SEARCH · PubMed Health

Results for “zebrafish”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed2003

Analysis of myostatin gene structure, expression and function in zebrafish.

Myostatin is a member of the TGF-beta family that functions as a negative regulator of skeletal muscle development and growth in mammals. Recently, Myostatin has also been identified in fish; however, its role in fish muscle development and growth remains unknown. We have reported here the isolation and characterization of myostatin genomic gene from zebrafish and analysis of its expression in zebrafish embryos, larvae and adult skeletal muscles. Our data showed that myostatin was weakly expressed in early stage zebrafish embryos, and strongly expressed in swimming larvae, juvenile and skeletal muscles of adult zebrafish. Transient expression analysis revealed that the 1.2 kb zebrafish myostatin 5' flanking sequence could direct green fluorescent protein (GFP) expression predominantly in muscle cells, suggesting that the myostatin 5' flanking sequence contained regulatory elements required for muscle expression. To determine the biological function of Myostatin in fish, we generated a transgenic line that overexpresses the Myostatin prodomain in zebrafish skeletal muscles using a muscle-specific promoter. The Myostatin prodomain could act as a dominant negative and inhibit Myostatin function in skeletal muscles. Transgenic zebrafish expressing the Myostatin prodomain exhibited no significant change in myogenic gene expression and differentiation of slow and fast muscle cells at their embryonic stage. The transgenic fish, however, exhibited an increased number of myofibers in skeletal muscles, but no significant difference in fiber size. Together, these data demonstrate that Myostatin plays an inhibitory role in hyperplastic muscle growth in zebrafish.

5' Flanking Region↗

PubMed2001

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

Animals↗

PubMed1990

The zebrafish homeobox gene hox-2.2: transcription unit, potential regulatory regions and in situ localization of transcripts.

The data presented in this report strongly suggest that the genome of the zebrafish, Brachydanio rerio, has a homeobox cluster which is equivalent to the murine Hox-2 locus. In support of this conclusion, we have found two closely linked zebrafish genes which are true homologues of the mouse Hox-2.1 and Hox-2.2 genes. Here we describe structural and functional properties of the zebrafish Hox-2.2 homologue hox-2.2. Furthermore, we have identified another zebrafish gene related to hox-2.2 which appears to correspond to the Hox-6.1 gene of the murine Hox-3 locus. In order to characterize the zebrafish hox-2.2 gene we have determined the genomic DNA sequence of a 3.4 kb SalI fragment. This revealed that the hox-2.2 transcription unit encodes a putative protein of 228 amino acids. The homeodomains of the murine Hox-2.2 and the zebrafish hox-2.2 proteins are almost identical and extensive sequence identity exists in other regions of the two proteins, which share 160 (70%) of the amino acid residues. Also in terms of expression, strong similarities were observed relative to the murine Hox-2.2 gene. Transcripts derived from zebrafish hox-2.2 start to accumulate when somite formation is initiated. Later in development these transcripts are detected mainly in the central nervous system. Reminiscent of Hox-2.2, the rostral boundary of zebrafish hox-2.2 expression is located in the posterior region of the hindbrain. Notably, untranslated regions of the hox-2.2 gene contain several short sequences closely related to a known homeodomain recognition sequence.

Amino Acid Sequence↗

PubMed1997

Differential regulation of chordin expression domains in mutant zebrafish.

Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.

Amino Acid Sequence↗

PubMed1996

Stable transfer of zebrafish chromosome segments into mouse cells.

Whole-cell fusion between zebrafish fibroblast-like ZF4 cells and mouse B78 melanoma cells resulted in hybrids containing one or a few zebrafish chromosome segments in a murine chromosomal background. Fluorescence in situ hybridization to hybrid cell metaphases with a zebrafish genomic DNA probe revealed that many hybrids contained zebrafish chromosome segments that were either inserted or translocated to a mouse chromosome, whereas other hybrids contained zebrafish chromosomes with no evidence of insertion or translocation. We have assigned hybrids to 17 linkage groups of the genetic map of the zebrafish genome. Our results demonstrate the feasibility of producing somatic cell hybrids between distantly related species. Zebrafish/mouse cell hybrids will provide a useful tool for the physical mapping of the zebrafish genome and for the cloning of genes affected in zebrafish mutants.

Animals↗

PubMed2006

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin.

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7 x 10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Animals↗

PubMed2004

The use of mature zebrafish (Danio rerio) as a model for human aging and disease.

Zebrafish (Danio rerio) have been extensively utilized for understanding mechanisms of development. These studies have led to a wealth of resources including genetic tools, informational databases, and husbandry methods. In spite of all these resources, zebrafish have been underutilized for exploring pathophysiology of disease and the aging process. Zebrafish offer several advantages over mammalian models for these studies, including the ability to perform saturation mutagenesis and the capability to contain thousands of animals in a small space. In this review, we will discuss the use of mature zebrafish as an animal model and provide specific examples to support this novel use of zebrafish. Examples include demonstrating that clinical pathology can be performed in mature zebrafish and that age-associated changes in heat shock response can be observed in zebrafish. These highlights demonstrate the utility of zebrafish as a model for disease and aging.

Aging↗

PubMed2004

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.

Amino Acid Sequence↗

PubMed2006

Characterization and expression patterns of the MAPK family in zebrafish.

The mitogen activated protein kinases (MAPK) family pathway is conserved in evolution through the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, proliferation, differentiation, survival and development. In this study we annotated and cloned members of the zebrafish MAPK gene-family, containing the ERK, JNK and p38 subfamilies. Their sequences were compared to orthologs of other vertebrates (human, mouse and rat) and the temporal and spatial expression levels of the zebrafish mapk genes were determined during early zebrafish development. Semi-quantitative reverse transcriptase-PCR analysis revealed that most mapk genes are expressed throughout zebrafish development. Erk2,3 and p38a were expressed at a constant level throughout zebrafish embryogenesis, whereas erk1,4,5,6,7 and p38b showed specific temporal expression patterns. The spatial expression patterns were obtained by whole mount in situ hybridization at 24 h post fertilization (hpf) and 48 hpf embryos. The expression patterns were localized in specific regions at both stages and were tightly regulated during embryogenesis. For p38b, no staining was detected at 24 and 48 hpf. However, its expression was demonstrated at blastula-stage. Together, we identified the zebrafish orthologs of the zebrafish MAPK gene family and determined their specific spatial and temporal expression and distribution patterns during zebrafish embryogenesis.

Amino Acid Sequence↗

PubMedDate not supplied

Developmental chlorpyrifos effects on hatchling zebrafish swimming behavior.

Chlorpyrifos (CPF), a widely used organophosphate insecticide and potent acetylcholinesterase inhibitor, interferes with neurobehavioral development. Rat models have been key in demonstrating that developmental CPF exposure causes learning deficits and locomotor activity alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the neurodevelopmental mechanisms underlying these persisting behavioral effects. Zebrafish (Danio rerio) with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant-impacted neurodevelopment. We have developed methods for assessing spatial discrimination learning in adult zebrafish and have documented persisting effects of developmental CPF exposure on swimming activity and learning after low and high doses of CPF (10 and 100 ng/ml) administered to zebrafish embryos on Days 1-5 postfertilization (pf). In the current study, we developed methods for behavioral assessment of CPF exposure on swimming activity in newly hatched zebrafish. An equal area segmented annular grid (concentric circles divided into quadrants through the diameter) was made in a 16-mm diameter cylinder. The test area was placed on a heating device secured to an Olympus SZH10 dissecting scope stage. Zebrafish embryos were exposed to 10 ng/ml CPF, 100 ng/ml CPF, or vehicle control (25 microl/ml DMSO) (n=8-10/treatment group). Each treatment group was kept in a total volume of 25 ml of egg water (60 mg/ml Instant Ocean) including DMSO with or without CPF mixed to above dilutions in an incubator set at 28.5 degrees C. CPF dilutions or vehicle were changed daily with exposure ending on Day 5 pf. Testing of larval zebrafish was performed on Days 6 and 9 pf. The fish were placed in the test cylinder with 1.5 ml of egg H(2)O (28.5 degrees C). After a 2-min acclimation period, the swimming activity of the fish was measured for a 3-min testing session. The 100 ng/ml CPF dose caused significant slowing of swimming activity on Days 6 and 9 pf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Developmental exposure to 10 ng/ml of CPF did not cause a significant change in locomotor activity during the period soon after hatching. CPF exposure during early development caused clear behavioral impairments detectable during the posthatching period. In a previous study, we found that early developmental CPF exposure caused behavioral alterations in zebrafish, which lasted throughout adulthood. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.

Animals↗

PubMed2004

Approaches to study neurogenesis in the zebrafish retina.

Similar to other vertebrate species, the zebrafish retina is simpler than other regions of the central nervous system (CNS). Relative simplicity, rapid development, and accessibility to genetic analysis make the zebrafish retina an excellent model system for the studies of neurogenesis in the vertebrate CNS. Numerous genetic screens have led to isolation of an impressive collection of mutations affecting the retina and the retinotectal projection in zebrafish. Mutant phenotypes are being studied using a rich variety of markers: antibodies, RNA probes, retrograde and anterograde tracers, as well as transgenic lines. Particularly impressive progress has been made in the characterization of the zebrafish genome. Consequently, positional and candidate cloning of mutant genes are now fairly easy to accomplish in zebrafish. Many mutant genes have, in fact, already been cloned and their analysis has provided important insights into the gene circuitry that regulates retinal neurogenesis. Genetic screens for visual system defects will continue in the future and progressively more sophisticated screening approaches will make it possible to detect a variety of subtle mutant phenotypes in retinal development. The remarkable evolutionary conservation of the vertebrate eye provides the basis for the use of the zebrafish retina as a model of human disorders. Some of the genetic defects of the zebrafish retina indeed resemble human retinopathies. As new techniques are being introduced and improved at a rapid pace, the zebrafish will continue to be an important organism for the studies of the vertebrate visual system.

Alleles↗

PubMed2003

The zebrafish as a vertebrate model of functional aging and very gradual senescence.

The zebrafish (Danio rerio) has been developed as a powerful model for genetic studies in developmental biology, which also gives insights into several diseases of adult humans such as cardiovascular disease and cancer. Because aging processes affect these and many other human diseases, it is important to compare zebrafish and other mammalian aging. However, the aging process of zebrafish remains largely unexplored, and little is known about its functional aging and senescence. In a survey of aging in zebrafish, we detected senescence-associated beta-galactosidase activity in skin and oxidized protein accumulation in muscle. However, we did not observe lipofuscin granules ('aging pigments'), which commonly accumulate in postmitotic cells of other vertebrates. This absence of lipofuscins may be consistent with the existence of continuously proliferating myocytes that incorporated BrdU in muscle tissues of aged zebrafish. Moreover, we demonstrated that zebrafish have constitutively abundant telomerase activity in somatic tissues from embryos to aged adults. Although some stress-associated markers are upregulated and minor histological changes are observed during the aging process of zebrafish, our studies together with other evidence of remarkable reproductive and regenerative abilities suggest that zebrafish show very gradual or sub-negligible senescence in vivo.

Aging↗

PubMed2001

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

Amino Acid Sequence↗

PubMed2001

Constant dark-rearing effects on visual adaptation of the zebrafish ERG.

Anatomical and electrophysiological studies have shown that zebrafish retinal neurons develop in a sequential manner. Several studies have examined the impact of restricted rearing environments on zebrafish visual development, but the results have been mixed. The purpose of this study was to examine the development of light adaptation properties of the zebrafish electroretinogram (ERG), and examine the effects of constant dark rearing on retinal development. Subjects were zebrafish, Danio rerio, reared under normal lighting conditions and zebrafish reared in constant dark from fertilization to 6 days postfertilization (dpf). Increment threshold functions were obtained from a- and b-wave ERG responses from normally reared subjects at different ages and from animals exposed to early constant dark rearing. Dark-reared subjects were tested immediately following constant dark exposure (6-9 dpf) and after exposure to normal cyclic lighting (11-13 dpf). Adult zebrafish were significantly more sensitive at lower background illuminations than were larvae zebrafish. Also, constant dark rearing had a differential effect on the a- and b-wave response measures. Constant dark rearing raised b-wave threshold uniformly across background illuminations, while only producing higher a-wave thresholds at low levels of illumination. These results are consistent with findings in studies on zebrafish retinal development, and may help explain some of the discrepancies across studies examining the effects of restricted rearing.

Aging↗

PubMed2001

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview.

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.

Animals↗

PubMed2003

Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase.

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5' and 3' regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential K(m) and V(max) values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 degrees C and 43 degrees C. Among ten different divalent metal cations tested, Fe2+ and Cd(2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2003

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.

Amino Acid Sequence↗