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PubMed2006

Polymerase chain reaction detection of Pseudoloma neurophilia, a common microsporidian of zebrafish (Danio rerio) reared in research laboratories.

One of the most prevalent pathogens found in zebrafish (Danio rerio) research facilities is the microsporidian parasite Pseudoloma neurophilia. Infections occur primarily in the spinal cord and are associated with emaciation and scoliotic changes. It is unclear why P. neurophilia is so widespread among research colonies, although transfer of infected animals and eggs between laboratories is a likely contributor. In addition to preventing the spread of this pathogen among facilities, it is desirable to have parasite-free fish for use in experiments. Therefore we have developed a polymerase chain reaction (PCR)-based diagnostic test for P. neurophilia. Compared with conventional diagnostic methods, PCR diagnosis is rapid, allows for screening of large numbers of fish, and can be applied to eggs, water filtrates, biofilms, and other samples. Using PCR primers specific to the small subunit ribosomal DNA of P. neurophilia, the test was consistently capable of detecting 10 spores per reaction and often as few as 0.1 spore per reaction, and it did not cross-react with other selected microsporidian species from fish. We recommend this PCR diagnostic assay for use by the research community to determine the presence (or absence) of P. neurophilia in colonies and for screening fish shipped between facilities, especially when parasite-free fish are required for experiments. Furthermore, we currently are using this PCR method to investigate the potential role of vertical transmission in the spread of P. neurophilia.

Animals↗
PubMed1990

Identification of spinal neurons in the embryonic and larval zebrafish.

Previous studies indicated that the developing fish spinal cord was a simple system containing a small number of distinguishable neuronal cell types (Eisen et al., Nature 320:269-271, '86; Kuwada, Science, 233:740-746, '86). To verify this we have characterized the cellular anatomy of the spinal cord of developing zebrafish in order to determine the number, identities, and organization of the spinal neurons. Spinal neurons were labeled by intracellular dye injections, application of an axonal tracer dye to all or subsets of the axonal tracts, and application of antibodies which recognize embryonic neurons. We found that nine classes of neurons could be identified based on soma size and position, pattern of dendrites, axonal trajectory, and time of axonogenesis. These are two classes of axial motor neurons, which have been previously characterized (Myers, J. Comp. Neurol. 236:555-561, '85), one class of sensory neurons, and six classes of interneurons. One of the interneuron classes could be subclassified as primary and secondary based on criteria similar to those used to classify the axial motor neurons into primary and secondary classes. The early cord (18-20 hours) is an extremely simple system and contains approximately 18 lateral cell bodies per hemisegment, which presumably are post-mitotic cells. By this stage, five of the neuronal classes have begun axonogenesis including the primary motor neurons, sensory neurons, and three classes of interneurons. By concentrating on these early stages when the cord is at its simplest, pathfinding by growth cones of known identities can be described in detail. Then it should be possible to test many different mechanisms which may guide growth cones in the vertebrate central nervous system (CNS).

Animals↗
PubMed2003

Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.

In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.

Adenoviridae↗
PubMed1994

Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly.

In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500-1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter. We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endocytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements. We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.

Animals↗
PubMed2006

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Amino Acid Sequence↗
PubMed2003

Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.

BACKGROUND: Drug-induced QT prolongation and torsades de pointes remain significant and often unpredictable clinical problems. Current in vitro preclinical assays are limited by biological simplicity, and in vivo models suffer from expense and low throughput. METHODS AND RESULTS: During a screen for the effects of 100 small molecules on the heart rate of the zebrafish, Danio rerio, we found that drugs that cause QT prolongation in humans consistently caused bradycardia and AV block in the zebrafish. Of 23 such drugs tested, 18 were positive in this initial screen. Poor absorption explained 4 of 5 false-negative results, as demonstrated by microinjection. Overall, 22 of 23 compounds that cause repolarization abnormalities were positive in this assay. Antisense "knockdown" of the zebrafish KCNH2 ortholog yielded bradycardia in a dose dependent manner confirming the effects of reduction of repolarizing potassium current in this model. Classical drug-drug interactions between erythromycin and cisapride, as well as cimetidine and terfenadine, were also reproduced. CONCLUSION: This simple high-throughput assay is a promising addition to the repertoire of preclinical tests for drug-induced repolarization abnormalities. The genetic tractability of the zebrafish will allow the exploration of heritable modifiers of such drug effects.

Animals↗
PubMed2005

High-throughput in vivo screening for bone anabolic compounds with zebrafish.

Osteoporosis and diseases of bone loss are a major public health problem for the present and the future since longevity and prevalence of the disease are increasing in all parts of the world. The bisphosphonates, widely used in the treatment of osteoporosis, act by inhibiting bone resorption. However, there are few agents that promote or increase bone formation in patients who have suffered substantial bone loss. To facilitate the identification of novel anabolic therapies, the authors have developed a rapid, high-throughput in vivo screen using larval zebrafish (Danio rerio) in which they are able to identify agents with anabolic effects in the skeleton within a 6-day time period. Vitamin D3 analogs and intermittent parathyroid hormone (PTH) result in dose-dependent increases in the formation of mineralized bone, whereas continuous exposure to PTH results in net bone loss. Because this model is fast, economical, and genetically tractable, it provides a powerful adjunct to mammalian models for the identification of new anabolic bone agents and offers the potential for genetic elucidation of pathways important in osteoblastic activity.

Animals↗
PubMed1999

Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis.

Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.

Animals↗
PubMed2004

Artemin-like immunoreactivity in the zebrafish, Danio rerio.

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.

Animals↗
PubMed2001

Moving towards the next generation.

In most organisms, primordial germ cells are set aside from the cells of the body early in development. To form an embryonic gonad, germ cells often have to migrate along complex routes through and along diverse tissues until they reach the somatic part of the gonad. Recent advances have been made in the genetic analysis of these early stages of germ line development. Here we review findings from Drosophila, zebrafish, and mouse; each organism provides unique insight into the mechanisms that determine germ cell fate and the cues that may guide their migration.

Animals↗
PubMed2005

Development of putative GABAergic neurons in the appendicularian urochordate Oikopleura dioica.

Studying the developing brain of urochordates can increase our understanding of brain evolution in the chordate lineage. To begin addressing regional patterns of neuronal differentiation in appendicularian urochordates, we examined the development of putative GABAergic neurons in Oikopleura dioica using GABA immunohistochemistry and in situ hybridization for the GABA-synthesizing enzyme GAD. First, we assessed the developmental dynamics of neuron number and organization in the cerebral and caudal ganglia. We then identified and mapped the positions of putative GABAergic neurons using confocal microscopy. We found GAD mRNA-positive and GABA-immunopositive neurons in the first brain nerves and the cerebral and caudal ganglia, but not in the caudal nerve cord. In both ganglia GAD mRNA-positive and GABA-immunopositive neurons are found in the same characteristic intraganglionic locations. The differentiation of these GABAergic markers occurs first in the first brain nerves and the cerebral ganglion and then with a several-hour delay in the caudal ganglion. In all three structures GAD mRNA expression appears 2-3 hours prior to GABA expression. In general, GABA is expressed by the same number of neurons as express GAD. Several discrepancies suggest differential regulation of the GABAergic phenotype in different neurons, however. Our results show that the GABAergic phenotype has a stereotyped pattern of expression along the anteroposterior axis of the CNS. Given recent genome sequencing and developmental patterning gene studies in this species, the GABAergic neurons in O. dioica provide a good model for assessing, at the invertebrate-vertebrate transition, the molecular mechanisms that specify the GABAergic phenotype.

Age Factors↗
PubMed2006

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin.

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7 x 10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Animals↗
PubMed2004

Modelling approaches for angiogenesis.

The development of a functional vasculature within a tumour is a requisite for its growth and progression. This fact has led to the design of therapies directed toward the tumour vasculature, aiming either to prevent the formation of new vessels (anti-angiogenic) or to damage existing vessels (vascular targeting). The development of agents with different mechanisms of action requires powerful preclinical models for the analysis and optimization of these therapies. This review concerns 'classical' assays of angiogenesis in vitro and in vivo, recent approaches to target identification (analysis of gene and protein expression), and the study of morphological and functional changes in the vasculature in vivo (imaging techniques). It mainly describes assays designed for anti-angiogenic compounds, indicating, where possible, their application to the study of vascular-targeting agents.

Angiogenesis Inhibitors↗