Báo cáo khoa học: Cellular retinol-binding protein type II (CRBPII) in adult zebrafish (Danio rerio) cDNA sequence, tissue-specific expression and gene linkage analysis pptx

Cellular retinol-binding protein type II (CRBPII) in adult zebrafish ( Danio rerio ) cDNA sequence, tissue-specific expression and gene linkage analysis Marianne C. Cameron 1 , Eileen M. Denovan-Wright 2 , Mukesh K. Sharma 1 and Jonathan M. Wright 1 1 Department of Biology, and 2 Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada We have determined the nucleotide sequence of a zebrafish cDNA clone that codes for a cellular retinol-binding protein type II (CRBPII). Radiation hybrid mapping revealed that the zebrafish and human CRBPII genes are located in syntenic groups. In situ hybridization and emulsion autora- diography localized the CRBPII mRNA to the intestine and the liver of adult zebrafish. CRBPII and intestinal fatty acid binding protein (I-FABP) mRNA was colocalized to the same regions along the anterior-posterior gradient of the zebrafish intestine. Similarly, CRBPII and I-FABP mRNA are colocalized in mammalian and chicken intestine. CRBPII mRNA, but not I-FABP mRNA, was detected in adult zebrafish liver which is in contrast to mammals where liver CRBPII mRNA levels are high during development but rapidly decrease to very low or undetectable levels following birth. CRBPII and I-FABP gene expression appears there- fore to be co-ordinately regulated in the zebrafish intestine as has been suggested for mammals and chicken, but CRBPII gene expression is markedly different in the liver of adult zebrafish compared to the livers of mammals. As such, retinol metabolism in zebrafish may differ from that of mammals and require continued production of CRBPII in adult liver. The primary sequence of the coding regions of fish and mammalian CRBPII genes, their relative chromo- somal location in syntenic groups and possibly portions of the control regions involved in regulation of CRBPII gene expression in the intestine appear therefore to have been conserved for more than 400 million years. Keywords: Danio rerio; fatty acid binding protein; cellular retinol binding protein; tissue-specific expression; retinol metabolism. Cellular retinol-binding proteins (CRBPs) are members of the intracellular lipid-binding protein family which includes the retinoic acid (CRABP) and fatty acid (FABP) binding proteins. This family consists of low molecular mass ( 14– 16 kDa) polypeptides that bind and transport retinoids, fatty acids, and bile salts [1,2]. Members of this protein superfamily have a common three dimensional shape described as a clamshell structure composed of two orthogonal b-sheets, each consisting of five antiparallel b-strands and two a-helices [3]. Hydrophobic ligands are held in the central cavity of the bivalve-like polypeptide. The three CRBPs, type I, II, and III, are named according to the order in which they were discovered in mammals. Their putative role in cell physiology is in the metabolism of retinol (vitamin A). Retinol and its derivates are important for vision, reproduction, metabolism, cellular differentiation and pattern formation during embryogenesis [4]. After absorption in the mammalian intestine, the enzyme b-carotene dioxygenase catalyzes the oxidative cleavage of b-carotene to retinal. Retinal is reduced to retinol by the enzyme retinal dehydrogenase. Retinol is then esterified by the microsomal enzyme lecithin:retinal acyltransferase (LRAT) to retinoic acid and packaged into chylomicrons for subsequent uptake by the liver. CRBPI and II bind retinal and retinol whereas CRBPIII binds retinol, but not retinal [4–7]. CRABPs bind and transport retinoic acid [4,5,7]. The CRBPs are thought to participate in ligand binding and regulate the metabolism of retinal and retinol while protecting the CRBP-bound ligands from nonspecific reactions [8]. Biochemical studies have shown that CRBPII- bound retinol serves as a substrate for the enzyme, LRAT, implicating CRBPII as a component in directing and channeling dietary retinol to nascent chylomicroms. Direct evidence for the role of CRBPI in vitamin A metabolism has been provided by transgenic knockout studies which demonstrated that CRBPI knockout mice are phenotypi- cally normal when fed a vitamin A-enriched diet, but when the diet is deficient in vitamin A, stores of retinyl esters are depleted over 5 months and the mice develop abnormalities consistent with postnatal hypovitaminosis A [9]. To date, there are no reports of CRBPII and CRBPIII gene knockouts and therefore direct evidence for their function in vitamin A metabolism remains speculative. CRBPII is restricted to the small intestine of adult rat [10], human [11], chicken [12,13] and pig [14]. Studies have shown that the small intestine has the highest levels of CRBPII mRNA in Correspondence to J. M. Wright, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H. Fax: + 902 494 3736, Tel.: + 902 494 6468, E-mail: jmwright@dal.ca Abbreviations: FABP, fatty acid binding protein; cDNA, comple- mentary DNA; CRBP, cellular retinol-binding protein; EST, expressed sequence tag; LG, linkage group; LRAT, lecithin:retinal acyltransferase; rbp2, zebrafish retinol binding protein 2 gene; RBP2, human retinol binding protein2 cellular gene. Note: web site available at http://is.dal.ca/biology2/index.html (Received 8 April 2002, revised 5 July 2002, accepted 5 August 2002) Eur. J. Biochem. 269, 4685–4692 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03170.x adult rats, with undetectable or low levels of CRBPII mRNA in other tissues [4,5]. Presumably, CRBPII is directly involved in the intestinal uptake and binding of retinol based on calculated rates of retinol uptake in a human intestinal cell culture model [15]. Members of the intracellular lipid-binding protein super- family are derived from at least 14 gene duplications [16]. Prior to the vertebrate/invertebrate split, the liver/intestinal/ ileal FABP and heart/adipose/mylein FABP clades diverged approximately 700 million years ago. It has been suggested that the CRBP genes diverged from the liver/intestinal/ileal FABP clade about 500 million years ago. The mammalian CRBPI and CRBPII genes presumably arose by gene duplication sometime after the divergence of amphibia from mammals as a single copy of the CRBP gene is found in Xenopus [17]. An alternative explaination, however, is that one of the duplicated copies of the CRBP gene may have been lost in the amphibian lineage. The structure and function of fatty acid and retinoid- binding proteins have been studied extensively in mammals, but only superficially in other taxa such as the teleost fishes. Vitamin A and its derivates are clearly important mediators of normal vertebrate development [4,8,9]. As zebrafish is promoted as a model experimental system for study of vertebrate development, an understanding of the function of CRBPs in vitamin A metabolism during zebrafish embryo- genesis would be of interest to developmental biologists. Moreover, comparative studies of CRBP gene expression in fishes and mammals may provide insight into the role(s) of these intracellular retinol- and retinal-binding proteins in vitamin A metabolism. As part of ongoing studies in our laboratories on the evolution, tissue-specific expression and gene regulation of members of the intracellular lipid-binding protein family in zebrafish [18–20], we have determined the nucleotide sequence of a cDNA clone and deduced the amino-acid sequence for a zebrafish CRBPII. Furthermore, we report the tissue-specific distribution of the CRBPII mRNA in adult zebrafish and assignment of the CRBPII gene to linkage group 15 in the zebrafish genome. MATERIALS AND METHODS Searches of the zebrafish EST database in GenBank identified a cDNA clone (GenBank accession number AI544932) that was similar to the 5¢ end of the rat cellular retinol-binding protein type II. This clone (fb69e02.y1) was purchased from Incyte Genomics Inc. and the complete nucleotide sequence was determined [18]. The deduced amino-acid sequence of the cDNA sequence was aligned with other intracellular lipid-binding protein sequences in GenBank using CLUSTALW [21] and an output of percentage sequence identity generated. DNA from the LN54 radiation hybrid panel [22] (zebrafish DNA in a mouse background) was used as template in PCR reactions to assign the linkage group for the CRBPII gene in the zebrafish genome (see Fig. 1 for primer location). PCR reactions contained 1X PCR buffer (MBI Fermentas), 1.5 m M MgCl 2 ,0.4l M sense primer (5¢-TTCGCCACCCGTAAGATC-3¢), 0.4 l M antisense primer (5¢-AAACTCCTCTCCAATGACG-3¢), 0.2 m M Fig. 1. Nucleotide sequence of a cDNA clone coding for a zebrafish CRBPII. The complete nucleotide sequence of the EST clone fb69e02.y1 was determined (GenBank accession number AF363957). The 549 bp sequence contained an open-reading frame of 405 nucleotides coding for a protein of 135 amino acids. The predicted amino-acid sequence of the zebrafish cDNA clone was most similar to mammalian CRBPIIs (see Fig. 2). The sequence complementary to the antisense oligonucleotide probe used for in situ hybridization analysis is underlined and in bold font. The position of the nucleotides corresponding to the 5¢ or complimentary to the 3¢ primers used for PCR amplification of the CRBPII cDNA probe used for Northern blot and radiation hybrid linkage mapping analysis are boxed and numbered Ô1Õ and Ô2Õ, respectively. The polyadenylation signal sequence, AATAAA, is italicized and in bold font. 4686 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002 dNTPs, 1.25 U Taq DNA polymerase and 100 ng of hybrid cell DNA. Control reactions contained 100 ng of either zebrafish or mouse parental cell line DNA or a 1 : 10 mixture of zebrafish and mouse parental cell line DNA. PCR conditions were 94 °C for 4 min followed by 32 cycles of 94 °Cfor30s,60°Cfor30s,72°C for 30 s and a final extension at 72 °C for 7 min Products were separated by agarose gel electrophoresis and the radiation hybrid panel was scored and then analyzed according to the directions at (http://mgcdh1.nichd.nih.gov:8000/zfrh/beta.cgi). PCR primers (5¢-CCAGCACATCCAGCTTC-3¢)and (5¢-GCCTGTTTGGAGCATTAG-3¢) (see Fig. 1 for pri- mer location) were used to amplify a 442-bp product from DNA of clone fb69e02.y1. This product was used as a hybridization probe for Northern blot analysis [23]. The size of the hybridizing mRNA was determined by comparing its electrophoretic mobility with molecular mass markers (0.24–9.5 kB RNA ladder, Gibco BRL). In situ hybridization was performed using an antisense oligonucleotide probe (see Fig. 1) to determine the pattern of CRBPII expression in adult zebrafish. Based on BLASTN searches of GenBank, the in situ hybridization probe did not exhibit significant sequence similarity to any other DNA sequence currently available in GenBank. Fourteen micrometer transverse, sagittal, and coronal sections of adult zebrafish were hybridized to DNA probes using previously described methods [24]. Following hybridization and post-hybridization washes, the sections were exposed to autoradiographic film. Emulsion autoradiography of the tissue sections that hybridized to the CRBPII antisense probe was performed to localize the in situ hybridization signal to the cellular level [24]. A hybridization probe corresponding to the sense strand of a portion of a zebrafish I-FABP mRNA, shown previously not to hybridize to any transcript in total zebrafish RNA [18], was used as a negative control for in situ hybridization studies. A probe complementaty to the coding strand of the zebrafish I-FABP mRNA [18] was used as a positive control for in situ hybridization and emulsion autoradio- graphy. Following emulsion autoradiography, the sections were stained with cresyl violet and viewed under bright- field and dark-field illumination [24]. RESULTS AND DISCUSSION The nucleotide sequence of a zebrafish EST clone reported in GenBank to have sequence similarity to the 5¢ end of CRBPII cDNAs from mammals and chicken was deter- mined (Fig. 1). Sequence from forward and reverse sequen- cing reactions was aligned and discrepancies were resolved by examination of the primary sequence data. The complete sequence (GenBank accession number AF363957), deter- mined from both strands, differed from that of the partial sequence of the EST clone reported in GenBank (accession number AI544932) at several positions. The cDNA sequence contained an open-reading frame of 405 bp encoding a protein of 135 amino acids. The percentage amino-acid sequence similarity between the open-reading encoded by the cDNA clone and the amino-acid sequences of intracellular lipid-binding proteins from zebrafish and other species indicate that the cDNA clone codes for the zebrafish CRBPII (Fig. 2). The zebrafish CRBPII protein was one amino acid longer than mammalian CRBPII and equal in length to chicken CRBPII. The molecular mass of the CRBPII protein in zebrafish, based on the predicted amino-acid sequence, is 15.8 kDa. The molecular mass of this zebrafish CRBPII is comparable to other members of the intracellular lipid-binding protein family which are all between 14 and 16 kDa [1,2]. The zebrafish CRBPII amino- acid sequence is most similar to the chicken CRBPII (76% identity). The zebrafish CRBPII amino-acid sequence exhibits 73–75% sequence similarity to mammalian CRBPIIs and less than 40% amino-acid sequence identity to other intracellular lipid-binding proteins. Cheng et al. [25] proposed that Arg106 and Arg126 present in some members of the lipid-binding protein family correspond to Gln109 and Gln129 in CRBPI and CRBPII. While all FABPs and CRBPs studied to date have the same tertiary structure, the amino-acid residues at positions 109 and 129 may determine ligand-binding specificity. Both Gln residues are found in the zebrafish CRBPII sequence at the comparable positions within the amino-acid alignment of other intracellular lipid-binding proteins (Fig. 2). Gln109 is not strictly conserved in all CRBPs, however, as the chicken CRBPII and mouse CRBPIII have a histidine residue at this position. Phylogenetic analyses of 51 intracellular lipid-binding proteins, from vertebrates and invertebrates, indicate that at least 14 gene duplications have occurred during the evolution of this multigene family [16]. As the amino-acid sequence of the zebrafish CRBPII reported here is most similar to CRBPIIs from other species, and not to CRBPI or other intracellular lipid-binding proteins from zebrafish (Fig. 2), the duplication of the ancestral CRBP gene that gave rise to the CRBPI and CRBPII genes most likely occurred before the divergence of the teleost fishes and mammals, approximately 400 mya. We have shown that the CRBPI gene exists in the zebrafish genome (M.K. Sharma & J.M. Wright, unpublished data). The mammalian CRBPI and CRBPII genes are linked on chromosome 9 in mouse and 3 in humans and share sequence similarity including the conserved Gln residues at positions 109 and 129 [25,26]. Radiation hybrid mapping studies [22] assigned the CRBPII gene to linkage group 15 (LOD score 19.8) in the zebrafish genome. (Primary data and the RH vector for linkage analysis is available upon request to the corresponding author). The CRBPII gene is flanked by the growth associated protein 43 (GAP 43) gene on one side and the chordin (CHRD) gene on the other in both zebrafish and in human (Table 1). This synteny suggests that a common linkage group was inherited from the ancestor of fishes and mammals. In mouse, however, the synteny has not been maintained as CRBPII is located on chromosome 9, while GAP 43 and CHRD are located on chromosome 16 (Table 1). This suggests a translocation/rearrangement of this region of the mouse genome after the divergence of fishes and mammals. The conservation of amino-acid sequence among all CRBPIIs and the evidence that zebrafish and human CRBPII genes are in the same syntenic group suggest that fish and mammals share a common ancestral CRBPII gene. Northern blot-hybridization of the zebrafish CRBPII cDNA to total RNA extracted from whole adult zebrafish detected a single mRNA transcript of  720 nucleotides (Fig. 3A). The difference in size between the mRNA transcript detected by Northern blot ( 720 nucleotides) Ó FEBS 2002 Expression of CRBPII in zebrafish (Eur. J. Biochem. 269) 4687 and the size of the cDNA sequence (549 nucleotides) suggests that the cDNA clone is probably lacking the complete poly A tail or part of the 5¢ untranslated region, or both. In situ hybridization analysis of adult zebrafish tissue sections revealed that the hybridization signal resulting from the specific annealing of the CRBPII antisense probe was confined to the intestine and, at relatively lower levels, to the zebrafish liver (Fig. 3B,C). Hybridization of the CRBPII- specific probe to the intestine is most evident in the transverse (Fig. 3B) and coronal (Fig. 3C) sections while hybridization to the liver is more clearly seen in the coronal sections (Fig. 3C). The radiolabel associated with the layer beneath the external skin appears to be non–specific interaction of the probe with this tissue as it is seen in all autoradiograms regardless of the hybridization probe employed, i.e. the CRBPII or I-FABP antisense probes or the I-FABP negative control sense probe (Figs 3B,C). The hybridization signal resulting from the specific annealing of the I-FABP antisense probe was confined to the intestine as previously reported [18]. As CRBPII and I-FABP mRNA have been colocalized in the mammalian and chicken proximal portion of the small intestine [27–29], we examined the distribution of CRBPII and I-FABP mRNA in adjacent tissue sections of adult zebrafish. Emulsion autoradiography of tissue sections that hybridized to the CRBPII and I-FABP antisense and negative control I-FABP sense probes was performed to localize the hybridization signal at the cellular level. The CRBPII mRNA was localized to the enterocytes in the microvilli of the intestine and to the hepatocytes of the liver (Fig. 4A,B). The I-FABP mRNA was similarly localized to the enterocytes of the intestine, but was not detected in the Fig. 2. Amino-acid sequence alignment of zebrafish CRBPII with other CRBPs, CRABPs, and FABPs. The amino-acid sequences of zebrafish CRBPII (ZbfshCRBPII; GenBank Accession number AF363957), chicken CRBPII (ChickCRBPII [43]), pig CRBPII (PigCRBPII; P50121), human CRBPII (HumanCRBPII; AAC50162), rat CRBPII (RatCRBPII; P06768), mouse CRBPIII (MouseCRBPIII; AA466092), rat CRBPI (RatCRBPI; P02696), rat CRABPII (RatCRABPII; P51673), human CRABPI (HumanCRABPI; P29762), zebrafish brain FABP (ZbfBFABP; Af237712), and zebrafish intestinal FABP (ZbfIFABP; AF180921) were aligned using CLUSTALW [21]. Dashes, indicating addition/deletion dif- ferences between the zebrafish CRBPII and other amino-acid sequences, were added to maximize alignment. Dots indicate identity between the amino-acid sequence of zebrafish CRBPII and other CRBPs, CRABPs and FABPs. The percentage amino-acid sequence similarity relative to the zebrafish CRBPII is indicated at the end of each sequence. Table 1. Zebrafish-human conserved syntenies. Zebrafish a Human b Mouse b LG Locus Accession Number Gene Chromosomal Position Gene Chromosomal location 15 gap43 L27645 GAP43 3q13.1-q13.2 Gap43 16 29.5 cM 15 rbp2 (CRBPII) AF363957 RBP2 3q23 Rbp2 9 57.0 cM 15 chd AF034606 CHRD 3q27 Chrd 16 14.0 cM a Wood et al. [45]; b LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi), NCBI. 4688 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002 liver (Fig. 4A,B) [18]. In the transverse sections, the positional difference of CRBPII mRNA demarcates the anterior and posterior parts of the intestine (Fig. 4A). In adjacent sections, the distribution of I-FABP-specific hybridization signal was the same as that observed for CRBPII (Fig. 4A). Due to the entwined positioning of the intestine within the zebrafish, the coronal sections display three cross-sections corresponding to different regions of the intestine (Fig. 4B). There was hybridization to only two of the three cross sections of the intestine for the CRBPII antisense and I-FABP antisense probes. The radiolabel associated with the centre of the intestine is present in all sections labeled with antisense and sense oligonucleotides indicating nonspecific binding of the probes to the contents of the gut. Evidence of I-FABP mRNA restricted to just the anterior of the zebrafish intestine was not previously observed by us possibly owing to the limited sections assayed [18]. It is possible that subtle differences in the amount of CRBPII and I-FABP mRNA exist along the anterior-posterior of the intestine that are not detectable using in situ hybridization and emulsion autoradiography. Moreover, using whole-mount in situ hybridization to zebrafish embryos, it was previously determined that I- FABP mRNA is first expressed in the intestinal tube 3 days postfertilization and, by 5 days postfertilization and on- ward, I-FABP mRNA is abundant in the anterior intestine but is not detectable in the posterior intestine [27]. In adult mammals, CRBPII mRNA levels gradually decrease along the anterior-posterior axis of the intestine [28,29]. The proximal intestine of mammals has a higher capacity to absorb retinol than does the distal portion [30,31]. Levin et al. [15] demonstrated that CRBPs are directly involved in retinol absorption in a human intestinal cell line and that the amount of CRBPI and CRBPII mRNA and protein is directly related to the rate of retinol absorption. It is believed that CRBPII directs retinal and retinol to the enzymes, retinal dehydrogenase and LRAT, respectively, in the intestine. CRBPII, microsomal retinal reductase and LRAT are colocalized in the mammalian intestine [32–34]. The CRBPII gradient in the intestine parallels the change in enzyme activity of LRAT and retinal reductase which is greater in the anterior than in the posterior of the intestine [34]. Thus, these findings are consistent with the proposed role of CRBPII in retinol metabolism. Similarly, previous studies in mammals have shown that there is a gradient of I-FABP expression along the horizontal axis as concentrations of I-FABP mRNA and proteins gradually decrease from high levels in the jejunum to negligible levels in the colon [35–38]. CRBPII and I-FABP expression is similar along the anterior- posterior axis in the intestine of mammals. Therefore, a corresponding trend for CRBPII and I-FABP expression in zebrafish is consistent with their expression in mammals. The abrupt termination of CRBPII expression along the anterior-posterior axis of the zebrafish intestine, however, contrasts with the gradual decrease in CRBPII and I-FABP expression pattern in mammals. In the 5¢-control regions of the mammalian CRBPII [39] and I-FABP [35] genes, a closely related cis-element that consists of nearly perfect tandem repeats, termed retinoid x response element (RXRE) [40] has been found. It is conceivable therefore that these two genes may both be regulated by the action of retinoid x receptor (RXR) binding to RXRE [41]. The similar distribution of CRBPII and I-FABP mRNA in the zebrafish intestine reported here may reflect the co-ordinate regulation of these genes by common intestinal transcriptional factors in zebrafish. In addition to being abundant in intestine, CRBPII is found in neonatal liver hepatocytes of the rat and chick [10,42]. In rat, however, the levels of CRBPII mRNA in the Fig. 3. CRBPII mRNA expression in the adult zebrafish. The complete coding sequence of the zebrafish CRBPII cDNA clone was amplified by PCR and used as a hybridization probe in Northern blot analysis of total cellular RNA isolated from adult zebrafish (A). The zebrafish CRBPII-specific probe hybridized to a transcript of approximately 720 nucleotides. The1.35 kb (upper line) and 0.24 kb (lower line) RNA molecular mass markers are shown on the left of the panel. In situ hybridization analysis was performed using a 3¢ end-labelled oligo- nucleotide complementary to an internal portion of the zebrafish CRBPII coding region (see Fig. 1). In situ hybridization of adjacent transverse (B) and coronal (C) sections of adult zebrafish to the the zebrafish CRBPII-specific and I-FABP-specific antisense probes are shown. An oligonucleotide corresponding to the sense strand of the I-FABP coding region was included as a negative control. This probe was previously shown to bind nonspecifically to adult zebrafish sec- tions in in situ hybridization analysis [18]. Labelled arrows point to the intestine (I) and liver (L) of the zebrafish. The hybridization signal resulting from the annealing of the CRBPII- and I-FABP-specific probes are intense in the intestine. A lower intensity of hybridization of the CRBPII-, but not the I-FABP-specific probes, was seen in the zebrafish liver. Ó FEBS 2002 Expression of CRBPII in zebrafish (Eur. J. Biochem. 269) 4689 liver decrease after birth and are undetectable in the adult liver [10]. In adult chicken, CRBPII mRNA is abundant in the intestine but is not detected in liver [43]. As stated above, it is thought that CRBPII directs retinal to the enzyme retinal dehydrogenase in the mammalian perinatal liver [42]. Takase et al. [42] have suggested that following birth, the absorptive cells of the intestine may be functionally immature and unable to convert b-carotene to retinal. b-carotene is transferred to the liver in neonatal mammals and converted to retinal by the enzyme b-carotene dioxyg- enase in hepatocytes. Later, the intestine matures and can convert b-carotene to retinal. High levels of hepatic CRBPII are then no longer necessary for the production of retinol and CRBPII levels decrease in adult liver. Chicken and rat are known to convert most ingested b-carotene to retinal and then to retinol in enterocytes such that lower levels of b-carotene and retinal are found in their circulation compared to the levels observed in humans [44]. These findings suggest that hepatic CRBPII may play a role in metabolizing hepatic b-carotene to retinal and the subse- quent esterification of the converted retinol only during the perinatal period in mammals [42]. The in situ hybridization and autoradiographic emulsion studies show that CRBPII mRNA is abundant in the liver of adult zebrafish (Fig. 4). This pattern of CRBPII expression therefore differs mark- edly from that observed in rat and chicken [10,42]. Retinol metabolism of fishes may differ from that of mammals and chicken in that large amounts of b-carotene continue to be transported to the adult liver of teleost fishes resulting in the need for high levels of CRBPII mRNA observed in the liver of adult zebrafish. CRBPII and I-FABP mRNA are colocalized in the fish and mammalian intestine and may be co-ordinately regu- lated by RXR acting at RXRE within the control regions of these genes. The differential expression of CRBPII and I-FABP in the adult zebrafish liver, however, suggests that other transcription factors may regulate CRBPII gene expression in the livers of adult zebrafish. In summary, the zebrafish CRBPII cDNA reported here has sequence similarity to CRBPs isolated from mammals. The patterns of gene expression for CRBPII and I-FABP in fishes and mammals suggest that there is co-ordinate regulation of these genes in the intestine, but not in the liver. This may reflect differences in retinol metabolism between adult teleost fishes and mammals. ACKNOWLEDGEMENTS We wish to thank Dr Marc Ekker for providing DNA samples from the LN54 collection of radiation hybrids. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to J. M. W. and a grant from the Canadian Institutes of Health Research to E. M. D-W. M. C. C. was the recipient of a Natural Sciences and Engineering Research Council Undergraduate Student Research Award and M. K. S. was the recipient of an Izaak Walton Killiam Memorial scholarship. REFERENCES 1. Banaszak, L., Winter, N., Xu, Z., Bernlohr, D.A., Cowan, S. & Jones, T.A. (1994) Lipid-binding proteins: a family of fatty acid and retinoid transport proteins. In Advances in Protein Chemistry Fig. 4. Autoradiograhpic emulsion of zebrafish sections hybridized to the CRBPII- and I-FABP-specific antisense and negative control I-FABP sense probes. Transverse (A) and coronal (B) zebrafish sections were hybridized to the I-FABP, CRBPII and negative control probes and directly exposed to autoradio- graphic emulsion. Following development of the emulsion, the sections were counterstained with cresyl violet and viewed under brightfield (BF) and darkfield (DF) illumination at 10· magnification. The intestine (I), liver (L) and pancreas (P) are indicated. The white granules viewed under darkfield illumination corres- ponded to the location of either the CRBPII or I-FABP mRNA as indicated. 4690 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Anfinsen, C.B., Edsall, J.T., Richards, F.M. & Eisenberg, D.S., eds), Vol. 45, pp. 89–151. Academic Press, San Diego. 2. Veerkamp, J.H., van Kuppevelt, T.H.M.S.M., Maatman, R.G.H.J. & Prinsen, C.F.M. (1993) Structural and functional aspects of cytoplasmic fatty-acid binding proteins. Prostaglandins Leukot. Essent. Fatty Acids 49, 887–906. 3. Sacchettini, J.C., Gordon, J.I. & Banaszak, L.J. (1989) Crystal structure of rat intestinal fatty acid-binding protein. J. Mol. Biol. 208, 327–339. 4. Sporn, M.B., Roberts, A.B. & Goodman, D.S. (1994) The Retinoids: Biology, Chemistry and Medicine, 2nd edn. Raven Press, New York, New York. 5. Li, E., Demmer, L.A., Sweetser, D.A., Ong, D.E. & Gordon, J.I. (1986) Rat cellular retinol-binding protein type II: Use of a cloned cDNA to define its primary structure, tissue-specific expression, and developmental regulation. Proc. Natl Acad. Sci. USA 83, 5779–5783. 6. MacDonald, P.N. & Ong, D.E. (1987) Binding specificites of cellular retinal-binding protein and cellular retinol-binding pro- tein, type II. J. Biol. Chem. 262, 10550–10556. 7. Vogel, S., Mendelsohn, C.L., Mertz, J.R., Piantedosi, R., Wald- burger, C., Gottesman, M.E. & Blaner, W.S. (2001) Character- ization of a new member of the fatty acid-binding protein family that binds all-trans retinol. J. Biol. Chem. 276, 1353–1360. 8. Troen,G.,Eskild,W.,Fromm,S.H.,DeLuca,L.M.,Ong,D.E., Wardlaw, S.A., Reppe, S. & Blomhoff, R. (1999) Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retionol-binding protein type I are not altered phenotypically. J. Nutr. 129, 1621–1627. 9. Ghyselinck, N.B., Bavik, C., Sapin, V., Mark, M., Bonnier, D., Hindelang,C.,Dierich,A.,Nilsson,C.B.,Hakansson,H.,Sau- vant, P., Azais-Braesco, V., Frasson, M., Picaud, S. & Chambon, P. (1999) Cellular retinol-binding protein I is essential for vitamin Ahomeostasis.EMBO J. 18, 4903–4914. 10. Ong, D.E. (1984) A novel retinol-binding protein from rat. J. Biol. Chem. 259, 1476–1482. 11. Ong, D.E. & Page, D.L. (1987) Cellular retinol-binding protein (type two) is abundant in human small intestine. J. Lipid Res. 28, 739–745. 12. Finlay, J.A. & DeLuca, H.F. (1988) Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D 3 -modulated protein from embryonic chick intestine. Biochemistry 27, 3381–3387. 13. Goda, T. & Takase, S. (1989) Purification, properties, and devel- opmental changes of cellular retinol-binding protein, type II, in chicken intestine. J. Nutr. Sci. Vitaminol. 35, 545–557. 14. Perozzi, G., Barlia, D., Murgia, C., Kelly, D., Begbie, R. & King, T.P. (1993) Expression of differentiated functions in the develop- ing porcine small intestine. J. Nutr. Biochem. 4, 669–705. 15. Leven, M.S. (1993) Cellular retinol-binding proteins are determi- nants of retinol uptake and metabolism in stably transfected caco- 2cells.J. Biol. Chem. 268, 8267–8276. 16. Schleicher, C.H., Cordoba, O.L., Santome, J.A. & Dell’Angelica, E.C. (1995) Molecular evolution of the multigene family of intracellular lipid-binding proteins. Biochem. Mol. Biol. Int. 36, 1117–1125. 17. Matarese, V., Stone, R.L., Waggoner, D.W. & Bernlohr, D.A. (1989) Intracellular fatty acid trafficking and the role of cytosolic lipid binding proteins. Prog. Lipid Res. 28, 245–272. 18. Pierce, M., Wang, Y., Denovan-Wright, E.M. & Wright, J.M. (2000) Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio). Biochem. Biophys. Acta. 1490, 175–183. 19. Denovan-Wright, E.M., Pierce, M., Sharma, M.K. & Wright, J.M. (2000) cDNA sequence and tissue-specific expression of a basic liver-type fatty acid binding protein in adult zebrafish (Danio rerio). Biochim. Biophys. Acta. 1492, 227–232. 20. Denovan-Wright, E.M., Pierce, M. & Wright, J.M. (2000) Nucleotide sequence of cDNA clones coding for a brain-type fatty acid binding protein and its tissue-specific expression in adult zebrafish (Danio rerio). Biochim. Biophys. Acta. 1492, 221–226. 21. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) ClustalW: improving the sensitivity of progressive multiple sequence align- ment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. 22. Hukriede, N.A., Joly, L., Tsang, M., Miles, J., Tellis, P., Epstein, J.A., Barbazuk, W.B., Li, F.N., Paw, B., Postlethwait, J.H., Hudson,T.J.,Zon,L.I.,McPherson,J.D.,Chevrette,M., Dawid, I.B., Johnson, S.L. & Ekker, M. (1999) Radiation hybrid mapping of the zebrafish genome. Proc. Natl Acad. Sci. USA 96, 9745–9750. 23. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 24. Denovan-Wright, E.M., Newton, R.A., Armstrong, J.N., Babity, J. & Robertson, H.A. (1998) Acute administration of cocaine, but not amphetamine, increase the level of synaptotagmin IV mRNA in the dorsal striatum of rat. Mol. Brain Res. 55, 350–354. 25. Cheng, L., Qian, S., Rothschield, C., Avignon, A., Lefkowith, J., Gordon, J. & Li, E. (1991) Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis. J. Biol. Chem. 266, 24404–24412. 26. Nilsson, M.H., Spurr, M.K., Lundvall, J., Rask, L. & Peterson, P.A. (1988) Human cellular retinol-binding protein gene organi- zation and chromosomal location. Eur. J. Biochem. 173, 35–44. 27. Andre, M., Ando, S., Ballagny, C., Durliat, M., Poupard, G., Briancon, C. & Babon, P.J. (2000) Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis. Int. J. Dev. Biol. 44, 249–252. 28. Ong, D.E. & Page, D.L. (1987) Cellular retionol-binding protein (type two) is abundant in human small intestine. J. Lipid Res. 28, 739–745. 29. Inagami, S. & Ong, D.E. (1992) Purification and partial char- acterization of cellular retinol-binding protein, type two, from human small intestine. J. Nutr. 122, 450–456. 30. Hollander, D. & Muralidhara, K.S. (1977) Vitamin A intestinal absorption in vivo: influence of luminal factors on transport. Am.J.Phsiol.232, E471–E477. 31. Said, H.M., Ong, D.E. & Redha, R. (1988) Intestinal uptake of retinol in suckling rats: characteristics and ontogeny. Pediatr. Res. 24, 481–485. 32. Kaddad, B.P. & Ong, D.E. (1988) Reduction of retinaldehyde bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine. J. Biol. Chem. 263, 12916–12919. 33. Ong, D.E. (1993) Retinoid metabolism during intestinal absorp- tion. J. Nutr. 123, 351–355. 34. Ong, D.E., Kakkad, B. & MacDonald, P.N. (1987) Acyl-CoA- independent esterification of retinol bound to cellular retinol- binding protein (type II) by microsome from rat intestine. J. Biol. Chem. 262, 2729–2736. 35. Sweetser, D.A., Birkenmeier, E.H., Klisak, I.J., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J. & Gordon, J.I. (1987) The human and rodent intestinal fatty acid-binding protein genes: a comparative analysis of their structure, expression, and linkage relationships. J. Biol. Chem. 262, 16060–16071. 36. Green, R.P., Cohn, S.M., Sacchettini, J.C., Jackson, K.E. & Gordon, J.I. (1992) The mouse intestinal fatty acid binding protein gene: nucleotide sequence, pattern of developmental and regional expression, and proposed structure of its protein product. DNA Cell Biol. 11, 31–41. 37. Sheilds, H.M., Bates, M.L., Bass, N.M., Best, C.J., Alpers, D.H. & Ockner, R.K. (1986) Light microscope immunocytochemical localization of hepatic and intestinal types of fatty acid binding proteins in rat small intestine. J. Lipid Res. 27, 549–557. Ó FEBS 2002 Expression of CRBPII in zebrafish (Eur. J. Biochem. 269) 4691 38. Iseki, S., Amano, O., Kanda, T., Fujii, H. & Ono, T. (1993) Expression and localization of intestinal 15 kDa protein in the rat. Mol. Cell. Biochem. 123, 113–120. 39. Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J. & Gordon, J.I. (1987) The cellular retinol-binding protein II gene. J. Biol. Chem. 262, 2458–2467. 40. Mangelsdorf, D.J., Umesono, K., Kliewer, S.A., Borgmeyer, U., Ong, E.S. & Evans, R.M. (1991) A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell 66, 555–561. 41. Goda, T., Yasutake, H. & Takase, S. (1994) Dietary fat regulates cellular retinol-binding protein II gene expression in rat jejunum. Biochim. Biophys. Acta. 1200, 34–40. 42. Takase, S., Suruga, K., Suzuki, R. & Goda, T. (1996) Relationship between perinatal appearance of cellular retinol-binding protein, type II and retinal reductase activity in chick liver. Life Sci. 58, 135–144. 43. Suruga, K., Goda, T., Igarashi, M., Kato, S., Masushige, S. & Takase, S. (1997) Cloning of chick cellular retinol-binding pro- tein, type II and comparison to that of some mammals: expression of the gene at different developmental stages, and possible involvement of RXRs and PPAR. Comp. Biochem. Physiol. 118, 859–869. 44. Nierenberg, D.W. & Stukel, T. (1987) Diurnal variation in plasma levels of retinol, tocopherol, and beta-carotene. Am.J.Med.Sci. 294, 187–190. 45. Woods, I.G., Kelly, P.D., Chu, F., Ngo-Hazelett, P., Yan, Y.L., Huang, H., Postlethwait, J.H. & Talbot, W.S. (2000) A comparative map of the zebrafish genome. Genome Res. 10, 1903– 1914. 4692 M. C. Cameron et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Cellular retinol-binding protein type II (CRBPII) in adult zebrafish ( Danio rerio ) cDNA sequence, tissue-specific expression and gene linkage analysis Marianne. fatty acid binding protein; cellular retinol binding protein; tissue-specific expression; retinol metabolism. Cellular retinol-binding proteins (CRBPs) are