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PubMed2002

Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio).

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.

Animals↗

PubMed2004

[Advanced methods of preparing pachytene bivalents and high resolution multiple bands of zebrafish (Danio rerio)].

The pachytene bivalents with high-resolution multiple G bands of zebrafish were obtained after the treatment with alkaline hypotonic solution and high concentration of chloroform fixative solution. When comparing six group chromosomes from different pachytene specimens, the characteristic and the number of bands were well matched. In order to systematize this technique and get stable result, we summarize the preparation procedure of the zebrafish bivalents. The 6-month-old to one-year-old zebrafish whose spermary appears ivory-white and opaque, is good material. The whole testis should be treated with hypotonic solution for 1.5 approximately 2 h at room temperature. Then, the testes were fixed for 20 min in high chloroform fixative solution (chloroform: methanol: acetic acid, 3: 6:1), and fixed in Carnoy's solution (methanol: acetic acid, 3:1) for two times. In addition, with the treatment of restriction endonuclease Alu I directed in situ nick translation, we successfully obtained well-resolved restrictive endonuclease banding of zebrafish bivalents, which was considered as G-like band patterns. The aging of the specimen is also important factor, should let them dry at room temperature for one week. The application of these methods in cytogenetics research of zebrafish and other fish can be expected. Construction of the steady technique system to prepare high resolution banding bivalents and idiogram of zebrafish is the basement to found stable and accurate framework for physical map.

Animals↗

PubMed2005

Expressed sequence tag analysis of zebrafish eye tissues for NEIBank.

PURPOSE: To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank. METHODS: mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were sequenced and analyzed using various bioinformatics programs. RESULTS: The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two gammaN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified. CONCLUSIONS: The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.

Animals↗

PubMed1995

Feeder cell cultures for zebrafish embryonal cells in vitro.

Use of fibroblast cells derived from mouse embryos as feeder layers was one of the major steps leading to the establishment of pluripotential mouse embryonal stem (ES) cells in culture. In attempts to obtain a culture of pluripotential ES cells from zebrafish, a culture of fibroblastoid cells, designated zebrafish embryo fibroblast (ZEF), was established from early gastrula stage zebrafish embryos for use as feeder layer. In primary cultures initiated from early embryos of zebrafish without feeder layers, melanocytes appeared on the second day of culture. In contrast, melanogenesis was markedly suppressed in cocultures containing confluent monolayers of ZEF or Buffalo rat liver (BRL) cells. BRL cells are commonly used feeder layer cells for mouse ES cells. Suppression of melanogenesis was not observed in primary cultures initiated in medium containing human recombinant differentiation-inhibiting activity (DIA) or in medium conditioned by cultures of BRL feeder cells. Proliferation of zebrafish embryonal cells was enhanced significantly in cocultures with either feeder layer. Zebrafish embryonal cells cocultured short-term on ZEF and BRL feeder layers gave rise to melanocytes and formed embryoid body-like structures when removed from feeder layers and cultured in suspension, suggesting that the cells remained pluripotent in culture.

Animals↗

PubMed1994

Modulation of electrical synaptic transmission in zebrafish retinal horizontal cells.

Electrical synaptic transmission is widespread in the vertebrate CNS and its modulation plays a critical role in altering the properties of coupled neural networks. In order to define further the mechanisms of electrical synaptic plasticity in the vertebrate retina, the electrophysiological characteristics of solitary horizontal cells and horizontal cell pairs from the zebrafish (Brachydanio rerio) were examined by whole-cell patch-clamp recordings from cells in primary cell culture. In solitary cells, the current-voltage relation exhibited inward current at potentials negative to -60 mV, a linear region of high resistance from -50 mV to 0 mV, and outward current positive to +20 mV. The inward current at negative potentials was blocked by substituting Cs+ for K+ in the extracellular medium, while the outward current at positive potentials was blocked by substitution of Cs+ for K+ in the pipette solution. Measurements of gap junctional conductance from electrically coupled cell pairs revealed that zebrafish horizontal cells expressed a mean junctional conductance of considerably smaller magnitude than other teleost retinal horizontal cells. Junctional conductance was found to be voltage dependent, exhibiting time-dependent closure with increasing transjunctional voltage. Voltage sensitivity was symmetrical about 0 mV junctional potential. At +/- 90 mV the ratio of steady state to peak current was approximately 0.5 and the time constant for inactivation of the junctional current was approximately 120 msec. Junctional conductance was also modulated by dopamine and cAMP. Pairs of horizontal cells responded to puff application of dopamine with a two- to threefold reduction in junctional conductance, but there was no discernible effect on extrajunctional conductances. The action of dopamine on coupling was mimicked by application of the dopamine agonist (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN) and the membrane permeable cAMP analog 8-bromo-cAMP. The selective D1 dopamine receptor antagonist SCH23390 blocked uncoupling by dopamine. These data provide a primary description of the electrophysiological characteristics of solitary horizontal cells and the electrical coupling between pairs of horizontal cells dissociated from the zebrafish retina. They indicate that zebrafish horizontal cells are distinct from the horizontal cells of other teleosts in their coupling characteristics. The results suggest that zebrafish horizontal cells exhibit differences in the regulation of synaptic assembly and maintenance that have important implications for the function of the zebrafish horizontal cell network in vivo.

Animals↗

PubMed1997

Delayed in vitro fertilization of zebrafish eggs in Hank's saline containing bovine serum albumin.

In zebrafish it is possible to create viable diploid fish whose genomic DNA is derived only from the female parent (parthenogenesis) or, as was more recently shown, only from the male (androgenesis). Androgenesis requires holding zebrafish eggs in an inactivated state in vitro for an hour or more. Previously this was achieved by placing the zebrafish eggs in ovary fluid obtained from rainbow trout (Onchorhynchus mykiss) or coho salmon (Onchorhynchus kisutch). Here we report that adding bovine serum albumin (BSA) to Hank's buffered saline prevents zebrafish egg activation in vitro. Of the zebrafish eggs placed in Hank's saline plus 0.5% BSA, 85% +/- 8.7% were fertilizable after incubation for one hour at room temperature (23 degrees C). Longer incubations are possible but with lower efficiency of fertilization. This technique not only could facilitate androgenesis, but also might be useful when making transgenics by microinjection, when performing antibody or RNA injections before fertilization, or for studying the mechanisms of egg activation in zebrafish.

Animals↗

PubMed2002

Cloning and characterization of replication protein A p32 complementary DNA in zebrafish (Danio rerio).

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (70, 32, and 14 kDa) that is an essential component of the DNA replication fork. A complementary DNA encoding zebrafish RPA 32-kDa subunit was isolated by screening a zebrafish embryo lambda APII cDNA library with a human RPA p32 cDNA probe. The zebrafish RPA p32 cDNA consisted of 1097 bp encoding 272 amino acid residues. The deduced amino acid sequence shows high similarity to mouse and human RPA p32. In vitro phosphorylation of zebrafish RPA protein by Cdc2 kinase was shown. A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography. In this article, cloning of the zebrafish RPA p32 cDNA is reported in relation to the study of DNA replication in fish.

Journal Article↗

PubMed2002

A simple spatial alternation task for assessing memory function in zebrafish.

A series of studies was initiated to examine learning and memory function in the zebrafish (Danio rerio) by using a simple spatial alternation paradigm for a food reward. Fish were fed on alternating sides of a divided fish tank, with a red card displayed on one side serving as a visual means of orientation. Although responses were recorded at cue (light tap on the tank), 5 s after cue (as food was delivered), and 5 s after food delivery, the learning test was choice of a correct side of the tank to receive food. Therefore, an accurate level of an animal's achievement of the spatial task was represented by responses at food delivery. Data collected from 11 separate experiments indicated that zebrafish learned to alternate for a food reward. Further, statistical analysis showed that the zebrafish learned the task in the first half of the experiment as exhibited by a calculated t1/2 of 13.9 trials. Zebrafish could recall the task after a short period of 10 days with no testing. The alternating behavior was extinguished by withholding the food reward. Thus, the spatial alternation task can be learned easily by zebrafish, and may be useful in addressing learning and memory functions in vertebrate animals using zebrafish as a model organism.

Journal Article↗

PubMed2002

Comparative analysis of somitogenesis related genes of the hairy/Enhancer of split class in Fugu and zebrafish.

BACKGROUND: Members of a class of bHLH transcription factors, namely the hairy (h), Enhancer of split (E(spl)) and hairy-related with YRPW motif (hey) (h/E(spl)/hey) genes are involved in vertebrate somitogenesis and some of them show cycling expression. By sequence comparison, identified orthologues of cycling somitogenesis genes from higher vertebrates do not show an appropriate expression pattern in zebrafish. The zebrafish genomic sequence is not available yet but the genome of Fugu rubripes was recently published. To allow comparative analysis, the currently known Her proteins from zebrafish were used to screen the genomic sequence database of Fugu rubripes. RESULTS: 20 h/E(spl)/hey-related genes were identified in Fugu, which is twice the number of corresponding zebrafish genes known so far. A novel class of c-Hairy proteins was identified in the genomes of Fugu and Tetraodon. A screen of the human genome database with the Fugu proteins yielded 10 h/E(spl)/hey-related genes. By analysing the upstream sequences of the c-hairy class genes in zebrafish, Fugu and Tetraodon highly similar sequence stretches were identified that harbour Suppressor of hairless paired binding sites (SPS). This motif was also discovered in the upstream sequences of the her1 gene in the examined fish species. Here, the Su(h) sites are separated by longer intervening sequences. CONCLUSIONS: Our study indicates that not all her homologues in zebrafish have been isolated. Comparison to the human genome suggests a selective duplication of h/E(spl) genes in pufferfish or loss of members of these genes during evolution to the human lineage.

Journal Article↗

PubMed2003

Modeling human hematopoietic and cardiovascular diseases in zebrafish.

Zebrafish have emerged as a useful vertebrate model system in which unbiased large-scale screens have revealed hundreds of mutations affecting vertebrate development. Many zebrafish mutants closely resemble known human disorders, thus providing intriguing prospects for uncovering the genetic basis of human diseases and for the development of pharmacologic agents that inhibit or correct the progression of developmental disorders. The rapid pace of advances in genomic sequencing and map construction, in addition to morpholino targeting and transgenic techniques, have facilitated the identification and analysis of genes associated with zebrafish mutants, thus promoting the development of zebrafish as a model for human disorders. This review aims to illustrate how the zebrafish has been used to identify unknown genes, to assign function to known genes, and to delineate genetic pathways, all contributing valuable leads toward understanding human pathophysiology.

Anemia↗

PubMed2005

Specification of the enveloping layer and lack of autoneuralization in zebrafish embryonic explants.

We have analyzed the roles of cell contact during determination of the outermost enveloping layer (EVL) and deeper neurectoderm in zebrafish embryos. Outer cells, but not deeper cells, are specified to express the EVL-specific marker, cyt1 by late blastula. EVL specification requires cell contact or close cell proximity, because cyt1 is not expressed after explant dissociation. The EVL may be homologous to the Xenopus epithelial layer, including the ventral larval epidermis. While Xenopus epidermal cytokeratin gene expression is activated by bone morphogenetic protein (BMP) signaling, zebrafish cyt1 is not responsive to BMPs. Zebrafish early gastrula ectodermal explants are specified to express the neural markers opl (zic1) and otx2, and this expression is prevented by BMP4. Dissociation of zebrafish explants prevents otx2 and opl expression, suggesting that neural specification in zebrafish requires cell contact or close cell proximity. This finding is in contrast to the case in Xenopus, where ectodermal dissociation leads to activation of neural gene expression, or autoneuralization. Our data suggest that distinct mechanisms direct development of homologous lineages in different vertebrates.

Amino Acid Sequence↗

PubMed2006

Low-molecular-weight vitellogenin 1-like proteins are components of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos.

A strong UV-damaged-DNA binding activity had been detected in the extracts of zebrafish embryos at 12 hr after fertilization by gel shift assay (Hsu et al. 2002. Fish Physiol Biochem 25:41-51). We attempted to study the components of this binding activity and their importance in DNA damage recognition. Among the proteins extracted from gel retardation complexes, a 30- and a 35-kDa polypeptide binding preferentially to 6-4photoproducts (6-4PPs) generated by UV irradiation were identified by peptide mass fingerprinting (PMF) as homologs of zebrafish vitellogenin I (zfVg1), a 150-kDa metalloprotein known as the precursor of yolk proteins in embryos. zfVg1-like polypeptides ranging from 25 to 105 kDa were detected in 12- and 96-hr-old zebrafish extracts by immunoblot analysis. Immunoblot analysis of affinity-captured proteins confirmed the preferential binding of the 30-35-kDa polypeptides to the 6-4PP probe, while 96-hr-old larval extracts containing very low levels of these two factors failed to recognize 6-4PPs. The presence of zfVg1-like factors was important in maintaining the embryonic UV-binding activity, as inclusion of a monoclonal anti-zfVg1 antibody in reaction mixtures caused a concentration-dependent reduction in 6-4PP-specific binding. In contrast, DNA damage recognition was not disturbed at all by an anti-HSP 70 antibody. The formation of 6-4PP-binding complexes was abolished after the addition of the metal chelating agent 1,10-phenanthroline (OP) to zebrafish extracts and the loss of UV-binding capacity correlated with the disappearance of the 35-kDa factor in OP-treated extracts. Our results demonstrated the ability of low-molecular-weight zfVg1-like proteins in zebrafish embryos to bind UV-damaged DNA and the expression of this embryonic UV-binding activity was metal dependent. Whether zfVg1-like UV-binding proteins are involved in repairing damaged DNA in embryos or in processing helical structures similar to UV-distorted DNA needs further investigation.

Animals↗

PubMed2004

Xenoestrogenic effects of ethinylestradiol in zebrafish (Danio rerio).

To assess the estrogenic effects of ethinylestradiol on zebrafish, zebrafish at different developmental stages (embryos, juveniles, and adults) were exposed to the synthetic hormone ethinylestradiol (EE2) in concentrations of 1, 10, and 100 ng/L for up to 33 days. Survival, hatching, length, weight, growth, condition, hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) production were examined. Exposure of zebrafish juveniles and embryos to 100 ng EE2/L for up to 33 days had significant effects on survival, growth, and hatching. Two VTG fragments with molecular weights of approximately 140 and 170 kDa were detected with protein electrophoresis and Western blotting in the blood of exposed males and exposed and unexposed females, as well as in whole-body homogenates of exposed and unexposed juveniles. Significantly higher VTG concentrations (compared to controls) were measured in adults exposed to 10 and 100 ng EE2/L for 14 days, but not in fish exposed to 1 ng EE2/L. This study demonstrated that (1) zebrafish juveniles, larvae, and embryos are sensitive to the toxic effects of the endocrine disrupter EE2; (2) the effects on VTG production in adults are detected after exposure to environmentally relevant concentrations of EE2; (3) unexposed juvenile zebrafish produce measurable concentrations of VTG.

Age Factors↗

PubMed2001

Molecular cloning, developmental expression, and hormonal regulation of zebrafish (Danio rerio) beta crystallin B1, a member of the superfamily of beta crystallin proteins.

The cDNA sequence of beta crystallin B1 was determined from zebrafish (Danio rerio) and compared to the corresponding genes of bovine, rat, chicken, human, and Xenopus. Multispecies comparison of superfamily diversity demonstrated beta crystallin B1 homology between zebrafish, bovine, chicken, and rat, but large distances to beta crystallin B2 and B3. Zebrafish cDNA has a size of 943 nucleotides and encodes a polypeptide of 233 amino acids. Zebrafish beta crystallin B1 shares 71.30, 75.86, and 71.00% similarities with bovine, chicken, and rat beta crystallin B1, respectively. Northern blot analysis revealed a single 0.9-kb beta crystallin B1 transcript which was expressed and progressively increased in the first 20 h of zebrafish embryogenesis. Whole-mount in situ hybridization revealed that the beta crystallin B1 transcript was only specifically expressed in the lens region of the eye. A starvation experiment revealed no variation in mRNA levels after 14 and 21 days. An experiment in which hormone was injected showed that the beta crystallin B1 transcript first increased 24 h after the injection of insulin-like growth factor I, insulin-like growth factor II, or growth hormone, then decreased 48 h after injection. The beta crystallin B1 transcript continuously increased after insulin was injected. Taken together, our results identify the early specific expression of beta crystallin B1 within the lens. Despite small differences, these results indicate that both the structure of the beta crystallin B1 protein and its involvement with regulation by growth factors appear to have been remarkably conserved.

Amino Acid Sequence↗

PubMedDate not supplied

Comprehensive analysis of blood coagulation pathways in teleostei: evolution of coagulation factor genes and identification of zebrafish factor VIIi.

It is not clear how the complex mammalian coagulation pathways evolved from an entirely dissimilar invertebrate coagulation cascade. Comprehensive analysis of pro-coagulant factors and their regulators is lacking in early vertebrates to discern the mechanism of evolution of these genes from the invertebrates. To elucidate the coagulation pathways found in early vertebrates, zebrafish cDNAs/gene orthologues for major coagulant, anticoagulant, and fibrinolytic proteins were identified and characterized by homology to mammalian sequences. We found that zebrafish carry all hemostatic genes present in mammals, providing evidence that the coagulation system of teleosts is nearly identical to mammals. Zebrafish factor VII and X genes were identified and analyzed to reveal a novel factor VII-like gene flanked by the factor VII and factor X genes. This gene encodes a protein homologous to factor VII, but lacks critical residues for factor VII activity. Expression of the factor VII-like protein (named factor VIIi) demonstrated that it functions as an inhibitor of blood coagulation in biochemical assays using zebrafish or human plasmas. Analysis of intergenic DNA between the zebrafish VII/VIIi/X gene cluster and a Drosophila trypsin gene cluster revealed significant homology, and based upon these data, we propose a model for a rapid evolution of coagulation factors from the invertebrates.

Amino Acid Sequence↗

PubMed1993

The DVR-1 (Vg1) transcript of zebrafish is maternally supplied and distributed throughout the embryo.

It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.

Amino Acid Sequence↗

PubMed1999

Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo.

The dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin in the early patterning of organisms other than Xenopus. We have studied the contribution of Noggin to the embryonic development of the zebrafish. While single-copy noggin genes have been characterized in several vertebrate species, we report that the zebrafish genome harbors three noggin homologues. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Finally we show that noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish.

Amino Acid Sequence↗

PubMed2000

Double-stranded RNA injection produces null phenotypes in zebrafish.

Zebrafish is a simple vertebrate that has many attributes that make it ideal for the study of developmental genetics. One feature that has been lacking in this model system is the ability to disable specifically targeted genes. Recently, double-stranded RNA has been used to silence gene expression in the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RNA that is specifically targeted to GFP. To determine that double-stranded RNA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pax6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. Furthermore, Zf-T gene expression could not be detected by in situ hybridization and the message was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish genes sonic hedgehog and floating head was altered in the embryos microinjected with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA targeted to Pax6.1 was associated with depressed expression of Pax6. 1 and resulted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures.

Animals↗