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At least 721 records · Page 40Linked to original sources

PubMed2003

Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish.

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage lambda causes transposition primarily into the vicinity of the lambda operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.

Animals↗

PubMed1992

Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea.

Recessive lethal mutations and mutations at the gol-1 locus were induced in the zebrafish by exposure of mature sperm to the alkylating agent ethyl nitrosourea (ENU). Embryonic lethal phenotypes were recognized among the parthenogenetic progeny of mutagenized animals or among the progeny of daughters of mutagenized animals. Novel specific locus mutations were identified by the failure of mutagenized chromosomes to complement pre-existing mutant alleles at the gol-1 locus. Each mutagenized individual harboured approximately 10 embryonic lethal mutations in its germ line and about 1 in 500 mutagenized animals harboured a new mutation at the gol-1 locus. Three lines of evidence indicate that the majority of mutations that were recovered following treatment of mature sperm with ENU were probably point mutations. First, the soma and germ lines of mutagenized animals were mosaic, as expected following simple alkylation of sperm DNA. Second, mutations induced by ENU at the gol-1 locus affected pigmentation but not viability, unlike the majority of mutations induced at this locus with gamma-irradiation. Third, the ratio of specific locus:recessive lethal mutations induced by ENU was approximately 50-fold lower than the ratio observed following mutagenesis with gamma-rays. Comparison of the incidence with which embryonic recessive lethal mutations were induced with the incidence with which specific locus mutations arose indicates that there are greater than 5000 genes essential to the development and viability of the zebrafish embryo.

Alkylation↗

PubMed2005

Specific RNAi mediated gene knockdown in zebrafish cell lines.

Here we demonstrate highly efficient RNA interference in ZFL, SJD and ZF4 cell lines derived from adult and embryonic zebrafish Danio rerio. Microinjection of siRNAs resulted in silencing in almost 100% of cells while transfection using cationic liposomes led to silencing in 30%. Use of siRNAs against zebrafish lamin A, lamin B2 and the motor protein Eg5, led to knockdown of the target genes with the specific phenotypes expected from prior studies in mammalian cells. In contrast injection of lamin A, GL2 and eGFP siRNAs into zebrafish embryos resulted in morphological defects, abnormal development and early death of most embryos. The results indicate unspecific responses to siRNAs in the embryo but a fully developed and active RNAi machinery in cell lines.

Animals↗

PubMed1993

Targeted ablation of diverse cell classes in the nervous system in vivo.

The study of both the function and development of complex neural systems would be greatly facilitated by a means for systematically blocking intercell communication. One way of preventing cells from signaling each other is to remove them from the system by ablation. Here we present a general technique for visualizing and ablating selected cell classes in vivo. The cells of interest are genetically engineered so that they can be selectively labeled with a photoactivatable dye and visualized in living preparations; the dye-labeled cells can then be photoablated. This approach is applicable to a broad range of cell types, in organisms amenable to gene transfer, and permits ablations to be performed at different developmental stages or in the adult. We demonstrate the use of this technique on several cell types in the mouse retina and cerebral cortex, and in the zebrafish embryo.

Affinity Labels↗

PubMed2000

A novel pbx family member expressed during early zebrafish embryogenesis forms trimeric complexes with Meis3 and Hoxb1b.

pbx genes encode homeodomain-containing transcriptional regulators that interact with other proteins to control embryogenesis and tumorigenesis. We present the characterization of a zebrafish pbx CDNA that appears to encode a novel family member, pbx4. pbx4 RNA is maternally deposited and is detected throughout the zebrafish embryo during blastula stages. It becomes excluded from ventroanterior structures at late gastrula stages and is detected within the forming central nervous system during segmentation stages. pbx4 expression overlaps with that of two other homeobox genes, hoxb1b and meis3, in the region of the presumptive caudal hindbrain during gastrula stages. In vitro binding experiments revealed that protein complexes containing Pbx4/Meis3 and Pbx4/Hoxb1b, but not Meis3/Hoxb1b could be generated. A novel trimeric complex containing Pbx4, Meis3, and Hoxb1b was also formed. We speculate that complexes with different combinations of Pbx4, Meis3, and Hoxb1b specify different developmental fates during vertebrate embryogenesis. Dev Dyn 2000;217:109-119.

Amino Acid Sequence↗

PubMed2003

Alk8 is required for neural crest cell formation and development of pharyngeal arch cartilages.

The type I TGFbeta family member receptor alk8 acts in bone morphogenetic protein (BMP) signaling pathways to establish dorsoventral patterning in the early zebrafish embryo. Here, we present evidence that alk8 is required for neural crest cell (NCC) formation and that alk8 signaling gradients direct the proper patterning of premigratory NCCs. We extend our previous functional studies of alk8 to demonstrate that ectopic expression of constitutively active and dominant negative Alk8, consistently results in more medially or laterally positioned premigratory NCCs, respectively. We also demonstrate that patterning defects in premigratory NCCs, induced by alk8 misexpression, correlate with subsequent defects in NCC-derived pharyngeal arch cartilages. Furthermore, an anteroposterior effect is revealed, where overexpression of Alk8 more severely affects anterior arch cartilages and decreased Alk8 activity more severely affects posterior arch cartilage formation. Ectopic expression studies of alk8 are supported by analyses of zygotic and maternal-zygotic laf/alk8 mutants and of several BMP pathway mutants. Pharyngeal mesodermal and endodermal defects in laf/alk8 mutants suggest additional roles for alk8 in patterning of these tissues. Our results provide insight into alk8-mediated BMP signaling gradients and the establishment of premigratory NCC mediolateral positioning, and extend the model for BMP patterning of the neural crest to include that of NCC-derived pharyngeal arch cartilages.

Activin Receptors, Type I↗

PubMed2001

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio).

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.

Alcohol Dehydrogenase↗

PubMedDate not supplied

Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development.

Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C(18), we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C(18) labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C(18) into the circulation of zebrafish embryos and identified the presence of DiI-C(18)-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.

Animals↗

PubMed2005

Vertebrate Smoothened functions at the primary cilium.

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

Amino Acid Motifs↗

PubMed1999

Maturation of neuromuscular transmission during early development in zebrafish.

We have examined the rapid development of synaptic transmission at the neuromuscular junction (NMJ) in zebrafish embryos and larvae by patch-clamp recording of spontaneous miniature endplate currents (mEPCs) and single acetylcholine receptor (AChR) channels. Embryonic (24-36 h) mEPCs recorded in vivo were small in amplitude (<50 pA). The rate of mEPCs increased in larvae (3.5-fold increase measured by 6 days), and these mEPCs were mostly of larger amplitude (10-fold on average) with (</=5-fold) faster kinetics. Intracellular labeling with Lucifer yellow indicated extensive coupling between muscle cells in both embryos and larvae (</=10 days). Blocking acetylcholinesterase (AChE) with eserine had no effect on mEPC kinetics in embryos at 1 day and only partially slowed (by approximately 1/2) the decay rate in larvae at 6 days. In acutely dissociated muscle cells, we observed the same two types of AChR with conductances of 45 and 60 pS and with similar, brief (<0.5 ms) mean open times in both embryos and larvae. We conclude that AChR properties are set early during development at these early stages; functional maturation of the NMJ is only partly shaped by expression of AChE and may also depend on postsynaptic AChR clustering and presynaptic maturation.

Animals↗

PubMed1991

Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis.

The paired box-containing (pax) gene family encodes a group of putative transcription factors differentially expressed during embryonic development. In this study, we describe the cloning and expression of a zebrafish gene pax[zf-b], which most probably is a direct homologue to the mouse Pax2 gene. The putative protein encoded by pax[zf-b] contains a paired box, an octapeptide, but no homeobox. However, a region of homology to the N-terminal half of paired-type homeoboxes is detected C-terminal to the pax[zf-b] paired domain. In zebrafish embryos, pax[zf-b] transcripts are first seen during the formation of the neural keel. At 9-10 h of development, two laterally located transverse stripes of cells expressing the gene appear in the rostral 1/3 of the embryo. The two areas subsequently move towards the midline and form the posterior portion of the midbrain. In the following stages of development, at 10-12 h, transcripts are detected in the otic placode, the Wolffian duct including the nephritic primodium and in the optic stalk. At a later time point, beginning at 14-15 h, single cells along the spinal cord, presumably interneurons, start to express the gene. The characteristic expression pattern of pax[zf-b] in the neural tube suggests an involvement of this gene in the regionalization of the midbrain as well as in the specification of neuronal cell fates at early embryonic stages.

Amino Acid Sequence↗

PubMed1998

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish.

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.

Amino Acid Sequence↗

PubMed1995

Anterior duplication of the Sonic hedgehog expression pattern in the pectoral fin buds of zebrafish treated with retinoic acid.

The Sonic hedgehog gene has been identified as a candidate for the signal mediating the function of the zone of polarizing activity (ZPA) during limb development in tetrapods. To better understand the early steps of development of paired fin buds in fish, we have analyzed the regulation of the zebrafish Sonic hedgehog gene (shh/vhh-1) in response to retinoic acid. Systemic administration of retinoic acid (RA) to zebrafish embryos during the initial stages of pectoral fin bud development resulted in the induction of ectopic expression of shh/vhh-1 on the anterior margin of the bud under the apical ectodermal ridge and in abnormal pectoral fin bud morphology. RA treatment also resulted in ectopic shh/vhh-1 expression in floor plate cells at the caudal end of the neural keel. These results suggest that the control of ZPA function during the initial stages of development of paired appendages has been conserved between fish and tetrapods.

Abnormalities, Drug-Induced↗

PubMed1992

Growth cone guidance in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have allowed researchers to make a detailed characterization of pathfinding by identifiable growth cones. The growth cones follow precise cell-specific pathways to their targets. Analyses of pathfinding in mutant and experimentally manipulated wild type embryos have shown that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant.

Animals↗

PubMed2002

A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish.

Inhibition of amyloid beta-peptide (Abeta) production by blocking gamma-secretase activity is at present one of the most promising therapeutic strategies to slow progression of Alzheimer's disease pathology. gamma-secretase inhibitors apparently block Abeta generation via interference with presenilin (PS) function. Besides being an essential component of the gamma-secretase complex, PS itself may be an aspartyl protease with gamma-secretase activity, which is not only required for Abeta production but also for a similar proteolytic process involved in Notch signaling. Here we demonstrate that treatment of zebrafish embryos with a known gamma-secretase inhibitor affects embryonic development in a manner indistinguishable from Notch signaling deficiencies at morphological, molecular and biochemical levels. This indicates severe side-effects of gamma-secretase inhibitors in any Notch-dependent cell fate decision and demonstrates that the zebrafish is an ideal vertebrate system to validate compounds that selectively affect Abeta production, but not Notch signaling, under in vivo conditions.

Alzheimer Disease↗

PubMed2006

Polarization and orientation of retinal ganglion cells in vivo.

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.

Animals↗

PubMed2000

Analysis of promoter activity of 5'-upstream regions of zebrafish olfactory receptor genes.

The vertebrate olfactory system receives and discriminates a great variety of odorants. Many lines of evidence suggest that individual olfactory neuron expresses a single type or limited types of the olfactory receptor genes. However, the mechanism of selection of a single gene in the olfactory receptor family remains unclear. In the present study, we utilized zebrafish to identify the promoter element of the olfactory receptor genes in their 5'-upstream regions. First, we isolated a number of zebrafish olfactory receptor genes. These olfactory receptor genes were specifically expressed in the olfactory tissue as visualized by whole mount in situ hybridization analysis. Time of onset of the expression of each receptor clone varied from 24 h to 48 h postfertilization. Then, we injected various constructs containing the 5'-upstream regions of the olfactory receptor genes connected to beta-galactosidase reporter gene into fertilized zebrafish embryos. Constructs from two independent olfactory receptor genes exerted beta-galactosidase (promoter) activity that is specifically upregulated in the olfactory tissue. Use of either longer or deleted constructs of these two genes diminished the promoter activity in the olfactory tissue. From these results we discuss the mechanism of the transcription of the olfactory receptor genes in the olfactory neurons.

Amino Acid Sequence↗

PubMed2004

WNT-frizzled signaling via cyclic GMP.

Wnt-Frizzled signaling is an essential aspect of early development, regulating cell fate, polarity, differentiation, and migration. In addition to the well-known Wnt/beta-catenin pathway characterized for Frizzled-1, there are other pathways regulated by Wnts that are not mediated by Frizzled-1 and do not lead to stabilization of beta-catenin and activation of the Lef/Tcf-sensitive transcription of genes. The first of these non-canonical pathways to be identified is the Wnt/Ca++ pathway in which Frizzled-2 activation leads to release of beta/gamma subunit complexes from heterotrimeric G-proteins (presumably Go and Gt) to activate phospholipase C and other effectors to stimulate a mobilization of intracellular Ca++. More recently a second, related pathway of Wnt-Frizzled signaling has been discovered that regulates the intracellular levels of cyclic GMP. Frizzled-2, established as a member of the family of 7TMS receptors that couple by heterotrimeric G-proteins to effectors, can signal via the G-protein Gt2, transducin, a G-protein prominent in phototransduction in the eye, to cyclic GMP phosphodiesterase. The discovery of the expression of Gt2 in embryonic cells was co-incident with the demonstration that inhibitors of cyclic GMP phosphodiesterase potently blocked various features of Frizzled-2 signaling in mouse embryonic F9 cells and in zebrafish embryos. The signal linkage map from Wnt to changes in intracellular cyclic GMP and development is the focus of this review. The molecular features of how changes in intracellular cyclic GMP concentrations control development remain to be elucidated.

3',5'-Cyclic-GMP Phosphodiesterases↗