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PubMed1999

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Animals↗

PubMed2001

The art and design of genetic screens: zebrafish.

Inventive genetic screens in zebrafish are revealing new genetic pathways that control vertebrate development, disease and behaviour. By exploiting the versatility of zebrafish, biological processes that had been previously obscured can be visualized and many of the responsible genes can be isolated. Coupled with gene knockdown and overexpression technologies, and small-molecule-induced phenotypes, genetic screens in zebrafish provide a powerful system by which to dissect vertebrate gene function and gene networks.

Animals↗

PubMed2006

Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish.

The small size and optical transparency of zebrafish embryos and larvae greatly facilitate modern intravital microscopic phenotyping of these experimentally tractable laboratory animals. Neither the experimentally derived dose-response relationships for chemicals commonly used in the mounting of live fish larvae, nor their effect on the stress of the animal, are currently available in the research literature. This is particularly problematic for IACUCs attempting to maintain the highest ethical standards of animal care in the face of a recent spate in investigator-initiated requests to use embryonic zebrafish as experimental models. The authors address this issue by describing the dose-dependent efficacy of several commonly used chemical mounting treatments and their effect on one stress parameter, embryo heart rate. The results of this study empirically define, for the first time, effective, minimally stressful treatments for immobilization and in vivo visualization during early zebrafish development.

Aminobenzoates↗

PubMed2005

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.

Amino Acid Sequence↗

PubMed2002

Cloned zebrafish by nuclear transfer from long-term-cultured cells.

Although mammals have been cloned from genetically manipulated cultured cells, a comparable achievement has not been realized in lower vertebrates. Here we report that fertile transgenic zebrafish can be obtained by nuclear transfer using embryonic fibroblast cells from long-term cultures. The donor nuclei, modified by retroviral insertions expressing green fluorescent protein (GFP), were transplanted into manually enucleated eggs. Overall, a 2% success rate was achieved, resulting in 11 adult transgenic zebrafish expressing GFP. These nuclear transplants produced fertile, diploid offspring, and their F1/F2 progeny continued to express GFP in a pattern identical to that of the founder fish. This finding demonstrates that slowly dividing nuclei from cultured cells can be reprogrammed to support rapid embryonic development and sets up a foundation for targeted genetic manipulation in zebrafish.

Animals↗

PubMed2003

Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.

The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.

Animals↗

PubMed2003

Cancer genetics and drug discovery in the zebrafish.

Fish have a long history of use in cancer toxicology studies, because they develop neoplasms that are histologically similar to human cancers. Because of considerable progress in zebrafish genetics and genomics over the past few years, the zebrafish system has provided many useful tools for studying basic biological processes. These tools include forward genetic screens, transgenic models, specific gene disruptions and small-molecule screens. By combining carcinogenesis assays, genetic analyses and small-molecule screening techniques, the zebrafish is emerging as a powerful system for identifying novel cancer genes and for cancer drug discovery.

Animals↗

PubMed2005

In vivo drug discovery in the zebrafish.

The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.

Animals↗

PubMed2005

The zebrafish: a new model of T-cell and thymic development.

T-cell and thymic development are processes that have been highly conserved throughout vertebrate evolution. Mammals, birds, reptiles and fish share common molecular signalling pathways that regulate the development of the adaptive immune system. This Review article focuses on defining the similarities and differences between zebrafish and mammalian T-cell immunobiology, and it highlights the advantages of using the zebrafish as a genetic model to uncover mutations that affect T-cell and thymic development. Finally, we summarize the use of the zebrafish as a new model for assessing stem-cell function and for drug discovery.

Animals↗

PubMed2006

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis.

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.

Animals↗

PubMed1998

Independent duplications of Bf and C3 complement genes in the zebrafish.

As part of an ongoing project aimed at the characterization of the MHC system in bony fishes, we have attempted to identify the class III region in the zebrafish (a teleost), a region that in higher vertebrates contains genes coding for complement proteins C2, Bf and C4. We obtained several genomic PAC clones by hybridization with a zebrafish Bf probe, previously identified in our laboratory, and searched these for the presence of other class III genes. We were able to obtain a second Bf-like gene, however, we were unable to detect any C2- or C4-like genes. By using highly degenerated primers, we extended our search to a hepatopancreas cDNA library and amplified from it clones corresponding to three different C3-like genes, and also the Bf genes, but not any C2- or C4-like genes. The zebrafish therefore contains two Bf and three C3 loci but apparently no C2 and C4 loci. Independent duplications of the Bf and C3 genes in bony fishes suggest that complement plays a prominent part in the immune response of this class of vertebrates.

Amino Acid Sequence↗

PubMed1997

The zebrafish thyroid hormone receptor alpha 1 is expressed during early embryogenesis and can function in transcriptional repression.

Nuclear receptors are a large family of ligand dependent transcription factors which participate in many diverse processes during development. In this report, we describe the cloning of the zebrafish thyroid hormone receptor alpha 1 (TR alpha 1) gene, the cellular counterpart of the viral oncogene v-erbA. TR alpha 1 is expressed during oogenesis and maternally supplied to the embryo. TR alpha 1 is expressed again after the mid blastula transition. By examining the effects of increased expression of TR alpha 1 on expression of a reporter gene which responds to both TR alpha 1 and retinoic acid receptors (RARs), we show that the zebrafish TR alpha 1 can act as a repressor during early zebrafish development before thyroid hormone is present in the embryo. In addition, our data suggest that TR alpha 1 can repress retinoic acid (RA)-signaling during early development. We propose that TR alpha 1 functions during early development as a transcriptional repressor, similar to the constitutive repressor activity of its viral counterpart v-erbA, which regulates anterior-posterior (A/P) patterning by repressing RA-signaling.

Amino Acid Sequence↗

PubMed2000

Type I and type II cytokeratin cDNAs from the zebrafish (Danio rerio) and expression patterns during early development.

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1,426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2,398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73%-91% and 87%-93% similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184-205 and 191-212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.

Aging↗

PubMed1999

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Activin Receptors, Type II↗

PubMed1998

Differential expression of voltage-gated K+ and Ca2+ currents in bipolar cells in the zebrafish retinal slice.

Whole-cell voltage-gated currents were recorded from bipolar cells in the zebrafish retinal slice. Two physiological populations of bipolar cells were identified. In the first, depolarizing voltage steps elicited a rapidly activating A-current that reached peak amplitude < or = 5 ms of step onset. IA was antagonized by external tetraethylammonium or 4-aminopyridine, and by intracellular caesium. The second population expressed a delayed rectifying potassium current (IK) that reached peak amplitude > or = 10 ms after step onset and did not inactivate. IK was antagonized by internal caesium and external tetraethylammonium. Bipolar cells expressing IK also expressed a time-dependent h-current at membrane potentials < -50 mV. Ih was sensitive to external caesium and barium, and was also reduced by Na+-free Ringer. In both groups, a calcium current (ICa) and a calcium-dependent potassium current (IK(Ca)) were identified. Depolarizing voltage steps > -50 mV activated ICa, which reached peak amplitude between -20 and -10 mV. ICa was eliminated in Ca+2-free Ringer and blocked by cadmium and cobalt, but not tetrodotoxin. In most cells, Ica was transient, activating rapidly at -50 mV. This current was antagonized by nickel. The remaining bipolar cells expressed a nifedipine-sensitive sustained current that activated between -40 and -30 mV, with both slower kinetics and smaller amplitude than transient ICa. IK(Ca) was elicited by membrane depolarizations > -20 mV. Bipolar cells in the zebrafish retinal slice preparation express an array of voltage-gated currents which contribute to non-linear I-V characteristics. The zebrafish retinal slice preparation is well-suited to patch clamp analyses of membrane mechanisms and provides a suitable model for studying genetic defects in visual system development.

Animals↗

PubMed1998

Development of the histaminergic neurons and expression of histidine decarboxylase mRNA in the zebrafish brain in the absence of all peripheral histaminergic systems.

The histamine-storing neural system in adult and developing zebrafish (Danio rerio) was studied with immunocytochemical and chromatographical methods. Furthermore, the gene for histidine decarboxylase was partially cloned and its expression mapped with in situ hybridization. The histamine-storing neurons were only seen in the caudal hypothalamus, around the posterior recess of the diencephalic ventricle. Almost all parts of the brain, except the cerebellum, contained at least some histamine-immunoreactive fibres. The ascending projections had the rostral part of the dorsal telencephalon as a major target. Descending projections terminated in the torus semicircularis, central grey and inferior olive. A prominent innervation of the optic tectum, which has not been reported in other fish, was seen. The in situ hybridization gave a strong signal in cells with the same anatomical position as the histamine-immunoreactive neurons. The first histamine-immunoreactive neurons appeared in the ventral hypothalamus at about 85 h post-fertilization, and at 90 h, immunoreactive fibres terminated in the dorsal telencephalon. The embryonic histamine production described in mammals was lacking in this species. Both immunocytochemical and chromatographical studies indicated that histamine is absent in all other parts of the zebrafish body, and no specific hybridization was seen in any other part of the fish than the hypothalamus. The zebrafish could therefore be a very useful model for pharmacological in vivo studies of the histaminergic system of the brain, since the powerful peripheral actions of histamine should be lacking in this species.

Age Factors↗

PubMed2003

Retinal function and morphology in two zebrafish models of oculo-renal syndromes.

We characterized visual system defects in two recessive zebrafish mutants oval and elipsa. These mutants share the syndromic phenotype of outer retinal dystrophy in conjunction with cystic renal disorder. We tested the function of the larval visual system in a behavioural assay, eliciting optokinetic eye movements by high-contrast motion stimulation while recording eye movements in parallel. Visual stimulation did not elicit eye movements in mutant larvae, while spontaneous eye movements could be observed. The retina proved to be unresponsive to light using electroretinography, indicative of a defect in the outer retina. Histological analysis of mutant retinas revealed progressive degeneration of photoreceptors, initiated in central retinal locations and spreading to more peripheral regions with increasing age. The inner retina remains unaffected by the mutation. Photoreceptors display cell type-specific immunoreactivity prior to apoptotic cell death, arguing for a dystrophic defect. Genomic mapping employing simple sequence-length polymorphisms located both mutations on different regions of zebrafish linkage group 9. These mutants may serve as accessible animal models of human outer retinal dystrophies, including oculo-renal diseases, and show the general usefulness of a behavioural genetic approach to study visual system development in the model vertebrate zebrafish.

Animals↗

PubMed1999

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration.

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗