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PubMed2005

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants.

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.

Animals↗

PubMed2004

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates.

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.

Amino Acid Sequence↗

PubMed2004

Cadherin-2 and cadherin-4 in developing, adult and regenerating zebrafish cerebellum.

Cadherins are cell adhesion molecules that regulate development of a variety of tissues and maintenance of adult structures. In this study, we examined expression of two zebrafish classical cadherins, cadherin-2 and cadherin-4, in the cerebellum of developing, normal adult, and regenerating adult zebrafish using in situ hybridization and immunohistochemical methods. Cadherin-2 was widely expressed by the cerebellum of embryonic (24-50-h post fertilization) and larval zebrafish (3-14 days). Cadherin-2 expression became much reduced in the adult cerebellum, but it was greatly up-regulated in the regenerating adult cerebellum. Cadherin-4 was not detected in the embryonic cerebellum, but it was expressed in the Purkinje cells of the larval and adult cerebellum. To gain insight into cadherin-2 role in the formation of the cerebellum, we analyzed embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO1), and found that the cerebellar development of the cdh2MO1-injected embryos was severely disrupted. This phenotype was confirmed by examining a cadherin-2 mutant, glass onion. Our results suggest that cadherins are crucial for the normal development of the zebrafish cerebellum, and they may also be involved in the regeneration of injured fish cerebellum.

Animals↗

PubMed2005

Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos.

The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.

Abnormalities, Drug-Induced↗

PubMedDate not supplied

Comparative aspects of zebrafish (Danio rerio) as a model for aging research.

The zebrafish has emerged over the past decade as a major model system for the study of development due to its invertebrate-like advantages coupled with its vertebrate biology. These features also make it a potentially valuable organism for gerontological research. The main advantages of zebrafish include its economical husbandry, small yet accessible size, high reproductive capacity, genetic tractability, and a large and growing biological database. Although zebrafish life span is longer than rodents, it shares the feasibility of large-scale mutational analysis with the extremely short-lived invertebrate models. This review compares zebrafish with the more widely used model organisms used for aging research, including yeast, worms, flies, mice, and humans.

Aging↗

PubMed2005

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion.

OBJECTIVE: Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models. MATERIALS AND METHODS: To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors. RESULTS: Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages. CONCLUSION: These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

Acute Disease↗

PubMed2005

Independent expansion of the keratin gene family in teleostean fish and mammals: an insight from phylogenetic analysis and radiation hybrid mapping of keratin genes in zebrafish.

The sequence and chromosomal distribution of keratin genes of zebrafish were compared with that of other fishes and mammals to provide an insight into the evolution of this gene family in vertebrates. By comparative sequence analysis and radiation hybrid mapping, we identified 16 type I and 7 type II keratin genes in the zebrafish genome. This contrasts with mammals, where type I and type II keratin genes are similar in number. The keratin genes are scattered in the fish genome, contrasting with the two clusters of keratin genes in mammalian genomes. Compared to genes from two species of pufferfish, the zebrafish type I keratin genes underwent an expansion by independent tandem duplications. Expression profiles based on EST counts suggest that some of the tandemly duplicated type I keratin genes from zebrafish either underwent sub-functionalization or acquired new expression domains. The chromosomal arrangement of keratins 8, keratin18, and a second type II keratin, as a cluster of three genes, has remained conserved in vertebrate evolution, except for duplication of the three-gene cluster in some teleosts. This contrasts with other members of the keratin gene family, which diverged independently between fish and mammals.

Animals↗

PubMed2005

Investigating chorion softening of zebrafish embryos with a microrobotic force sensing system.

The zebrafish is a model organism for addressing questions of vertebrate embryo development. In this paper, the softening phenomenon of the chorion envelope of zebrafish embryos at different developmental stages was mechanically quantitated by using a microrobotic force sensing system. The microrobotic system integrates a piezoelectric cellular force sensor to measure the required forces for penetrating the chorion envelope. Magnitude of penetration forces was found to decrease as an embryo develops. The results mechanically quantitate "chorion softening" in zebrafish embryos due to protease activities subtly modifying the chorion structure, providing an understanding of zebrafish embryo development.

Animals↗

PubMed2004

Development of a respiratory burst assay using zebrafish kidneys and embryos.

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.

Animals↗

PubMed2005

Neuronal development and migration in zebrafish hindbrain explants.

The zebrafish embryo is an excellent system for studying dynamic processes such as cell migration during vertebrate development. Dynamic analysis of neuronal migration in the zebrafish hindbrain has been hampered by morphogenetic movements in vivo, and by the impermeability of embryos. We have applied a recently reported technique of embryo explant culture to the analysis of neuronal development and migration in the zebrafish hindbrain. We show that hindbrain explants prepared at the somitogenesis stage undergo normal morphogenesis for at least 14 h in culture. Importantly, several aspects of hindbrain development such as patterning, neurogenesis, axon guidance, and neuronal migration are largely unaffected, inspite of increased cell death in explanted tissue. These results suggest that hindbrain explant culture can be employed effectively in zebrafish to analyze neuronal migration and other dynamic processes using pharmacological and imaging techniques.

Animals↗

PubMed2005

Characterization of four receptor cDNAs: PAC1, VPAC1, a novel PAC1 and a partial GHRH in zebrafish.

To understand the role of growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) and to examine the functional significance of the co-expression of GHRH and PACAP in fish, their receptors were characterized in zebrafish. Three cDNAs encoding the PAC(1) receptor, the VPAC(1) receptor, and the partial GHRH receptor were identified from zebrafish. Functional expression of the PAC(1) and VPAC(1) receptors revealed that both are potently coupled to the adenylyl cyclase pathway, but only the PAC(1) receptor is coupled to the phospholipase C pathway. Transcripts for all three receptors were widely distributed, often in an overlapping pattern in the adult zebrafish. Also, one splice variant of the partial GHRH receptor and three splice variants of the PAC(1) receptor were identified from adult zebrafish. The long GHRH receptor transcript contained a 27 amino acid insert in transmembrane domain 5 encoding a premature stop codon leading to a truncated receptor protein. For the PAC(1) receptor, two of the splice variants corresponded to the hop1 and hop2 variants characterized in mammals. The third splice variant identified from the gill encoded a novel 107 bp insert containing a premature stop codon. Therefore, PACAP and GHRH have widespread, overlapping target sites suggesting a coordinated role for these hormones in evolution.

Adenylyl Cyclases↗

PubMed2005

Intestinal growth and differentiation in zebrafish.

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

Animals↗

PubMed2005

E-cadherin is required for gastrulation cell movements in zebrafish.

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.

Alleles↗

PubMed2005

Expression of the somatolactin beta gene during zebrafish embryonic development.

Somatolactin (Sl) is a pituitary hormone closely related to prolactin (Prl) and growth hormone that was until now only found in various fish species. We isolated the cDNA coding for zebrafish Slbeta and we identified the gene encoding this hormone. We also obtained a 1kb genomic fragment corresponding to the slbeta upstream promoter region. Furthermore, the slbeta expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Slbeta mRNA is first detected in a single cell at the anterior border of the neural plate starting at 23h post fertilization (hpf). Slbeta-expressing cells also express the transcription factor pit1 and are located close to prl-expressing cells. Using combined fluorescent in situ hybridization, we show that slbeta- and prl-expressing cells are clearly distinct at 29 hpf. Starting at 30 hpf, the number of slbeta positive cells increases and their location becomes more clearly distinct from lactotrope cells, in a more posterior position. At later stages (48 hpf), slbeta expression was observed posterior to growth hormone expression, again in a distinct cell type. We show that zebrafish mutants aal, as well as mutants in the pit1 gene, are deficient in slbeta expression. In conclusion, slbeta expression defines a new, additional cell type in zebrafish pituitary that depends on pit1 and aal for its differentiation.

Animals↗

PubMed2004

Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.

The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.

Animals↗

PubMed2006

Pentachlorophenol treatment in vivo elevates point mutation rate in zebrafish p53 gene.

Pentachlorophenol (PCP), a probable human carcinogen, has been heavily used as an aseptic and a biocide throughout the world, and is widely present in the environment. Recent survey in Germany revealed that the average PCP amount in the urine of general German populations was 1.04 microg/L while the peak concentration could reach up to 19.1 microg/L. PCP was reported to cause DNA damage, but whether it can be involved in inducing point mutations in genome is unknown. To determine the genotoxicity of PCP on vertebrate, we first performed acute toxicity test on zebrafish for the effect of PCP exposure. The LC50 values of zebrafish exposed to PCP at 24, 48, 72 and 96 h were determined to be 0.196, 0.130, 0.130 and 0.130 mg/L, respectively. We then treated zebrafish with PCP for 10 days at 0 (control), 0.5, 5 and 50 microg/L, respectively, to determine whether PCP could be involved in inducing point mutations. Employing denaturing high-performance liquid chromatography analysis and DNA sequencing, we demonstrated that exposure of PCP to zebrafish at a concentration as low as 5 microg/L for 10 days elevates point mutation rate in p53 gene in liver cells. This is the first direct evidence revealing that PCP can elevate point mutation rate in the vertebrate genomes. The result implies PCP might be involved in carcinogenesis by elevating point mutation rate in the somatic genomes.

Animals↗

PubMed2004

S100 protein-like immunoreactivity in the crypt olfactory neurons of the adult zebrafish.

The olfactory epithelium of some teleosts, including zebrafish, contains three types of olfactory sensory neurons. Because zebrafish has become an ideal model for the study of neurogenesis in the olfactory system, it is of capital importance the identification of specific markers for different neuronal populations. In this study we used immunohistochemistry to analyze the distribution of S100 protein-like in the adult zebrafish olfactory epithelium. Surprisingly, specific S100 protein-like immunostaining was detected exclusively in crypt neurons, whereas ciliated and microvillous neurons were not reactive, and the supporting glial cells as well. The pattern of immunostaining was exclusively cytoplasmic without apparent polarity within the soma, and the intensity of immunostaining was not related with the maturative stage of the neurons. The role of S100 protein in crypt olfactory neurons is unknown, although it is probably associated with the capacity of these cells to respond to chemical stimuli. In any case, it represents an excellent marker to identify crypt olfactory neurons in zebrafish.

Animals↗

PubMed2005

Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.

Rodent seizure models have significantly contributed to our basic understanding of epilepsy. However, medically intractable forms of epilepsy persist and the fundamental mechanisms underlying this disease remain unclear. Here we show that seizures can be elicited in a simple vertebrate system e.g. zebrafish larvae (Danio rerio). Exposure to a common convulsant agent (pentylenetetrazole, PTZ) induced a stereotyped and concentration-dependent sequence of behavioral changes culminating in clonus-like convulsions. Extracellular recordings from fish optic tectum revealed ictal and interictal-like electrographic discharges after application of PTZ, which could be blocked by tetrodotoxin or glutamate receptor antagonists. Epileptiform discharges were suppressed by commonly used antiepileptic drugs, valproate and diazepam, in a concentration-dependent manner. Up-regulation of c-fos expression was also observed in CNS structures of zebrafish exposed to PTZ. Taken together, these results demonstrate that chemically-induced seizures in zebrafish exhibit behavioral, electrographic, and molecular changes that would be expected from a rodent seizure model. Therefore, zebrafish larvae represent a powerful new system to study the underlying basis of seizure generation, epilepsy and epileptogenesis.

Animals↗