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At least 1,189 records · Page 66Linked to original sources
PubMed1998

Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish.

Sonic hedgehog (Shh) is a secreted protein that is involved in the organization and patterning of several tissues in vertebrates. We show that the zebrafish sonic-you (syu) gene, a member of a group of five genes required for somite patterning, is encoding Shh. Embryos mutant for a deletion of syu display defects in patterning of the somites, the lateral floor plate cells, the pectoral fins, the axons of motorneurons and the retinal ganglion cells. In contrast to mouse embryos lacking Shh activity, syu mutant embryos do form medial floor plate cells and motorneurons. Since ectopic overexpression of shh in zebrafish embryos does not induce ectopic medial floor plate cells, we conclude that shh is neither required nor sufficient to induce this cell type in the zebrafish.

Animals↗
PubMed2002

Expression of zebrafish cyp11a1 as a maternal transcript and in yolk syncytial layer.

Cyp11a1 (P450scc, cholesterol side-chain cleavage enzyme) is the first enzyme for the synthesis of all steroid hormones. The regulation of steroid synthesis has been extensively investigated, except during embryogenesis. To study steroidogenesis in embryos, we have isolated the zebrafish cyp11a1 gene, which consists of 11 exons. Reverse transcription-polymerase chain reaction analysis indicates that zebrafish cyp11a1 is expressed temporally in two waves during embryonic stages and when sexual differentiation begins. It is expressed in adult brain, testicular Leydig cells, and the granulosa/theca layer of the ovary. In addition, zebrafish cyp11a1 is expressed in oocytes, and is inherited as a maternal transcript in early embryos. Throughout zebrafish epiboly and segmentation stages, cyp11a1 is expressed in the yolk syncytial layer. At 36 h post fertilization, cyp11a1 transcript is located ventral to the third somite, where the primordial interrenal gland is located. In summary, zebrafish cyp11a1 is expressed in the cytoplasm of oocytes, as a maternal transcript, and in yolk syncytial layer during early embryogenesis.

Amino Acid Sequence↗
PubMed1994

Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos.

goosecoid is an immediate early gene expressed at the dorsal blastoporal lip of the Xenopus gastrula. Microinjection experiments have suggested a direct role for goosecoid in organizing the dorsoventral axis of the frog embryo. Here we characterize the zebrafish homologue of goosecoid (gsc) and compare its expression to that of Brachyury or no tail (ntl), another immediate early gene required in developing mesoderm. We show that gsc exhibits two independent phases of expression: an early one in cells anterior to the presumptive notochord, but not in cells of the notochord itself, and a later one in neural crest derivatives in the larval head. Zygotic gsc transcripts are detected soon after the midblastula transition, and at the blastula stage form a gradient with a maximum at the dorsal side. Use of gsc as a dorsal marker allowed us to demonstrate that ntl expression is initially activated at the dorsal side of the blastula. At this early stage, gsc and ntl show overlapping domains of expression and are co-expressed in cells at the dorsal midline of the early gastrula. However, gsc- and ntl-expressing cells become separated in the course of gastrulation, with gsc being expressed in the axial hypoblast (prechordal plate) anterior to the ntl-expressing presumptive notochord cells. Studies with mutant embryos suggest that gsc is independent of ntl function in vivo.

Amino Acid Sequence↗
PubMed2005

Making waves in cancer research: new models in the zebrafish.

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.

Animals↗
PubMed1994

Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture.

1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.

Animals↗
PubMed2004

The calcium pump of the endoplasmic reticulum plays a role in midline signaling during early zebrafish development.

During early vertebrate development, a signaling network is activated along the midline of the embryo. This signaling network induces the neural tube floor plate and ventral brain regions. In turn, induction of the ventral brain region is important for bilateral division of the forebrain and bilateral separation of the eyes. The present study provides direct evidence for a role of the endoplasmic reticulum Ca(2+) pump in zebrafish midline signaling. The endoplasmic reticulum Ca(2+) pump was inhibited in zebrafish embryos using thapsigargin or cyclopiazonic acid. Inhibition of the endoplasmic reticulum Ca(2+) pump during early gastrulation induces cyclopia, mimicking defects observed in cyclops, squint, one-eyed pinhead, and silberblick mutant embryos. In contrast, inhibition of the endoplasmic reticulum Ca(2+) pump during mid-gastrulation does not induce cyclopia, but does induce tail defects, mimicking defects observed in no-tail mutant embryos. This study is the first to relate thapsigargin and cyclopiazonic acid with induction of cyclopia. In addition, obtained results provide new information on the roles of Ca(2+) in embryonic development and may lead to new insights on the mechanisms underlying holoprosencephaly, a relatively common brain defect in human development.

Animals↗
PubMed2004

Developmental biology of zebrafish.

The biology and genetics of zebrafish has become a very popular system in developmental biology especially because of their suitability for genetics and the transparent embryo of this small tropical fish. Studies reported briefly here demonstrate that one can use gene expression in the zebrafish embryo to find new components of important and well-studied developmental mechanisms and signal transduction pathways. The study of zebrafish developmental genetics has proven invaluable in determining many aspects of vertebrate development. Further use of this model organism promises to generate many interesting and useful data.

Animals↗
PubMed1997

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Animals↗
PubMed2005

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis.

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.

Animals↗
PubMed2003

Expression analysis of a tyrosinase promoter sequence in zebrafish.

Sequence comparisons and functional analysis of the 5' upstream regions of tyrosinase genes have revealed the importance of cis-regulatory elements acting to control the spatiotemporal expression of tyrosinase in the melanocytes and retinal pigmented epithelium of developing embryos. To date there are no reports addressing the control of tyrosinase gene transcription in zebrafish, a vertebrate model organism of increasing importance. To exploit the tyrosinase gene as a marker in zebrafish we set out to clone its promoter and analyse its regulation during embryogenesis. Amplification of a zebrafish tyrosinase complementary DNA fragment by reverse transcriptase polymerase chain reaction allowed us to isolate and sequence a 1041 nt genomic DNA fragment that includes a transcription initiation site and 73 nt of the open reading frame. Bioinformatic analysis of this genomic sequence revealed five E-box motifs, including one CATGTG type E-box present in a putative initiation region. These are conserved positive regulatory elements in vertebrate tyrosinase promoters. We show that a region of 814 nt upstream from the translation start site of the zebrafish tyrosinase gene can drive expression in retinal pigmented epithelium in transiently transgenic zebrafish embryos but that its activity is not restricted to melanin-producing cells. This region is unable to drive transcription in human melanoma cell lines. Ectopic expression from this zebrafish tyrosinase promoter fragment is probably due to the absence of positive and negative cis-regulatory elements, such as a tyrosinase distal element, which is known to function as a pigment cell-specific enhancer.

Animals↗
PubMed2004

Regulation of vertebrate eye development by Rx genes.

The paired-like homeobox-containing gene Rx has a critical role in the eye development of several vertebrate species including Xenopus, mouse, chicken, medaka, zebrafish and human. Rx is initially expressed in the anterior neural region of developing embryos, and later in the retina and ventral hypothalamus. Abnormal regulation or function of Rx results in severe abnormalities of eye formation. Overexpression of Rx in Xenopus and zebrafish embryos leads to overproliferation of retinal cells. A targeted elimination of Rx in mice results in a lack of eye formation. Mutations in Rx genes are the cause of the mouse mutation eyeless (ey1), the medaka temperature sensitive mutation eyeless (el) and the zebrafish mutation chokh. In humans, mutations in Rx lead to anophthalmia. All of these studies indicate that Rx genes are key factors in vertebrate eye formation. Because these results cannot be easily reconciled with the most popular dogmas of the field, we offer our interpretation of eye development and evolution.

Animals↗
PubMed1997

Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.

Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.

Amino Acid Sequence↗
PubMed2000

Effective targeted gene 'knockdown' in zebrafish.

The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.

Animals↗
PubMed2001

The zebrafish as a novel system for functional genomics and therapeutic development applications.

Unconventional antisense technology has entered the mainstream for both therapeutic and functional genomics applications in a variety of biological settings. Further development of this approach has been hampered by the high cost and limited information obtained with standard bioassays and animal models. The embryo of the zebrafish Danio rerio offers both biologists and technologists a new strategy that rapidly garners efficacy, toxicity and specificity data in an in vivo setting. This system has been used to optimize current antisense targeting methods, and it provides an ideal initial assay system for the development of new chemistries or other new gene targeting approaches.

Animals↗
PubMed2000

Heat-inducible expression of a reporter gene detected by transient assay in zebrafish.

Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing beta-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35-36 degrees C up to 39 degrees C with an apparent decrease at 40 degrees C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5' UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish.

Animals↗
PubMed2001

Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene.

We have cloned and characterized a tilapia (Oreochromis mossambicus) L18 ribosomal protein gene, including the complete transcribed region and 488 bp of upstream regulatory sequences. We have also isolated two L18 cDNAs from another tilapia (Oreochromis niloticus) with a few conservative nucleotide differences. Our results suggest the presence of two genes in both species. Reporter constructs were tested for transient expression in CV1 cells and in microinjected zebrafish and tilapia embryos. The tilapia L18 promoter was able to drive expression of the reporter gene in all three experiments, with no apparent preference for a particular tissue. The tilapia L18 promoter is therefore likely to be a powerful tool to drive tissue-independent gene expression in fish.

Amino Acid Sequence↗