527 bp in length and coded for 22, 60, 34, and 16 amino acids, respectively (fig. 1). The three introns of the zebrafish rbp1b gene were 3,108 bp, 966 bp, and 3,226 bp in length, respectively, accounting for 89% of the whole-gene sequence. The zebrafish rbp2b gene was much smaller than rbp1b and spanned 1,262 bp of genomic DNA. The rbp2b gene also consisted of four exons (109 bp, 179 bp, 102 bp, and 185 bp) and three introns (140 bp, 365 bp, and 182 bp). The four exons of rbp2b coded for 24, 60, 34, and 17 amino acids, respectively. The nucleotides at the splice sites of each exon/intron boundary for both rbp1b and rbp2b conformed to the GT-AG rule (Breathnach and Chambon 1981).
The transcription start site of the zebrafish rbp1b gene was defined by 5' RLM-RACE, which generated a single product of the intact 5' cDNA end with the 7-methyl G cap. A negative control in which tobacco acid pyrophosphatase (TAP) treatment was omitted yielded no RACE product (data not shown). The 5' RLM-RACE product was cloned and sequenced, and the transcription start site was mapped to the nucleotide 92 bp upstream of the rbp1b ATG initiation codon (fig. 1). In the same way, the transcription start site of rbp2b was shown to be located 36 bp upstream of the rbp2b initiation codon (fig. 1).
Inspection of the 5' upstream sequence of the zebrafish rbp1b and rbp2b genes with MatInspector Professional version 6.2 (Quandt et al. 1995) revealed a TATA box at position –25 of the rbp2b gene sequence, whereas no TATA box was found within the proximal promoter region of rbp1b gene (fig. 1). A TATA box is present in the promoter of the zebrafish rbp2a gene and the mammalian rbp2 gene but not in the rbp1a gene of zebrafish or the single-copy rbp1 genes from other vertebrate species (Liu et al. 2004; Ong, Newcomer, and Chytil 1994).
Characterization of the Zebrafish rbp1b and rbp2b cDNAs
To further characterize the transcripts of the zebrafish rbp1b and rbp2b genes, we isolated their cDNAs. In addition to 5' RLM-RACE, we performed 3' RACE using gene-specific sense primers corresponding to a sequence in the 5' UTR (fig. 1). A single 3' RACE product of expected size was generated, cloned and sequenced for each gene. The complete nucleotide sequence of the zebrafish rbp1b cDNA (GenBank accession number AY395732), determined by combining the 5' RLM-RACE and 3' RACE sequences, was 971 nt in length, excluding the poly(A) tail. The rbp2b cDNA (GenBank accession number AY619686) was 575 nt, excluding the poly(A) sequence. Putative polyadenylation signals were identified 15 and 16 nt upstream of the poly(A) initiation site of the zebrafish rbp1b and rbp2b cDNA sequences, respectively. Alignment of the zebrafish rbp1b cDNA sequence with the gene sequence revealed a GAG insertion in the cDNA sequence at position 144 to 146. The extra GAG in the cDNA sequence resulted in an insertion of a glutamic acid in the CRBPIb amino acid sequence relative to the amino acid sequence deduced from the rbp1b genomic sequence. This discrepancy between the cDNA and genomic sequence is not likely to be a PCR artifact or a sequencing error but rather an intraspecies genetic variation, because independent clones from separate 3' RACE and 5' RLM-RACE reactions contained this extra GAG. The rbp1b gene sequence and an expressed sequence tag (EST) from GenBank (accession number BI886241) did not contain these three nucleotides. At the present time, we do not know whether this variation has any functional implications. Except for the difference of the GAG triplet, the rest of the coding sequence of the zebrafish rbp1b gene and the cloned cDNA was identical. The nucleotide sequence of the zebrafish rbp2b cDNA is identical to its genomic coding sequence.
The deduced amino acid sequence from the zebrafish rbp1b cDNA sequence consisted of 133 amino acids and the CRBPIb protein has a molecular mass of 15.2 kDa and theoretical isoelectric point of 4.94. Alignment of the zebrafish CRBPIb sequence with mammalian CRBPs and CRABPs showed highest sequence identity with that of the mammalian CRBPIs (74%) and the zebrafish CRBPIa (66%), followed by mammalian and zebrafish CRBPIIs (56%), human CRBPIII (54%), human CRBPIV (51%), mammalian CRABPIs (33%), and mammalian CRABPIIs (29% to 33%). The deduced amino acid sequence from the zebrafish rbp2b cDNA sequence consisted of 135 amino acids, with an estimated molecular mass of 15.6 kDa and a theoretical isoelectric point of 7.18. The deduced zebrafish CRBPIIb protein had sequence identity of 67% to 70% with mammalian CRBPIIs, 64% with the zebrafish CRBPIIa, 52% to 53% with mammalian CRBPIs, 53% with human CRBPIV, 50% with human CRBPIII, approximate 30% with mammalian CRABPIIs and approximately 27% with mammalian CRABPIs. The divergence of the amino acid sequences between the zebrafish CRBPIa and CRBPIb as well as between CRBPIIa and CRBPIIb is shown in figure 2.
FIG. 2.— Alignment of the deduced amino acid sequences of the zebrafish CRBPI and CRBPII duplicate gene pairs. (A) CRBPIa (GenBank accession number AAQ54326) aligned with CRBPIb (accession number AAR31829); (B) CRBPIIa (accession number AAL38648) aligned with CRBPIIb (accession number AAT40241). Dots indicate amino acid identity and dashes represent gaps.
Gene Phylogeny of the Zebrafish rbp1 and rbp2 Duplicates
Phylogenetic analysis was performed to reveal the evolutionary relationship among the newly identified zebrafish rbp1b, rbp2b, and other cellular retinoid-binding protein genes from mammals and fishes using their primary amino acid sequences (fig. 3). The zebrafish CRBPIb clustered with the mammalian CRBPIs in the same clade (bootstrap value 821/1000 [fig. 3]) and the zebrafish CRBPIa and CRBPIb formed a subclade from the mammalian CRBPIs. The zebrafish CRBPIIb clustered with the mammalian and fish CRBPII clade with a bootstrap value of 801/1000 (fig. 3). The results of phylogenetic analysis indicated that the genes for the zebrafish CRBPIb and CRBPIIb are orthologous to the genes for the mammalian CRBPI and CRBPII.
FIG. 3.— Phylogenetic tree of cellular retinoid-binding proteins. The bootstrap neighbor-joining phylogenetic tree was constructed with ClustalX (Thompson et al. 1997) using the zebrafish (Zf) intestinal type fatty acid–binding protein (I-FABP) amino acid sequence (GenBank accession number AAF00925) as an outgroup. The bootstrap values (based on number per 1,000 duplicates) are indicated on the nodes. Other amino acid sequences used in this analysis include zebrafish CRBPIa (accession number AAQ54326) and CRBPIb (accession number AAR31829), human (Hm) CRBPI (accession number P09455), mouse (Ms) CRBPI (accession number Q00915), rat (Rt) CRBPI (accession number P02696), zebrafish CRBPIIa (accession number AAL38648) and CRBPIIb (accession number AAT40241), human CRBPII (accession number P50120), mouse CRBPII (accession number Q08652), rat CRBPII (accession number P06768), human CRBPIII (accession number P82980), human CRBPIV (accession number AAN61071), human CRABPI (accession number P29762), mouse CRABPI (accession number P02695), human CRABPII (accession number P29373), mouse CRABPII (accession number P22935), and rat CRABPII (accession number P51673). Scale bar = 0.1 substitutions per site.
Linkage Mapping and Conserved Syntenies of the Zebrafish rbp1b and rbp2b Genes
The zebrafish rbp1b and rbp2b genes were both assigned by radiation hybrid mapping to linkage group 2 (LG 2) of the zebrafish genome. The zebrafish rbp1b was mapped to a locus on LG 2 at a distance of 5.23 cR from the marker Z211023, and rbp2b was located 6.19 cR from Z21490 [GenBank] (mapping data is available on request). The mapping distance between rbp1b and rbp2b on LG 2, estimated by their flanked markers, was approximately 6.7 cR. The close linkage relationship of the zebrafish rbp1b and rbp2b revealed by radiation hybrid mapping here is in agreement with their actual distance in the genomic sequence mentioned above (10 kb apart). Using the map of zebrafish LG 2 (Woods et al. 2000) and data from LocusLink (http://www.ncbi.nlm.nih.gov/locuslink), we found that the zebrafish rbp1b and rbp2b genes on LG 2, like rbp1a (Liu et al. 2004) and rbp2a (Cameron et al. 2002), have conserved synteny with the human RBP1 and RBP2 genes on chromosome 3 and mouse Rbp1 and Rbp2 genes on chromosome 9 (fig. 4)
FIG. 4.— Comparison of syntenic relationship of rbp1 and rbp2 genes from zebrafish, human, and mouse. Gene duplicates of the zebrafish rbp1and rbp2 genes have conserved syntenies (left box) with the human RBP1 and RBP2 genes on chromosome 3 (middle box) and the mouse Rbp1 and Rbp2 genes on chromosome 9 (right box). Orthologous rbp1 and rbp2 gene symbols are in bold. The order of the human syntenic genes on human chromosome 3 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 and mouse chromosomes and the last common ancestor's chromosome (lower box) are listed in the order appearing on the human chromosome 3. Hypothesized evolutionary events leading to the syntenic relationships of the zebrafish, human, and mouse rbp1 and rbp2 genes are indicated at the nodes of the evolutionary tree.
The Zebrafish rbp1b and rbp2b mRNA Distribution During Development
We examined the spatial and temporal distribution of the zebrafish rbp1b and rbp2b transcripts during embryonic and larval stages of development using whole-mount in situ hybridization with an antisense RNA probe synthesized from the zebrafish rbp1b and rbp2b cDNA clones (fig. 5). The zebrafish rbp1b mRNA was not detected in the early developing zebrafish until 5 dpf. In the 5 dpf larvae, rbp1b transcripts were restricted to the gall bladder (fig. 5A). Hybridization signals were not observed in any other embryonic or larval tissues. The zebrafish rbp2b mRNA was detected at 48 hpf stage in the liver and yolk syncytial layer (YSL [fig 5B, 1–2]). At 5 dpf stage, rbp2b transcripts were restricted to the liver (fig. 5B, 3–4)
FIG. 5.— Detection of zebrafish rbp1b and rbp2b mRNA during development by whole-mount in situ hybridization. (A) Whole-mount in situ hybridization of 5-day-old zebrafish larvae. rbp1b-specific antisense RNA probe detected rbp1b mRNA in the developing gall bladder (Gb) of the larvae at 5 dpf. (A, 1) Lateral view, head to the left. (A, 2) Cross section through the gall bladder. (A, 3) Sagittal section through the gall bladder. (A, 4) Dorsal view, head to the left. Ib, intestine bulb; Nc, notochord; Nt, neural tube. (B) Abundant rbp2b mRNA was observed in the liver (L) of 48 hpf (B, 1 and B, 2) and 5-dpf larvae (B, 3 and B, 4). The 48-hpf larval yolk syncytial layer (YSL) also showed lower levels of rbp2b mRNA distribution (B, 1 and B, 2). (B, 1 and B, 3) Lateral view, head to the left. (B, 2 and B, 4) Dorsal view, head to the left. Nonspecific staining was observed in the myotomes of 5-dpf larvae (B, 3).
Tissue Distribution of rbp1 and rbp2 Transcripts in Adult Zebrafish
The distribution of rbp1b and rbp2b mRNA in adult tissues was investigated by RT-PCR using gene-specific primers and compared with the patterns of their sister duplicate copies, rbp1a and rbp2a (fig. 6). The rbp1b-specific primers generated RT-PCR products of the expected size only from RNA of the ovary but not from RNA of all other tissues examined, including the liver, skin, intestine, brain, heart, muscle, and testis, whereas a relatively low level of rbp1a mRNA was detected in a number of tissues, including the liver, ovary, intestine, brain, and testis after 35 cycles of PCR amplification (fig. 6). Both rbp2a-specific and rbp2b-specific primers produced RT-PCR products in the liver and intestine, but the relative intensity of rbp2b product in the intestine was relatively low compared with that of rbp2a RT-PCR product (fig. 6). RT-PCR products were generated from RNA of all the tissues examined with specific primers corresponding to the cDNA sequence of the constitutively expressed receptor for activated C kinase (RACK1) gene (Hamilton and Wright 1999). No RT-PCR products were detected from the negative controls that lacked the reverse-transcribed cDNA templates for all genes assayed (fig. 6).
FIG. 6.— Tissue-specific distribution of the transcripts from rbp1 and rbp2 duplicates in adult zebrafish detected by RT-PCR. Tissue-specific RT-PCR products were generated from total RNA extracted from various adult zebrafish tissues (indicated on the top) using primers corresponding to the cDNA for CRBPIa, CRBPIb, CRBPIIa, and CRBPIIb. An RT-PCR product corresponding to the constitutively expressed receptor for activated C kinase (RACK1) was generated from RNA in all samples. A negative PCR control (–) did not contain cDNA template and did not generate any RT-PCR product.
RT-PCR analysis demonstrated that the expression of rbp1b was restricted to ovary. To determine the cell specificity of rbp1b gene expression in the ovary, we first performed in situ hybridization analysis of whole-zebrafish tissue sections using an antisense oligonucleotide probe corresponding to a nucleotide sequence of the 3' UTR of the zebrafish rbp1b cDNA (fig. 1). Strong punctuate hybridization signal was specifically detected in the zebrafish ovary but not in any other tissues (fig. 7A, 1) (data not shown). Nonspecific background was observed after hybridization to a sense probe throughout the tissue section (fig. 7A, 2). To identify the cells expressing rbp1b mRNA in the ovary, we conducted emulsion autoradiography of the in situ hybridization tissue sections (fig. 7B). Silver grains corresponding to rbp1b-specific hybridization were densely clustered over the primary (stage I) oocytes (fig. 7B, 1–3) but not in the stage II or III oocytes (fig. 7B, 2). The rbp1b-specific hybridization was not observed in any other tissues, including muscle (fig. 7B, 1 and 3) and liver (fig. 7B, 3) .
FIG. 7.— Specific distribution of zebrafish rbp1 mRNA in the primary oocytes detected by tissue section in situ hybridization and autoradiography. (A) In situ hybridization detected rbp1b mRNA in the ovary (arrow) on the tissue section hybridized with an rbp1b cDNA-specific antisense oligonucleotide probe (A, 1) but not with the sense probe (A, 2). (B) Bright and dark field showed the presence of rbp1b mRNA (indicated by silver grains) at the cellular level by emulsion autoradiography. Silver grains were observed in the stage I primary oocytes (arrows, B, 1–3), but not in stage II, stage III oocytes (B, 2), or the muscle (M) and liver (L).
Discussion
Previously, we reported the sequence and expression patterns of rbp1a and rbp2a (Cameron et al. 2002; Liu et al. 2004). Here, we have described the duplicated genes rbp1b and rbp2b encoding CRBPI and CRBPII from zebrafish and their spatiotemporal expression during development and in adulthood. It appears zebrafish, unlike mammals, has two rbp1 genes, rbp1a and rbp1b, and two rbp2 genes, rbp2a and rbp2b.
In humans, the RBP1 and RBP2 genes are closely linked and both genes have been physically mapped on human chromosome 3 at 3q23 (De Baere et al. 1998a, 1998b). By examining the human genomic DNA sequence, we found these two genes reside on the same contig sequence (GenBank accession number NT005832) and they are approximately 60 kb apart. In the zebrafish, we also identified a DNA assembly sequence from a single clone (GenBank accession number AL953896) that harbors both rbp1b and rbp2b genes separated by approximately 10 kb of DNA. As expected, these two genes were assigned to the same linkage group, LG 2, and the distance between them is 6.7 cR. The zebrafish rbp1a and rbp2a genes were assigned to LG 15 (Cameron et al. 2002) and LG 16 (Liu et al. 2004), respectively, and each of them has conserved syntenies with the human and mouse RBPI and RBPII genes. In the zebrafish genome, another pair of duplicate sister genes, atp1b3a and atp1b3b, also located on LG 2 and LG 15, syntenic with the zebrafish rbp2b and rbp2a gene, respectively. In humans and mouse, the orthologous gene for ATP1B3 (located at 3q22-q23 in humans and LG 9 51cM in mouse) is also closely linked to the RBPII gene (3q23 in humans and LG 9 57 cM in mouse) (Malik et al. 1998; Besirli, Gong, and Lomax 1998). This finding indicates that these two zebrafish rbp2 genes arose and were retained in the zebrafish genome after chromosomal duplication or whole-genome duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003; Taylor et al. 2003). It is likely that after the chromosome or genome duplication in the fish lineage, the syntenic relationship of rbp1 and rbp2 genes in fishes was preserved on one of the duplicate chromosomes (rbp1b and rbp2b) but was not retained on the other (rbp1a and rbp2a), presumably because of translocation or chromosomal fission (fig. 4).
Currently, three theories have been proposed for the evolutionary fate of duplicated genes. First, the nonfunctionalization theory states that one of the gene duplicates becomes silenced because of degenerative mutations and becomes a pseudogene or is eventually lost from the genome. Second, the neofunctionalization theory states that one of the gene duplicates acquires a new and beneficial function, while the other duplicate retains the function of the ancestral gene, such that both genes are preserved. Third, a duplication-degeneration-complementation (DDC) model was recently proposed (Force et al. 1999, and references therein), which states that, in addition to the nonfunctionalization and neofunctionalization fate of duplicate genes, subfunctionalization might be an important mechanism for preservation of duplicated genes. After duplication, degenerative mutations in the cis regulatory elements results in partitioning of the original functions into each duplicate gene. As such, the ancestral function is divided between the duplicates, which functionally complement each other and, thus, the duplicate genes are retained in the host genome (Force et al. 1999).
The mammalian rbp1 genes are expressed in a number of adult tissues. In rats, CRBPI is abundantly distributed in the adult liver and kidney and, to a lesser extent, in the lung, testis, spleen, eye, ovary, uterus, and intestine (Ong, Newcomer, and Chytil 1994), whereas in humans, the ovary contains the highest amount of rbp1 gene transcripts and protein, but the levels of CRBPI are relatively low in human kidney and lung (Fex and Johannesson 1984; Ong and Page 1986; Folli et al. 2001). The relative levels of CRBPI in mammalian adult tissues, therefore, vary among species. Expression of the mammalian and the zebrafish rbp1 genes supports the hypothesis that the ancestral rbp1 gene was expressed in multiple adult tissues, including ovary, liver and in the early developing CNS, and retina. It is clear that the duplicate zebrafish rbp1 genes complement each other to retain some of the ancestral rbp1 functions, where rbp1b transcripts have a specialized distribution in the oocytes and rbp1a transcripts are expressed in the developing CNS and retina (Liu et al. 2004). One feature of the zebrafish rbp1 duplicated genes is that they were subfunctionalized both spatially and temporally, which is not stated by the DDC model of duplicate gene retention (Force et al. 1999). Temporal subfunctionalization might be a common feature for developmentally regulated gene duplicates. Thummel et al. (2004) have noted temporal subfunctionalization of the hoxc13a and hoxc13b genes in zebrafish, where hoxc13a is maternally expressed, and hoxc13b is expressed later in development. Neither rbp1a nor rbp1b is abundantly expressed in the adult liver and do not, therefore, complement each other to retain this ancestral subfunction after duplication. By contrast, both zebrafish rbp2a (Cameron et al. 2002, Liu et al. 2004) and rbp2b are expressed in the adult liver, despite the fact that none of the mammalian rbp2 genes studied to date are expressed in the liver. As such, our data suggests that the ancestral subfunction in the liver for rbp1 has been acquired by the duplicates of a closely related paralogous gene, rbp2, after duplication. We propose that "subfunction shuffling" between duplicates of paralogous genes might be a mechanism for preservation of the duplicates from unifunctional genes, such as the zebrafish rbp2 genes (fig. 8). At present, it is not possible to determine how cis-acting elements are shuffled. However, we speculate that mutation or unequal crossing-over between paralogs could result in the loss of cis-element function. Alternatively, a new subfunction to one of the duplicate gene pair may be acquired by "gain-of-function," such as transposon insertion of a new cis-acting element into the regulatory region of one of the duplicate genes .
FIG. 8.— A model for subfunction shuffling after gene duplication and subfunctionalization. Boxes numbered 1 to 4 denote cis regulatory elements controlling unique functions. Solid boxes denote functional cis elements, whereas open boxes denote null mutated cis elements. Arrows after cis elements denote transcription start sites of genes. Duplication of the ancestral gene (duplication 1) gives rise to a pair of paralogous genes, one multifunctional (paralog 1) and the other unifunctional (paralog 2) through subfunctionalization. After the second round of duplication (duplication 2), the subfunctions of paralog 1 and paralog 2 are shuffled, which results in the acquisition of subfunction 3 from duplicates of paralog 1 to a duplicate of paralog 2 (paralog 2b). Both duplicates from the unifunctional paralog 2 (paralog 2a and 2b) are then preserved.
Egg yolk from oviparous animals is one of the richest sources of vitamin A. In fish and other oviparous animals, retinol must be taken up from the serum by the rapidly growing oocytes and then stored and metabolized before functioning during embryonic development. Retinoids have been detected in high levels in unfertilized zebrafish eggs and during embryogenesis (Costaridis et al. 1996). Holo-CRBPI (retinol-CRBPI complex) is the substrate for lecithin:retinol acyltransferase (LRAT), an enzyme catalyzing the esterification of retinol with long-chain fatty acids to form retinyl ester for storage, whereas Apo-CRBPI strongly inhibits the esterification reaction (Herr and Ong 1992). Because of the significant and distinct features of retinoid mobilization and metabolism in the fish ovary, it is reasonable to speculate that rbp1b has evolved and subfunctionalized to play a particular role in retinol uptake, metabolism, and storage in the fish ovary.
Acknowledgements
This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada (to J.M.W.), from the Canadian Institutes of Health Research (to E. D.-W.), from the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, H?pital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, National Institute of Health (to C.T. and B.T.), and an Izaak Walton Killam Memorial Scholarship (to R.-Z.L). We thank Marc Ekker for the DNA from the LN54 radiation hybrid panel for linkage mapping. We also thank Mukesh Sharma, Violaine Alunni, Aline Lux, Vincent Heyer, and Agnes Degrave for their help during the experimental stages of this work.
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Correspondence: E-mail: jmwright@dal.ca.
Abstract
There are single copies of the genes encoding the cellular retinol-binding protein type I and II (CRBPI and CRBPII) in the human and rodent genomes. We have identified duplicate genes for both CRBPI and CRBPII in the zebrafish (Danio rerio) genome (rbp1b and rbp2b). The zebrafish rbp1b and rbp2b have conserved gene structures, amino acid sequence similarities, gene phylogenies, and syntenic relationships with their mammalian orthologs and zebrafish paralogs, rbp1a and rbp2a. Like the mammalian genes for CRBPI and CRBPII, the zebrafish rbp1b and rbp2b genes are closely linked on a single linkage group. Comparative analysis suggests that the duplicate genes of rbp1 and rbp2 in the zebrafish genome may have arisen by chromosomal or whole-genome duplication. During embryonic development, rbp1b transcripts were detected in the gall bladder of 5-day postfertilization (5 dpf) larvae. The rbp2b mRNA was abundant in the developing liver through 48 hours postfertilization (48 hpf) to 5 dpf. Using reverse transcription-polymerase chain reaction (RT-PCR), rbp1b transcripts were detected in the ovary, and rbp2b mRNA was observed predominantly in the adult liver. Tissue section in situ hybridization and emulsion autoradiography localized rbp1b mRNA to primary oocytes within the zebrafish ovary. The differential mRNA distribution patterns of the rbp1a, rbp1b, rbp2a, and rbp2b genes in the developing and adult zebrafish suggest that shuffling of subfunctions among duplicate copies of paralogous genes may be a mechanism for the retention of duplicated genes in vertebrates.
Key Words: gene duplication ? gene structure ? phylogeny ? genome evolution ? gene expression ? subfunctionalization
Introduction
Retinoids play crucial roles in organogenesis during embryonic development and, thereafter, in the maintenance of a wide range of biological processes, including vision, reproduction, growth, and immunity (Napoli 1996; Ross 2000). It has long been recognized that retinoids are essential micronutrients for normal reproductive function of animals (Thompson, Howell, and Pit 1964). Recent experiments show that retinoids may serve as important regulators in oogenesis and oocytes maturation in vertebrates (Livera et al. 2000; Morita and Tilly 1999; Whaley et al. 2000).
Retinoids are hydrophobic molecules, and their transport in the cytoplasm is thought to be facilitated by a group of intracellular carrier proteins, which specifically bind different isomeric retinoids and target them to various intracellular enzymes or receptors for retinoid metabolism and eventual actualization of their physiological activities. During the past decade, increasing evidence has suggested the involvement of intracellular retinoid-binding proteins in retinoid biological action and metabolism (reviewed by Napoli [1999], Noy [2000] and Adida and Spener 2002). 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 (reviewed by Ong, Newcomer, and Chytil [1994]. Glatz and Vusse [1996], and Bernlohr et al. [1997]). In mammals, two different types of cellular retinoid-binding proteins with distinct ligand-binding properties have been identified, the cellular retinol-binding proteins (CRBPs) and the cellular retinoic acid–binding proteins (CRABPs). Four types of CRBP, including CRBPI, CRBPII, CRBPIII and CRBPIV, are encoded by different genes in mammals (Bashor, Toft, and Chytil 1973; Ong, Newcomer, and Chytil 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002). Although ligand-binding affinity (Kd) differs among different CRBP types, all CRBPs have affinity for retinol and retinal but do not bind retinoic acid or retinyl esters (Ong 1984; Folli et al. 2001; Vogel et al. 2001; Folli et al. 2002).
To date, only a single copy of the gene coding for either CRBPI or CRBPII has been identified in mammalian genomes. In previous studies, the gene structure, cDNA sequence, linkage relationship, and expression of the genes coding for cellular retinol-binding protein type I (hereafter referred to as rbp1a) and type II (rbp2a) from zebrafish has been determined (Cameron et al. 2002; Liu et al. 2004). Here, we report the discovery of a pair of duplicate genes coding for CRBPI (rbp1b) and CRBPII (rbp2b) in the zebrafish genome. Phylogenetic analysis, conserved gene structure, and linkage relationship suggest that the zebrafish rbp1b and rbp2b are orthologs of the mammalian genes for CRBPI and CRBPII, respectively, and arose by genome-wide duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003). Comparative analysis of the mRNA distribution patterns for the gene duplicates of rbp1 and rbp2 in developing and adult zebrafish indicated that the closely related rbp paralogs have different subfunctions, and that "subfunction shuffling" among duplicates of paralogous genes might be an important mechanism for preservation of duplicated genes.
Materials and Methods
5' RNA Ligase Mediated–Rapid Amplification of cDNA Ends (5' RLM-RACE)
5' RLM-RACE was performed as previously described (Liu et al. 2003a) to amplify the 5' end of the zebrafish rbp1b and rbp2b transcripts and map their transcription start sites. Nested antisense primers used for the 5' RLM-RACE were designed within the coding sequence of the zebrafish rbp1b and rbp2b gene, respectively (as1 and as2 for rbp1b and as3 and as4 for rbp2b in figure 1). The 5' RACE products were cloned and sequenced.
FIG. 1.— Structures of the zebrafish rbp1b and rbp2b genes. Exons are shown in uppercase letters with the coding sequences of each exon underlined and the deduced amino acid sequence indicated below. Numbers on the right indicate nucleotide positions in the gene sequence. The initiation site for transcription is marked by a star and numbered as +1, and a putative polyadenylation signal is double underlined. A potential TATA box in the 5' upstream region of rbp2b is in bold and boxed. Nucleotide sequences corresponding to sense (s) and antisense (as) PCR primers and the tissue section in situ hybridization probe (p) used in this study are boxed and indicated. The position of a potential GAG insertion variant in exon 1 of rbp1b gene is marked by "". The zebrafish rbp1b and rbp2b gene sequences were identified from a zebrafish genomic DNA sequence assembly of clone CH211 to 11908 (GenBank accession number AL953896).
3' RACE
To obtain the complete cDNA sequence encoded by the zebrafish rbp1b and rbp2b genes, 3' RACE was employed (Liu et al. 2003b). The nested sense primer sequences used for 3' RACE are shown in figure 1 (s1 and s2 for rbp1b and s4 for rbp2b). Three clones of each gene were sequenced and the complete cDNA sequences of rbp1b and rbp2b genes were determined by aligning the sequences of the 5' RLM-RACE and 3' RACE cDNA clones.
Phylogenetic Analysis
Phylogenetic analysis of the zebrafish rbp1b, rbp2b, and the other fish and mammalian cellular retinoid-binding protein genes was performed with ClustalX (Thompson et al. 1997), and the bootstrap neighbor-joining phylogenetic tree was constructed using the zebrafish intestinal-type fatty acid–binding protein sequence (I-FABP) as an outgroup.
Radiation Hybrid Mapping
Radiation hybrids of the LN54 panel (Hukriede et al. 1999) were used to assign the rbp1b and rbp2b genes to a specific zebrafish linkage group. The sequences of the primers employed to amplify the genomic DNA from cell hybrids of LN54 panel are shown in figure 1 (s3 and as2 for rbp1b and s4 and as3 for rbp2b), and the PCR conditions were the same as previously described (Liu et al. 2003a).
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Conditions for RT-PCR used to determine the tissue-specific distribution of the rbp1b and rbp2b transcripts in adult zebrafish were the same as those previously described (Liu et al. 2003a). Gene-specific primers used for RT-PCR are shown in figure1 (s3 and as1 for rbp1b and s4 and as4 for rbp2b), and reaction products were size-fractionated by agarose gel electrophoresis. The tissue-specific mRNA distribution patterns of rbp1b and rbp2b were compared with that of rbp1a and rbp2a (Liu et al. 2004).
Tissue Section in situ Hybridization and Emulsion Autoradiography
Zebrafish tissue section in situ hybridization and emulsion autoradiography were performed as previously described (Liu et al. 2003b). The antisense oligonucleotide was complementary to the zebrafish rbp1b cDNA sequence (p in figure 1).
Whole-Mount in situ Hybridization to Embryos
To reveal the spatio-temporal distribution of the zebrafish rbp1b and rbp2b transcripts in developing zebrafish, whole-mount in situ hybridization was performed as described by C. Thisse and B. Thisse at the Web site: http://zfin.org/zf_info/zfbook/chapt9/9.82. Antisense RNA probes were prepared from the zebrafish rbp1b and rbp2b cDNA generated by 3' RACE.
Results
Structure of the Zebrafish rbp1b and rbp2b Genes
A zebrafish genomic DNA sequence record (GenBank accession number AL953896) was identified in GenBank by a tBlastN search for sequences corresponding to the published CRBPI and CRBPII amino acid sequence (Calderone et al. 2002; Cameron et al. 2002; Liu et al. 2004). The identified genomic DNA sequence contained two segments corresponding to the potential duplicated copies of the zebrafish rbp1 gene (rbp1b) and rbp2 gene (rbp2b), respectively. The distance between them is approximately 10 kb. The gene size and structure (fig. 1) of rbp1b and rbp2b were further defined after aligning their corresponding cDNA sequences (see below) with the genomic DNA sequences. The zebrafish rbp1b gene spanned 8,267 bp from the transcription start site (see below) to the polyadenylation site and consisted of four exons separated by three introns. The four exons of rbp1b were 159 bp, 179 bp, 102 bp, and 527 bp in length and coded for 22, 60, 34, and 16 amino acids, respectively (fig. 1). The three introns of the zebrafish rbp1b gene were 3,108 bp, 966 bp, and 3,226 bp in length, respectively, accounting for 89% of the whole-gene sequence. The zebrafish rbp2b gene was much smaller than rbp1b and spanned 1,262 bp of genomic DNA. The rbp2b gene also consisted of four exons (109 bp, 179 bp, 102 bp, and 185 bp) and three introns (140 bp, 365 bp, and 182 bp). The four exons of rbp2b coded for 24, 60, 34, and 17 amino acids, respectively. The nucleotides at the splice sites of each exon/intron boundary for both rbp1b and rbp2b conformed to the GT-AG rule (Breathnach and Chambon 1981).
The transcription start site of the zebrafish rbp1b gene was defined by 5' RLM-RACE, which generated a single product of the intact 5' cDNA end with the 7-methyl G cap. A negative control in which tobacco acid pyrophosphatase (TAP) treatment was omitted yielded no RACE product (data not shown). The 5' RLM-RACE product was cloned and sequenced, and the transcription start site was mapped to the nucleotide 92 bp upstream of the rbp1b ATG initiation codon (fig. 1). In the same way, the transcription start site of rbp2b was shown to be located 36 bp upstream of the rbp2b initiation codon (fig. 1).
Inspection of the 5' upstream sequence of the zebrafish rbp1b and rbp2b genes with MatInspector Professional version 6.2 (Quandt et al. 1995) revealed a TATA box at position –25 of the rbp2b gene sequence, whereas no TATA box was found within the proximal promoter region of rbp1b gene (fig. 1). A TATA box is present in the promoter of the zebrafish rbp2a gene and the mammalian rbp2 gene but not in the rbp1a gene of zebrafish or the single-copy rbp1 genes from other vertebrate species (Liu et al. 2004; Ong, Newcomer, and Chytil 1994).
Characterization of the Zebrafish rbp1b and rbp2b cDNAs
To further characterize the transcripts of the zebrafish rbp1b and rbp2b genes, we isolated their cDNAs. In addition to 5' RLM-RACE, we performed 3' RACE using gene-specific sense primers corresponding to a sequence in the 5' UTR (fig. 1). A single 3' RACE product of expected size was generated, cloned and sequenced for each gene. The complete nucleotide sequence of the zebrafish rbp1b cDNA (GenBank accession number AY395732), determined by combining the 5' RLM-RACE and 3' RACE sequences, was 971 nt in length, excluding the poly(A) tail. The rbp2b cDNA (GenBank accession number AY619686) was 575 nt, excluding the poly(A) sequence. Putative polyadenylation signals were identified 15 and 16 nt upstream of the poly(A) initiation site of the zebrafish rbp1b and rbp2b cDNA sequences, respectively. Alignment of the zebrafish rbp1b cDNA sequence with the gene sequence revealed a GAG insertion in the cDNA sequence at position 144 to 146. The extra GAG in the cDNA sequence resulted in an insertion of a glutamic acid in the CRBPIb amino acid sequence relative to the amino acid sequence deduced from the rbp1b genomic sequence. This discrepancy between the cDNA and genomic sequence is not likely to be a PCR artifact or a sequencing error but rather an intraspecies genetic variation, because independent clones from separate 3' RACE and 5' RLM-RACE reactions contained this extra GAG. The rbp1b gene sequence and an expressed sequence tag (EST) from GenBank (accession number BI886241) did not contain these three nucleotides. At the present time, we do not know whether this variation has any functional implications. Except for the difference of the GAG triplet, the rest of the coding sequence of the zebrafish rbp1b gene and the cloned cDNA was identical. The nucleotide sequence of the zebrafish rbp2b cDNA is identical to its genomic coding sequence.
The deduced amino acid sequence from the zebrafish rbp1b cDNA sequence consisted of 133 amino acids and the CRBPIb protein has a molecular mass of 15.2 kDa and theoretical isoelectric point of 4.94. Alignment of the zebrafish CRBPIb sequence with mammalian CRBPs and CRABPs showed highest sequence identity with that of the mammalian CRBPIs (74%) and the zebrafish CRBPIa (66%), followed by mammalian and zebrafish CRBPIIs (56%), human CRBPIII (54%), human CRBPIV (51%), mammalian CRABPIs (33%), and mammalian CRABPIIs (29% to 33%). The deduced amino acid sequence from the zebrafish rbp2b cDNA sequence consisted of 135 amino acids, with an estimated molecular mass of 15.6 kDa and a theoretical isoelectric point of 7.18. The deduced zebrafish CRBPIIb protein had sequence identity of 67% to 70% with mammalian CRBPIIs, 64% with the zebrafish CRBPIIa, 52% to 53% with mammalian CRBPIs, 53% with human CRBPIV, 50% with human CRBPIII, approximate 30% with mammalian CRABPIIs and approximately 27% with mammalian CRABPIs. The divergence of the amino acid sequences between the zebrafish CRBPIa and CRBPIb as well as between CRBPIIa and CRBPIIb is shown in figure 2.
FIG. 2.— Alignment of the deduced amino acid sequences of the zebrafish CRBPI and CRBPII duplicate gene pairs. (A) CRBPIa (GenBank accession number AAQ54326) aligned with CRBPIb (accession number AAR31829); (B) CRBPIIa (accession number AAL38648) aligned with CRBPIIb (accession number AAT40241). Dots indicate amino acid identity and dashes represent gaps.
Gene Phylogeny of the Zebrafish rbp1 and rbp2 Duplicates
Phylogenetic analysis was performed to reveal the evolutionary relationship among the newly identified zebrafish rbp1b, rbp2b, and other cellular retinoid-binding protein genes from mammals and fishes using their primary amino acid sequences (fig. 3). The zebrafish CRBPIb clustered with the mammalian CRBPIs in the same clade (bootstrap value 821/1000 [fig. 3]) and the zebrafish CRBPIa and CRBPIb formed a subclade from the mammalian CRBPIs. The zebrafish CRBPIIb clustered with the mammalian and fish CRBPII clade with a bootstrap value of 801/1000 (fig. 3). The results of phylogenetic analysis indicated that the genes for the zebrafish CRBPIb and CRBPIIb are orthologous to the genes for the mammalian CRBPI and CRBPII.
FIG. 3.— Phylogenetic tree of cellular retinoid-binding proteins. The bootstrap neighbor-joining phylogenetic tree was constructed with ClustalX (Thompson et al. 1997) using the zebrafish (Zf) intestinal type fatty acid–binding protein (I-FABP) amino acid sequence (GenBank accession number AAF00925) as an outgroup. The bootstrap values (based on number per 1,000 duplicates) are indicated on the nodes. Other amino acid sequences used in this analysis include zebrafish CRBPIa (accession number AAQ54326) and CRBPIb (accession number AAR31829), human (Hm) CRBPI (accession number P09455), mouse (Ms) CRBPI (accession number Q00915), rat (Rt) CRBPI (accession number P02696), zebrafish CRBPIIa (accession number AAL38648) and CRBPIIb (accession number AAT40241), human CRBPII (accession number P50120), mouse CRBPII (accession number Q08652), rat CRBPII (accession number P06768), human CRBPIII (accession number P82980), human CRBPIV (accession number AAN61071), human CRABPI (accession number P29762), mouse CRABPI (accession number P02695), human CRABPII (accession number P29373), mouse CRABPII (accession number P22935), and rat CRABPII (accession number P51673). Scale bar = 0.1 substitutions per site.
Linkage Mapping and Conserved Syntenies of the Zebrafish rbp1b and rbp2b Genes
The zebrafish rbp1b and rbp2b genes were both assigned by radiation hybrid mapping to linkage group 2 (LG 2) of the zebrafish genome. The zebrafish rbp1b was mapped to a locus on LG 2 at a distance of 5.23 cR from the marker Z211023, and rbp2b was located 6.19 cR from Z21490 [GenBank] (mapping data is available on request). The mapping distance between rbp1b and rbp2b on LG 2, estimated by their flanked markers, was approximately 6.7 cR. The close linkage relationship of the zebrafish rbp1b and rbp2b revealed by radiation hybrid mapping here is in agreement with their actual distance in the genomic sequence mentioned above (10 kb apart). Using the map of zebrafish LG 2 (Woods et al. 2000) and data from LocusLink (http://www.ncbi.nlm.nih.gov/locuslink), we found that the zebrafish rbp1b and rbp2b genes on LG 2, like rbp1a (Liu et al. 2004) and rbp2a (Cameron et al. 2002), have conserved synteny with the human RBP1 and RBP2 genes on chromosome 3 and mouse Rbp1 and Rbp2 genes on chromosome 9 (fig. 4)
FIG. 4.— Comparison of syntenic relationship of rbp1 and rbp2 genes from zebrafish, human, and mouse. Gene duplicates of the zebrafish rbp1and rbp2 genes have conserved syntenies (left box) with the human RBP1 and RBP2 genes on chromosome 3 (middle box) and the mouse Rbp1 and Rbp2 genes on chromosome 9 (right box). Orthologous rbp1 and rbp2 gene symbols are in bold. The order of the human syntenic genes on human chromosome 3 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 and mouse chromosomes and the last common ancestor's chromosome (lower box) are listed in the order appearing on the human chromosome 3. Hypothesized evolutionary events leading to the syntenic relationships of the zebrafish, human, and mouse rbp1 and rbp2 genes are indicated at the nodes of the evolutionary tree.
The Zebrafish rbp1b and rbp2b mRNA Distribution During Development
We examined the spatial and temporal distribution of the zebrafish rbp1b and rbp2b transcripts during embryonic and larval stages of development using whole-mount in situ hybridization with an antisense RNA probe synthesized from the zebrafish rbp1b and rbp2b cDNA clones (fig. 5). The zebrafish rbp1b mRNA was not detected in the early developing zebrafish until 5 dpf. In the 5 dpf larvae, rbp1b transcripts were restricted to the gall bladder (fig. 5A). Hybridization signals were not observed in any other embryonic or larval tissues. The zebrafish rbp2b mRNA was detected at 48 hpf stage in the liver and yolk syncytial layer (YSL [fig 5B, 1–2]). At 5 dpf stage, rbp2b transcripts were restricted to the liver (fig. 5B, 3–4)
FIG. 5.— Detection of zebrafish rbp1b and rbp2b mRNA during development by whole-mount in situ hybridization. (A) Whole-mount in situ hybridization of 5-day-old zebrafish larvae. rbp1b-specific antisense RNA probe detected rbp1b mRNA in the developing gall bladder (Gb) of the larvae at 5 dpf. (A, 1) Lateral view, head to the left. (A, 2) Cross section through the gall bladder. (A, 3) Sagittal section through the gall bladder. (A, 4) Dorsal view, head to the left. Ib, intestine bulb; Nc, notochord; Nt, neural tube. (B) Abundant rbp2b mRNA was observed in the liver (L) of 48 hpf (B, 1 and B, 2) and 5-dpf larvae (B, 3 and B, 4). The 48-hpf larval yolk syncytial layer (YSL) also showed lower levels of rbp2b mRNA distribution (B, 1 and B, 2). (B, 1 and B, 3) Lateral view, head to the left. (B, 2 and B, 4) Dorsal view, head to the left. Nonspecific staining was observed in the myotomes of 5-dpf larvae (B, 3).
Tissue Distribution of rbp1 and rbp2 Transcripts in Adult Zebrafish
The distribution of rbp1b and rbp2b mRNA in adult tissues was investigated by RT-PCR using gene-specific primers and compared with the patterns of their sister duplicate copies, rbp1a and rbp2a (fig. 6). The rbp1b-specific primers generated RT-PCR products of the expected size only from RNA of the ovary but not from RNA of all other tissues examined, including the liver, skin, intestine, brain, heart, muscle, and testis, whereas a relatively low level of rbp1a mRNA was detected in a number of tissues, including the liver, ovary, intestine, brain, and testis after 35 cycles of PCR amplification (fig. 6). Both rbp2a-specific and rbp2b-specific primers produced RT-PCR products in the liver and intestine, but the relative intensity of rbp2b product in the intestine was relatively low compared with that of rbp2a RT-PCR product (fig. 6). RT-PCR products were generated from RNA of all the tissues examined with specific primers corresponding to the cDNA sequence of the constitutively expressed receptor for activated C kinase (RACK1) gene (Hamilton and Wright 1999). No RT-PCR products were detected from the negative controls that lacked the reverse-transcribed cDNA templates for all genes assayed (fig. 6).
FIG. 6.— Tissue-specific distribution of the transcripts from rbp1 and rbp2 duplicates in adult zebrafish detected by RT-PCR. Tissue-specific RT-PCR products were generated from total RNA extracted from various adult zebrafish tissues (indicated on the top) using primers corresponding to the cDNA for CRBPIa, CRBPIb, CRBPIIa, and CRBPIIb. An RT-PCR product corresponding to the constitutively expressed receptor for activated C kinase (RACK1) was generated from RNA in all samples. A negative PCR control (–) did not contain cDNA template and did not generate any RT-PCR product.
RT-PCR analysis demonstrated that the expression of rbp1b was restricted to ovary. To determine the cell specificity of rbp1b gene expression in the ovary, we first performed in situ hybridization analysis of whole-zebrafish tissue sections using an antisense oligonucleotide probe corresponding to a nucleotide sequence of the 3' UTR of the zebrafish rbp1b cDNA (fig. 1). Strong punctuate hybridization signal was specifically detected in the zebrafish ovary but not in any other tissues (fig. 7A, 1) (data not shown). Nonspecific background was observed after hybridization to a sense probe throughout the tissue section (fig. 7A, 2). To identify the cells expressing rbp1b mRNA in the ovary, we conducted emulsion autoradiography of the in situ hybridization tissue sections (fig. 7B). Silver grains corresponding to rbp1b-specific hybridization were densely clustered over the primary (stage I) oocytes (fig. 7B, 1–3) but not in the stage II or III oocytes (fig. 7B, 2). The rbp1b-specific hybridization was not observed in any other tissues, including muscle (fig. 7B, 1 and 3) and liver (fig. 7B, 3) .
FIG. 7.— Specific distribution of zebrafish rbp1 mRNA in the primary oocytes detected by tissue section in situ hybridization and autoradiography. (A) In situ hybridization detected rbp1b mRNA in the ovary (arrow) on the tissue section hybridized with an rbp1b cDNA-specific antisense oligonucleotide probe (A, 1) but not with the sense probe (A, 2). (B) Bright and dark field showed the presence of rbp1b mRNA (indicated by silver grains) at the cellular level by emulsion autoradiography. Silver grains were observed in the stage I primary oocytes (arrows, B, 1–3), but not in stage II, stage III oocytes (B, 2), or the muscle (M) and liver (L).
Discussion
Previously, we reported the sequence and expression patterns of rbp1a and rbp2a (Cameron et al. 2002; Liu et al. 2004). Here, we have described the duplicated genes rbp1b and rbp2b encoding CRBPI and CRBPII from zebrafish and their spatiotemporal expression during development and in adulthood. It appears zebrafish, unlike mammals, has two rbp1 genes, rbp1a and rbp1b, and two rbp2 genes, rbp2a and rbp2b.
In humans, the RBP1 and RBP2 genes are closely linked and both genes have been physically mapped on human chromosome 3 at 3q23 (De Baere et al. 1998a, 1998b). By examining the human genomic DNA sequence, we found these two genes reside on the same contig sequence (GenBank accession number NT005832) and they are approximately 60 kb apart. In the zebrafish, we also identified a DNA assembly sequence from a single clone (GenBank accession number AL953896) that harbors both rbp1b and rbp2b genes separated by approximately 10 kb of DNA. As expected, these two genes were assigned to the same linkage group, LG 2, and the distance between them is 6.7 cR. The zebrafish rbp1a and rbp2a genes were assigned to LG 15 (Cameron et al. 2002) and LG 16 (Liu et al. 2004), respectively, and each of them has conserved syntenies with the human and mouse RBPI and RBPII genes. In the zebrafish genome, another pair of duplicate sister genes, atp1b3a and atp1b3b, also located on LG 2 and LG 15, syntenic with the zebrafish rbp2b and rbp2a gene, respectively. In humans and mouse, the orthologous gene for ATP1B3 (located at 3q22-q23 in humans and LG 9 51cM in mouse) is also closely linked to the RBPII gene (3q23 in humans and LG 9 57 cM in mouse) (Malik et al. 1998; Besirli, Gong, and Lomax 1998). This finding indicates that these two zebrafish rbp2 genes arose and were retained in the zebrafish genome after chromosomal duplication or whole-genome duplication in the euteleost lineage some 250 to 400 MYA (Van de Peer, Taylor, and Meyer 2003; Taylor et al. 2003). It is likely that after the chromosome or genome duplication in the fish lineage, the syntenic relationship of rbp1 and rbp2 genes in fishes was preserved on one of the duplicate chromosomes (rbp1b and rbp2b) but was not retained on the other (rbp1a and rbp2a), presumably because of translocation or chromosomal fission (fig. 4).
Currently, three theories have been proposed for the evolutionary fate of duplicated genes. First, the nonfunctionalization theory states that one of the gene duplicates becomes silenced because of degenerative mutations and becomes a pseudogene or is eventually lost from the genome. Second, the neofunctionalization theory states that one of the gene duplicates acquires a new and beneficial function, while the other duplicate retains the function of the ancestral gene, such that both genes are preserved. Third, a duplication-degeneration-complementation (DDC) model was recently proposed (Force et al. 1999, and references therein), which states that, in addition to the nonfunctionalization and neofunctionalization fate of duplicate genes, subfunctionalization might be an important mechanism for preservation of duplicated genes. After duplication, degenerative mutations in the cis regulatory elements results in partitioning of the original functions into each duplicate gene. As such, the ancestral function is divided between the duplicates, which functionally complement each other and, thus, the duplicate genes are retained in the host genome (Force et al. 1999).
The mammalian rbp1 genes are expressed in a number of adult tissues. In rats, CRBPI is abundantly distributed in the adult liver and kidney and, to a lesser extent, in the lung, testis, spleen, eye, ovary, uterus, and intestine (Ong, Newcomer, and Chytil 1994), whereas in humans, the ovary contains the highest amount of rbp1 gene transcripts and protein, but the levels of CRBPI are relatively low in human kidney and lung (Fex and Johannesson 1984; Ong and Page 1986; Folli et al. 2001). The relative levels of CRBPI in mammalian adult tissues, therefore, vary among species. Expression of the mammalian and the zebrafish rbp1 genes supports the hypothesis that the ancestral rbp1 gene was expressed in multiple adult tissues, including ovary, liver and in the early developing CNS, and retina. It is clear that the duplicate zebrafish rbp1 genes complement each other to retain some of the ancestral rbp1 functions, where rbp1b transcripts have a specialized distribution in the oocytes and rbp1a transcripts are expressed in the developing CNS and retina (Liu et al. 2004). One feature of the zebrafish rbp1 duplicated genes is that they were subfunctionalized both spatially and temporally, which is not stated by the DDC model of duplicate gene retention (Force et al. 1999). Temporal subfunctionalization might be a common feature for developmentally regulated gene duplicates. Thummel et al. (2004) have noted temporal subfunctionalization of the hoxc13a and hoxc13b genes in zebrafish, where hoxc13a is maternally expressed, and hoxc13b is expressed later in development. Neither rbp1a nor rbp1b is abundantly expressed in the adult liver and do not, therefore, complement each other to retain this ancestral subfunction after duplication. By contrast, both zebrafish rbp2a (Cameron et al. 2002, Liu et al. 2004) and rbp2b are expressed in the adult liver, despite the fact that none of the mammalian rbp2 genes studied to date are expressed in the liver. As such, our data suggests that the ancestral subfunction in the liver for rbp1 has been acquired by the duplicates of a closely related paralogous gene, rbp2, after duplication. We propose that "subfunction shuffling" between duplicates of paralogous genes might be a mechanism for preservation of the duplicates from unifunctional genes, such as the zebrafish rbp2 genes (fig. 8). At present, it is not possible to determine how cis-acting elements are shuffled. However, we speculate that mutation or unequal crossing-over between paralogs could result in the loss of cis-element function. Alternatively, a new subfunction to one of the duplicate gene pair may be acquired by "gain-of-function," such as transposon insertion of a new cis-acting element into the regulatory region of one of the duplicate genes .
FIG. 8.— A model for subfunction shuffling after gene duplication and subfunctionalization. Boxes numbered 1 to 4 denote cis regulatory elements controlling unique functions. Solid boxes denote functional cis elements, whereas open boxes denote null mutated cis elements. Arrows after cis elements denote transcription start sites of genes. Duplication of the ancestral gene (duplication 1) gives rise to a pair of paralogous genes, one multifunctional (paralog 1) and the other unifunctional (paralog 2) through subfunctionalization. After the second round of duplication (duplication 2), the subfunctions of paralog 1 and paralog 2 are shuffled, which results in the acquisition of subfunction 3 from duplicates of paralog 1 to a duplicate of paralog 2 (paralog 2b). Both duplicates from the unifunctional paralog 2 (paralog 2a and 2b) are then preserved.
Egg yolk from oviparous animals is one of the richest sources of vitamin A. In fish and other oviparous animals, retinol must be taken up from the serum by the rapidly growing oocytes and then stored and metabolized before functioning during embryonic development. Retinoids have been detected in high levels in unfertilized zebrafish eggs and during embryogenesis (Costaridis et al. 1996). Holo-CRBPI (retinol-CRBPI complex) is the substrate for lecithin:retinol acyltransferase (LRAT), an enzyme catalyzing the esterification of retinol with long-chain fatty acids to form retinyl ester for storage, whereas Apo-CRBPI strongly inhibits the esterification reaction (Herr and Ong 1992). Because of the significant and distinct features of retinoid mobilization and metabolism in the fish ovary, it is reasonable to speculate that rbp1b has evolved and subfunctionalized to play a particular role in retinol uptake, metabolism, and storage in the fish ovary.
Acknowledgements
This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada (to J.M.W.), from the Canadian Institutes of Health Research (to E. D.-W.), from the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, H?pital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, National Institute of Health (to C.T. and B.T.), and an Izaak Walton Killam Memorial Scholarship (to R.-Z.L). We thank Marc Ekker for the DNA from the LN54 radiation hybrid panel for linkage mapping. We also thank Mukesh Sharma, Violaine Alunni, Aline Lux, Vincent Heyer, and Agnes Degrave for their help during the experimental stages of this work.
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