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At least 721 records · Page 40Linked to original sources

PubMed1993

Cloning of the beta 2-microglobulin gene in the zebrafish.

The beta 2-microglobulin (beta 2m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the alpha chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the beta 2m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3' untranslated (UT) region, and at least part of the 5'UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish beta 2m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey beta 2m (codons at positions 85 and 86 have been deleted in the Brre-B2m gene). The amino acid and nucleotide sequence similarities between zebrafish and human beta 2m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known beta 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the beta strands (some 47% of the beta-strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.

Amino Acid Sequence↗

PubMed1993

Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos.

During the last decade, zebrafish (Brachydanio rerio) have emerged as a novel and attractive system to study embryogenesis and organogenesis in vertebrates. The main reason is that both extensive genetic studies and detailed embryologic analysis are possible using this small tropical fresh water teleost. However, in vitro analysis using cell culture or molecular genetics are still far less advanced than in other vertebrate systems. Here we report the generation and characterization of a fibroblast like cell line, ZF4, derived from 1-day-old zebrafish embryos. The hyperploid cell line has been stable in multiple passages for more than 2 yr now and is the first zebrafish cell line that can be maintained in conventional medium containing mammalian serum. Using a series of plasmids for expression of a marker gene, we evaluate in ZF4 cells the relative strength of expression from several different viral, fish, and mammalian promoters. Stable integration can be obtained by using G418 selection. We hope that our cell line will be a useful tool for the analysis of gene regulation in zebrafish.

Animals↗

PubMed2006

Identification of a promoter element within the zebrafish colXalpha1 gene responsive to runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2alphaA2.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Amino Acid Sequence↗

PubMed2001

Characterization of the calcium-release channel/ryanodine receptor from zebrafish skeletal muscle.

Calcium (Ca2+)-mediated signaling is fueled by two sources for Ca2+: Ca2+ can enter through Ca2+ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca2+ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca2+ concentrations of the RyR isoform that is inhibited by high Ca2+ concentrations (teleost alpha RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca2+ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish.

Animals↗

PubMed2005

Evolution of transcriptional enhancers in the immunoglobulin heavy-chain gene: functional characteristics of the zebrafish Emu3' enhancer.

Past studies of the channel catfish immunoglobulin heavy-chain (IgH) locus indicates that it lacks an Emu enhancer in the J(H)-Cmu1 intron but does have an enhancer, termed Emu3', in the mu-delta intergenic region. The positioning of the catfish enhancer downstream of the mu-chain exons is predicted to be unfavorable for antibody-affinity maturation in catfish, and would also have been an impediment to the evolution of class switch recombination, had it existed in early tetrapods. To determine if this downstream enhancer is a general feature of teleost fish, we have identified the location of the transcriptional enhancer in the zebrafish IgH locus. We find that zebrafish, like catfish, only have an Emu3'-like enhancer that has cross-species activity, but which is B-cell-specific in its activity. A 300-bp region of the zebrafish enhancer shares sequence homology with the core of the catfish Emu3', although there has been loss and gain of specific octamer enhancer motifs. Mutagenesis studies demonstrate that the zebrafish IgH enhancer depends on a pair of E-box motifs that are found in the enhancer core. Similarly spaced E-box motifs appear to exist in the Emu3' enhancer regions of other teleost fish, suggesting this is a common feature among fish IgH enhancers. We discuss how this distal positioning of the enhancer may influence affinity maturation in extant teleosts as well as the evolution of this process in the early tetrapods.

Amino Acid Motifs↗

PubMed2003

The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos.

MMP activities are controlled by a combination of proteolytic pro-enzyme activation steps and inhibition by endogenous inhibitors like alpha2-macroglobulin and the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are the key inhibitors in tissue. The expression of both MMPs and TIMPs is controlled during tissue remodeling to maintain a balance in the turnover of extracellular matrix. Disruption of this balance may result in a broad spectrum of diseases. Additionally, TIMP-2 has been reported to have growth factor activities. To further study the function of TIMP-2 in development, we utilized zebrafish as an experimental model system. We have successfully isolated a TIMP-2 homologue from zebrafish (zTIMP-2). This zebrafish TIMP-2 showed high similarity to human TIMP-2 with all critical features conserved. Whole-mount in situ analysis showed that zTIMP-2 was expressed as early as the one-cell stage indicating a maternal origin. This expression continued through later stages of development. RT-PCR analysis confirmed the early expression pattern from the 16-cell stage through blastula, gastrula and 24-h stages. In addition, at the protein level, immunoreactive zTIMP-2 was detected using antibody against recombinant human TIMP-2. RFP-reporter analysis indicated that TIMP-2 can be secreted into the extracellular space where ECM is forming. Functional studies showed that the balance of TIMP-2 expression is important to normal development as reflected by the fact that both blockage of TIMP-2 translation using antisense morpholino oligonculeotides or increased translation of TIMP-2 using a mRNA microinjection approach resulted in abnormal zebrafish development. This is in contrast to murine knockout studies that indicate that TIMP-2 does not have a major role in mouse embryogenesis.

Amino Acid Sequence↗

PubMed2000

Monoclonal antibody stains oligodendrocytes and Schwann cells in zebrafish (Danio rerio).

We prepared a monoclonal antibody that recognizes oligodendrocytes and Schwann cells in zebrafish. On immunoblots, the antibody mainly recognized three protein bands of 34 kDa in a membrane fraction from adult zebrafish brain. Medaka fish (Oryzias latipes) also possessed the same protein bands in a membrane fraction. The antibody did not stain neurons, but stained cells in fiber tracts and cranial and spinal nerves. In order to determine the nature of these cells, the staining pattern of the monoclonal antibody was compared with that of a myelin basic protein antiserum. Both antibodies stained oligodendrocytes and Schwann cells in fixed sections from the adult zebrafish. Both antigens were also co-localized in cultured glial cells. Taken together, these results indicate that the new monoclonal antibody recognizes myelinating glial cells in zebrafish and will be useful for the analysis of piscine glia.

Animals↗

PubMed2000

The zebrafish pronephros: a genetic system for studies of kidney development.

The zebrafish, as a model system for vertebrate development, offers distinct experimental advantages for studies of organogenesis. The simplicity of the zebrafish pronephros, the feasibility of isolating large numbers of mutants, and the growth in infrastructure for genomics makes the zebrafish an attractive system for the analysis of kidney development. Mutants affecting several aspects of nephrogenesis, including differentiation of the intermediate mesoderm, nephron patterning, epithelial polarity, and angiogenesis, have been isolated. Analysis of mutant phenotypes and the cloning of mutant genes has revealed: (1) a role for bone morphogenetic proteins in patterning the ventral mesoderm, (2) an essential role for the pax2.1 gene in pronephric development, (3) multiple loci required for establishing epithelial polarity in the pronephric duct, (4) a central role for podocytes in directing glomerulogenesis, and (5) 15 loci associated with cystic maldevelopment in the pronephros. The striking similarities of pronephric cell types to those found in higher vertebrates, as well as the conservation of kidney-specific gene expression patterns, suggest that insights gained from studies in zebrafish will be broadly applicable to cell differentiation in the kidney.

Animals↗

PubMed2004

Zebrafish cDNA encoding multifunctional Fatty Acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids.

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the GenBank database identified several expressed sequence tag sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript ZfELO, encoding a polypeptide of 291 amino acid residues, was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame shared high similarity with the elongases of Caenorhabditis elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish open reading frame conferred the ability to lengthen the chain of a range of C18, C20, and C22 polyunsaturated fatty acids, indicating not only that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is feasible, but also that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously.

Acetyltransferases↗

PubMed2005

Characterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafish.

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5' flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.

Animals↗

PubMed2005

Homer 1b/c expression correlates with zebrafish olfactory system development.

The zebrafish, (Danio rerio) is an important model organism for the analysis of molecular mechanisms that govern neuronal circuit development. The neuronal circuitry that mediates olfaction is crucial for the development and survival of all teleost fishes. In concert with other sensory systems, olfaction is functional at early stages in zebrafish development and mediates important behavioral and survival strategies in the developing larva. Odorant cues are transduced by an array of signaling molecules from receptors in olfactory sensory neurons. The scaffolding protein family known as Homer is well placed to orchestrate this signaling cascade by interacting with and coupling membrane bound receptors to cytosolic signaling partners. To date, Homer has not been demonstrated in the zebrafish. Here we report that the Homer 1b/c isoform was prominent in the olfactory system from the earliest stages of differentiation. We describe the spatial and temporal distribution of Homer in the zebrafish olfactory system. At 24 hours post fertilization (hpf), Homer expression delineated the boundary of the presumptive olfactory placode. Subsequent expression steadily increased throughout the developing olfactory placode, with a prominent localization to the dendritic knobs of the olfactory sensory neurons. Homer expression in the developing olfactory bulb was punctate and prominent in the glomeruli, displaying an apparent synaptic localization. This work supports the hypothesis that Homer is an important molecule in neuronal circuit development, necessary for crucial behaviors required for development and survival.

Animals↗

PubMed2005

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system.

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp (green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.

Animals↗

PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗

PubMed2005

Chromosomal organization, evolutionary relationship, and expression of zebrafish GnRH family members.

Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24-26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.

Animals↗

PubMed1994

The development of neurotrophin receptor Trk immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The purpose of this study was to examine the cellular distribution of the Trk family of neurotrophin receptors in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Semithin sections from zebrafish retinae were examined immunohistochemically for the presence of Trk polypeptides using commercially available antisera that cross-react with the fish. Cross-reactivity was confirmed by Western blot. Trk polypeptides were detected at about 1 day of age on the surfaces of retinal neuroblasts and faint Trk immunoreactivity was observed in the primordial optic nerve at 1.5 days. By 2 days the optic nerve was clearly positive for Trk and at 2.5 days Trk immunoreactivity was found in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers, as well as in the optic nerve. At 3 days and 4 days the location of Trk immunoreactivity was unchanged but by 4 days it had diminished in intensity. In the adult zebrafish retina Trk immunoreactivity was found in the same locations as in the embryonic fish, as well as in a population of cells in the middle of the inner nuclear layer and in photoreceptors. We conclude that Trk neurotrophin receptors are present in the zebrafish eye during development and that their persistence in the adult may support the continuous neural reorganization that accompanies the growth of the eye in the fish.

Animals↗

PubMed1995

Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain.

The complete amino-acid sequences of two zebrafish emx homeoproteins containing a homeodomain homologous to that of the Drosophila empty spiracles head gap gene product have been determined by cloning and sequencing of cDNAs. The zebrafish emx1 and emx2 homeoproteins consist of 233 and 247 amino acids, respectively. The zebrafish emx1 and emx2 mRNAs are present at 12 h after fertilization, when the presumptive brain is in a simple tubular structure, and before first primary neurons appear. During brain development, the emx1 mRNA is localized in the dorsal telencephalon, whereas the emx2 mRNA is distributed in the dorsal telencephalon, parts of the diencephalon and the otocyst. The differential expression patterns of the two emx homeoprotein mRNAs may define the subdivisions of the zebrafish telencephalon.

Amino Acid Sequence↗

PubMed1995

Wnt4 affects morphogenesis when misexpressed in the zebrafish embryo.

The Wnts are a family of secreted glycoproteins involved in cell-cell signalling and pattern formation during development, although the extent to which various Wnts are functionally equivalent remains unclear. We have cloned zebrafish (Danio rerio) wnt4, characterized its expression, and compared its activity relative to other Wnts. The wnt4 transcript is first detected early in somitogenesis, in the dorsocaudal region of the forebrain, and then appears in the dorsal and lateral regions of the caudal hindbrain and neural keel. During somitogenesis, wnt4 appears in the floor plate, and this expression is absent in cyclops mutants, which lack floor plate. wnt4 is also expressed in the developing pronephros and gill slit. To characterize the biological activity of wnt4, synthetic zebrafish wnt4 mRNA was injected into embryos of zebrafish and Xenopus laevis. The phenotypic effects of misexpression in the zebrafish include cyclopia, misfolding in the brain, and an anteriorly forking notochord. Comparison of the phenotypes arising from misexpression of wnt4 and Xwnt-5A in both organisms suggests close parallels in the response to these Wnts. Our data suggest that wnt4, like Xwnt-5A, inhibits cell movements, and that these Wnts define a functional class distinct from the class which includes Wnt-1, Xwnt-3A and Xwnt-8.

Amino Acid Sequence↗

PubMed1995

Axis formation in zebrafish.

Recent advances in our understanding of axis formation and patterning in zebrafish relate the developmental mode of this aspiring genetic model organism to higher vertebrates. The effect of UV irradiation and lithium treatment, as well as detailed early lineage analyses, have shed some light on dorsoventral axis formation. However, the molecular mechanism of axis formation, as well as the identity of a fish Nieuwkoop center, are still open issues. A Vg1 homolog is expressed in zebrafish, and activin as well as the mouse nodal gene product have been demonstrated to induce mesoderm and ectopic axes, respectively, in zebrafish. The zebrafish organizer is defined by the expression domains of goosecoid, axial, and lim1. The cyclops gene is involved in maintaining goosecoid expression in axial mesoderm of the head. Large mutagenesis screens provide the basis for a genetic analysis of axis formation.

Animals↗