Retention of the duplicated cellular retinoic acid-binding protein 1 genes (crabp1a and crabp1b) in the zebrafish genome by subfunctionalization of tissue-specific expression.

The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals. We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b. These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs. The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively. Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus. Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication. Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis. crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages. In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization. The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome.

Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development.

The cellular retinoic acid-binding protein 2 (CRABP2) is believed to be involved in regulating access of retinoic acid to nuclear retinoic acid receptors. We have determined the cDNA sequence and the genomic organization of the duplicated crabp2 gene (crabp2b) in zebrafish. The crabp2b cDNA was 522bp in length and encodes a polypeptide consisting of 146 amino acids. Radiation hybrid mapping assigned the crabp2b gene to zebrafish linkage group 19. The comparison of the mapped human CRABP2 gene, zebrafish crabp2a and zebrafish crabp2b genes revealed that human chromosome 1 has a syntenic relationship to zebrafish linkage groups 16 and 19. Reverse transcription-polymerase chain reaction (RT-PCR) detected crabp2b mRNA in total RNA extracted from whole adult zebrafish, but not in any of the adult zebrafish tissues examined. The crabp2a mRNA was detected in total RNA extracted from whole adult zebrafish, adult zebrafish muscle, testes, and skin and to a lesser extent in heart, ovary and brain. No crabp2a mRNA-specific product was detected in kidney, liver or intestine of the adult zebrafish. Whole mount in situ hybridization detected crabp2b and crabp2a mRNA in a number of structures known to require retinoic acid signaling during embryonic development. The crabp2b mRNA was detected in the central nervous system, branchial arches, pectoral fins, retina (dorsal to the lens), epidermis and otic vesicle of the developing zebrafish. The crabp2a transcripts were detected by whole mount in situ hybridization in the central nervous system, epidermis, proliferative zone of the retina, intestinal bulb, oesophagus, pectoral fins and branchial arches during zebrafish embryonic development.

Spatio-temporal distribution of cellular retinol-binding protein gene transcripts (CRBPI and CRBPII) in the developing and adult zebrafish (Danio rerio).

We have cloned and determined the nucleotide sequence of the cDNA coding for a cellular retinol-binding protein type I (CRBPI) from zebrafish. The deduced amino acid sequence of the zebrafish CRBPI showed highest sequence identity ( approximately 59%) to the mammalian CRBPIs of the intracellular lipid-binding protein (iLBP) multigene family. Phylogenetic analysis clustered the zebrafish CRBPI to the CRBPI clade. The zebrafish CRBPI gene (rbp1) and CRBPII gene (rbp2) both consist of four exons separated by three introns, identical to all other iLBP genes in vertebrates. Two transcription start sites were identified in the rbp1 promoter and a single transcription start site was identified for rbp2. Radiation hybrid mapping assigned the zebrafish rbp1 gene to linkage group 16 and conserved syntenic genes were found by comparative analysis of mammalian orthologous rbp1 genes. RT-PCR detected mRNA transcripts in the adult intestine, liver, brain, ovary and testis for rbp1 gene and in the intestine and liver for rbp2 gene. Whole mount in situ hybridization of zebrafish embryos revealed rbp1 mRNA expression in the developing zebrafish central nervous system at specific sites that are known to have abundant retinoic acid distribution and significant retinoic acid action. Whole mount in situ hybridization also showed that the zebrafish rbp2 mRNA was localized specifically in the embryonic intestinal bulb and the developing intestine during the larval stage, implying a novel function for the rbp2 gene product during organogenesis and development of the zebrafish intestine.

A cellular retinoic acid-binding protein from zebrafish (Danio rerio): cDNA sequence, phylogenetic analysis, mRNA expression, and gene linkage mapping.

We report the sequence of a cDNA clone coding for a cellular retinoic acid-binding protein (CRABP) in zebrafish. The encoded polypeptide is 142 amino acids in length with an estimated molecular mass of 15.8 kDa and a calculated isoelectric point of 5.2. The zebrafish CRABP exhibits highest sequence identity to the pufferfish CRABPIIa (83%) and CRABPIIb (79%), and human CRABPII (74%) than to any other member of the intracellular lipid-binding protein (ILBP) family. A phylogenetic tree for different members of the ILBP multigene family including fatty acid-binding proteins (FABPs), cellular retinol-binding proteins (CRBPs) and CRABPs shows that the cloned zebrafish cDNA encodes a protein that clusters with CRABPs from other species and not with CRBPs and FABPs. Reverse-transcription polymerase chain reactions (RT-PCR), using oligonucleotide primers specific to the zebrafish CRABP cDNA made from total RNA of embryos collected at various developmental stages, did not detect the CRABP mRNA until 12 h post-fertilization. In adult zebrafish, CRABP mRNA was detected by RT-PCR in total RNA extracted from muscle, testes and skin, barely detectable in heart, ovary and brain and undetectable in liver, kidney and intestine. Quantitative RT-PCR (qRT-PCR) revealed a similar tissue-specific distribution for zebrafish CRABP mRNA with highest levels of CRABP mRNA in muscle followed by testes, skin, ovary and much lower levels in heart. Radiation hybrid mapping assigned the CRABP gene to linkage group 16 in the zebrafish genome. Comparison of the mapped zebrafish CRABP and human CRABPII genes revealed that zebrafish linkage group 16 has a syntenic relationship with human chromosome 1. Based on phylogenetic analysis and the syntenic relationship to the CRABPII gene in human, the zebrafish cDNA clone appears to code for a type II CRABP.