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 1,477 records · Page 82Linked to original sources

PubMed2004

Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm.

Transgenic modification of sperm before fertilization has distinct advantages over conventional transgenic methods. The primary advantage is that the mosaicism inherent in those other techniques is avoided. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation from spermatogonia to functional sperm can occur in vitro, provides the opportunity for genetic modification of sperm in vitro. Here, we report the production of transgenic zebrafish from cultured sperm. The sperm were differentiated from premeiotic germ cells infected with a pseudotyped retrovirus in vitro. The collected sperm were used to perform successful in vitro fertilizations, and transgenic embryos were identified. The transgenic fish transmitted the proviral integration to the next generation in a Mendelian fashion. We report the generation of a transgenic animal by cultured sperm and open the door to many exciting possibilities for the rapid generation of transgenic lines in model organisms such as zebrafish or other animal systems that are otherwise intractable to transgenesis.

Animals↗

PubMed2005

tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.

TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53(N168K) and tp53(M214K)). Although homozygous tp53(N168K) mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37 degrees C, cells in the tp53(M214K) embryos failed to undergo apoptosis in response to gamma radiation at both 28 and 37 degrees C. Unlike wild-type control embryos, irradiated tp53(M214K) embryos also failed to up-regulate p21 and did not arrest at the G(1)/S checkpoint. Beginning at 8.5 months of age, 28% of tp53(M214K) mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.

Alleles↗

PubMed2006

The zebrafish homologue of mammalian chimerin Rac-GAPs is implicated in epiboly progression during development.

In this paper, we report an in vivo model for the chimerins, a family of Rac GTPase-activating proteins (Rac-GAPs) that are uniquely regulated by the lipid second messenger diacylglycerol and have been implicated in the control of actin dynamics, migration, and proliferation. We cloned the zebrafish homologue of mammalian alpha2-chimerin (chn1) and determined that it possesses Rac-GAP activity and a C1 domain with phorbol ester/diacylglycerol-binding capability. chn1 morpholino knockdown embryos exhibit severe abnormalities, including the development of round somites, lack of yolk extension, and a kinked posterior notochord. These zebrafish morphants show Rac hyperactivation and progress faster through epiboly, leading to tailbud-stage embryos that have a narrow axis and an enlarged tailbud with expanded bmp4 and shh expression. Phenotypic rescue was achieved by mRNA microinjection of chn1 or an active chimerin Rac-GAP domain into the yolk syncytial layer but not by a chn1 mutant deficient in Rac-GAP activity, suggesting that the lack of chn1 Rac-GAP activity in the yolk syncytial layer was causative of the misbalance in morphogenetic movements. Our results reveal a crucial role for chn1 in early development and implicate Rac as a key regulator of morphogenetic movements during zebrafish epiboly.

Animals↗

PubMed2006

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and/or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wild-type recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2/Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.

Animals↗

PubMed2002

Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies.

Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is a hallmark of myelodysplastic syndrome and acute myeloid leukemia. Although the genes affected by these changes have not been identified, two critically deleted regions (CDRs) are well established. We have identified 76 zebrafish cDNAs orthologous to genes located in these 5q CDRs. Radiation hybrid mapping revealed that 33 of the 76 zebrafish orthologs are clustered in a genomic region on linkage group 14 (LG14). Fifteen others are located on LG21, and two on LG10. Although there are large blocks of conserved syntenies, the gene order between human and zebrafish is extensively inverted and transposed. Thus, intrachromosomal rearrangements and inversions appear to have occurred more frequently than translocations during evolution from a common chordate ancestor. Interestingly, of the 33 orthologs located on LG14, three have duplicates on LG21, suggesting that the duplication event occurred early in the evolution of teleosts. Murine orthologs of human 5q CDR genes are distributed among three chromosomes, 18, 11, and 13. The order of genes within the three syntenic mouse chromosomes appears to be more colinear with the human order, suggesting that translocations occurred more frequently than inversions during mammalian evolution. Our comparative map should enhance understanding of the evolution of the del(5q) chromosomal region. Mutant fish harboring deletions affecting the 5q CDR syntenic region may provide useful animal models for investigating the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia.

Animals↗

PubMed2002

Developmental expression of functional cyclooxygenases in zebrafish.

Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E(2) is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.

Amino Acid Sequence↗

PubMed2002

Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish.

Neuropilin-1 (NRP1) is a cell-surface receptor for both vascular endothelial growth factor(165) (VEGF(165)) and class 3 semaphorins that is expressed by neurons and endothelial cells. NRP1 is required for normal developmental angiogenesis in mice. The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries. The zebrafish NRP1 gene (znrp1) was isolated and when overexpressed in cells, zNRP1 protein was a functional receptor for human VEGF(165). Whole-mount in situ hybridization showed that transcripts for znrp1 during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Morpholino-mediated knockdown of zNRP1 in embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels. Circulation via trunk axial vessels was not affected. Embryos treated with VEGF receptor-2 kinase inhibitor had a similar intersegmental vessel defect suggesting that knockdown of zNRP1 reduces VEGF activity. To determine whether NRP1 and VEGF activities were interdependent in vivo, zNRP1 and VEGF morpholinos were coinjected into embryos at concentrations that individually did not significantly inhibit blood vessel development. The result was a potent inhibition of blood cell circulation via both intersegmental and axial vessels demonstrating that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results provide the first physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway.

Animals↗

PubMed1992

Major histocompatibility complex class II genes of zebrafish.

Twenty cDNA clones derived from beta-chain-encoding class II genes of the zebrafish (Brachydanio rerio) major histocompatibility complex (MHC) have been sequenced. They fall into three groups identifying three loci of expressed genes. The length and organization of these genes are similar to those of their mammalian homologs. Amplification by polymerase chain reaction and sequencing of genomic DNA from zebrafish collected at different locations in India indicate the existence of a fourth group of sequences (fourth locus). A high degree of polymorphism at the B. rerio MHC loci and concentration of variability to the putative peptide-binding region of the beta 1-domain-encoding part of the gene are also indicated. Large genetic distances between alleles suggest trans-specific evolution of fish MHC polymorphism. Zebrafish genes appear to be derived from a different ancestor than the various class II gene families of other vertebrates. In spite of great sequence divergence between fish and mammalian MHC genes, there seems to be a striking conservation in their overall organization.

Animals↗

PubMed1996

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

3T3 Cells↗

PubMed1996

The RNA subunit of ribonuclease P from the zebrafish, Danio rerio.

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

Animals↗

PubMed1999

Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis.

We have developed a simple fluorescent assay for detection of phospholipase A2 (PLA2) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA2 inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca2+-dependent cytosolic PLA2 (cPLA2) subtype. Embryonic cPLA2 activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA2, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.

Animals↗

PubMed2001

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Acetylcholinesterase↗

PubMed2004

Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure, expression pattern, and ligand binding.

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.

Animals↗

PubMed2004

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Animals↗

PubMed2002

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.

Animals↗

PubMed2002

Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?

Morpholino phosphorodiamidate anti-sense oligonucleotides (MPOs) have recently emerged as a tool for gene-specific knockdown. MPOs have a great potential for both therapeutic applications and functional genomics. In particular, zebrafish are well suited for gene function studies using MPOs owing to their rapid external development, transparent embryos and the ease of delivery of the intervening MPOs. This paper describes principles of MPO action and the application of MPOs for gene function studies and therapeutics. In the field of functional genomics, the MPO strategy has been most successfully used to study gene function in zebrafish. Over 35 mutations have been successfully phenocopied and over 30 novel gene functions have been analysed in zebrafish using MPOs during the last two years. The essential controls that are required to avoid misinterpretation of experimental data when using MPOs for gene function analysis will also be described.

Amides↗

PubMed2004

Behavioral responses of newly hatched zebrafish (Danio rerio) to amino acid chemostimulants.

The zebrafish chemosensory systems of olfaction, taste and solitary chemosensory cells (SCCs) are established during the first week after fertilization (a.f.). These systems presumably support the early development of feeding behaviors required as yolk supplies diminish over the same period. Yet there is no previous data reporting early chemosensory responses in zebrafish. We therefore assayed the chemosensory behavior of newly hatched zebrafish on days 3, 4 and 5 a.f. Responses were compared between fish exposed to water alone versus water containing a mixture of 12 amino acids (100 microM each) flowing through a 50 ml test chamber at 4 ml/min; computer-assisted motion analysis was used to quantify responses. Behavioral responses were first observed at day 4 a.f.; the number of fish swimming, their swimming speeds, and their net-to-gross displacement (NGDR) all increased significantly in response to amino acid stimulation. Because taste buds first appear 4-5 days a.f. and the SCCs may not respond to amino acids, these initial chemosensory responses of day 4 fish may be mediated by already established olfactory neurons. The onset of chemosensitivity in day 4 fish corresponded with an easily recognizable developmental phenotype of inactive floating; day 3 fish were inactive and resting on the bottom while day 5 fish were active and moving through the water column. The ease of identifying responsive day 4 fish suggests these animals may be useful for characterizing odorant sensitivity or developmental plasticity or for screening for chemosensory mutations.

Amino Acids↗

PubMed2003

Gene duplication and spectral diversification of cone visual pigments of zebrafish.

Zebrafish is becoming a powerful animal model for the study of vision but the genomic organization and variation of its visual opsins have not been fully characterized. We show here that zebrafish has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes in the genome, among which LWS-2, RH2-2, and RH2-3 are novel. SWS2, LWS-1, and LWS-2 are located in tandem and RH2-1, RH2-2, RH2-3, and RH2-4 form another tandem gene cluster. The peak absorption spectra (lambdamax) of the reconstituted photopigments from the opsin cDNAs differed markedly among them: 558 nm (LWS-1), 548 nm (LWS-2), 467 nm (RH2-1), 476 nm (RH2-2), 488 nm (RH2-3), 505 nm (RH2-4), 355 nm (SWS1), 416 nm (SWS2), and 501 nm (RH1, rod opsin). The quantitative RT-PCR revealed a considerable difference among the opsin genes in the expression level in the retina. The expression of the two red opsin genes and of three green opsin genes, RH2-1, RH2-3, and RH2-4, is significantly lower than that of RH2-2, SWS1, and SWS2. These findings must contribute to our comprehensive understanding of visual capabilities of zebrafish and the evolution of the fish visual system and should become a basis of further studies on expression and developmental regulation of the opsin genes.

Amino Acid Sequence↗