Cellular retinol-binding protein type II (CRBPII) in adult zebrafish (Danio rerio). cDNA sequence, tissue-specific expression and gene linkage analysis.

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 autoradiography 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 therefore 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 chromosomal 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.     

Identification of zebrafish A2 adenosine receptors and expression in developing embryos

The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson’s disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62–74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.     

Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio)

We have cloned a cDNA from zebrafish (Danio rerio) that contains an open-reading frame of 132 amino acids coding for a fatty acid binding protein (FABP) of approximately 15 kDa. Multiple sequence alignment revealed extensive amino acid identity between this zebrafish FABP and intestinal-like FABPs (I-FABP) from other species. The zebrafish I-FABP cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases PstI Bg/II or EcoRI suggesting that a single copy of the I-FABP gene is present in the zebrafish genome. An oligonucleotide probe complementary to the zebrafish I-FABP mRNA hybridized to an mRNA of approximately 800 bases in Northern blot analysis. In situ hybridization revealed that the I-FABP mRNA was expressed exclusively in the intestine of the adult zebrafish.     

Gene expression patterns of the ALP family during zebrafish development

The actinin-associated LIM protein (ALP) genes belong to the PDZ/LIM protein family which is characterized by the presence of both a PDZ and a LIM domain. The ALP subfamily in mammals has four members: ALP, Elfin, Mystique and RIL. In this study, we have annotated and cloned the zebrafish ALP gene family and identified a zebrafish-specific fifth member of the family, the alp-like gene. We compared the zebrafish sequences to their human and mouse orthologues. A phylogenetic analysis based on the amino acid sequences showed the overall high degree of conservation within the family. We describe here the expression patterns for all five ALP family genes during zebrafish development. Whole mount in situ hybridization results revealed common and distinct expression patterns for the five genes. With the exception of elfin, all genes were expressed as maternal RNAs at early developmental stages. Gene expression for all of them appeared regulated and localized in specific regions at the eight different developmental stages studied. Expression for all five genes was observed in the central nervous system (CNS), which led us to further investigate brain-specific expression in sections of embryos at 2 days of development. In summary, we identified the zebrafish orthologues of the ALP family and determined their gene expression patterns during zebrafish embryogenesis. Finally, we compare our results to the limited expression data available for this gene family during mammalian development.     

cDNA cloning and mRNA expression of Transformer 2 (Tra 2) in chicken embryo

We report the cloning of a chicken Transformer 2 (Tra 2) cDNA that encodes a protein of 289 amino acids which are 97.9% identical to those of mammalian splicing factor, Tra 2. Tra 2 mRNA was expressed in chicken embryonic tissues and was observed as a band of 1.5 kb by Northern blot analysis. Whole mount in situ hybridization showed an mRNA expression of Tra 2 in telencephalon, mandible, hyoid arch, wing and leg buds as early as day 3.5 of incubation. These results suggest that the Tra 2 gene may play a role in organogenesis in the chicken embryo.     

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.     

Shared Segment Analysis and Next-Generation Sequencing Implicates the Retinoic Acid Signaling Pathway in Total Anomalous Pulmonary Venous Return (TAPVR)

Most isolated congenital heart defects are thought to be sporadic and are often ascribed to multifactorial mechanisms with poorly understood genetics. Total Anomalous Pulmonary Venous Return (TAPVR) occurs in 1 in 15,000 live-born infants and occurs either in isolation or as part of a syndrome involving aberrant left-right development. Previously, we reported causative links between TAVPR and the PDGFRA gene. TAPVR has also been linked to the ANKRD1/CARP genes. However, these genes only explain a small fraction of the heritability of the condition. By examination of phased single nucleotide polymorphism genotype data from 5 distantly related TAPVR patients we identified a single 25 cM shared, Identical by Descent genomic segment on the short arm of chromosome 12 shared by 3 of the patients and their obligate-carrier parents. Whole genome sequence (WGS) analysis identified a non-synonymous variant within the shared segment in the retinol binding protein 5 (RBP5) gene. The RBP5 variant is predicted to be deleterious and is overrepresented in the TAPVR population. Gene expression and functional analysis of the zebrafish orthologue, rbp7, supports the notion that RBP5 is a TAPVR susceptibility gene. Additional sequence analysis also uncovered deleterious variants in genes associated with retinoic acid signaling, including NODAL and retinol dehydrogenase 10. These data indicate that genetic variation in the retinoic acid signaling pathway confers, in part, susceptibility to TAPVR.