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PubMed2005

Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis.

The development and function of vertebrate ovary are primarily controlled by the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), from the pituitary. However, most of the activities of FSH and LH are mediated or modulated by a variety of locally produced factors that form an intimate regulatory network within and between the follicles. As a top vertebrate model for genetic and developmental studies, the zebrafish has caught tremendous attention in the past two decades; however, its utility has quickly been extended to other areas including physiology. In the past few years, a variety of peptide growth factors have been identified and characterized in the zebrafish ovary including activin and epidermal growth factor (EGF), and lines of evidence point to the existence of an ovarian network of communication involving these factors. This article provides the state of the art of zebrafish as a model for analyzing ovarian development and its regulation.

Animals↗

PubMed2006

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

Animals↗

PubMed2004

Zebrafish beta tubulin 1 expression is limited to the nervous system throughout development, and in the adult brain is restricted to a subset of proliferative regions.

Tubulin, the building block of microtubules, consists of an alpha and beta subunit, each in itself a family of several highly homologous isotypes. Abundance, tissue specificity, developmental regulation, and possibly function vary between isotypes. Six isotypes of beta tubulin (class I to class VI) have been cloned from several vertebrate species. Class I beta tubulin is believed to be widely expressed, but has not been studied by in situ hybridization in any vertebrate species so far. We have cloned a beta tubulin from zebrafish that appears most similar to other vertebrate class I tubulins and name it zbeta1 tubulin, accordingly. We report a distinct expression pattern of zbeta1 tubulin in the zebrafish embryo in restricted regions of the peripheral and central nervous system that comprise early-differentiating neurons. The expression pattern changes during development and in the adult zebrafish expression mostly is confined to a subset of proliferative zones that include the subependymal zone around the telencephalic ventricle, zones in the preoptic and hypothalamic area and in the olfactory epithelium. Thus, zbeta1 tubulin is expressed with remarkable selectivity during neuronal differentiation and neurogenesis in the embryonic and adult nervous system, respectively.

Animals↗

PubMed2006

Characterization of retinoid-X receptor genes rxra, rxrba, rxrbb and rxrg during zebrafish development.

During development of vertebrate embryos, retinoic acid plays a variety of roles that are mediated by binding to retinoic acid receptors (Rars) and their heterodimerization partners, the retinoid receptors (Rxrs). Here, we characterize the expression patterns of four zebrafish rxr genes during development and provide an analysis of the phylogenetic relationships between zebrafish and tetrapod Rxr genes based on sequence similarities and conserved syntenies. This analysis prompted the renaming of several of the zebrafish rxr genes to match their tetrapod orthologs. Understanding phylogenetic relationships among Rxr genes and their expression patterns during development provides a foundation for future studies of Rxr functions.

Animals↗

PubMed2005

Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons.

Parkinson's disease is characterized by a severe loss of dopaminergic neurons resulting in a range of motor deficits. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause a similar loss of dopaminergic neurons in the human midbrain with corresponding Parkinsonian symptoms. Several animal species have also shown sensitivity to MPTP, including primates, mice, goldfish, and, most recently, zebrafish. This study demonstrates that the effect of MPTP on dopaminergic neurons in zebrafish larvae is mediated by the same pathways that have been demonstrated in mammalian species. MPTP-induced neurodegeneration was prevented by co-incubation with either the monoamine oxidase-B (MAO-B) inhibitor l-deprenyl or the dopamine transporter (DAT) inhibitor nomifensine. Furthermore, targeted inactivation of the DAT gene by antisense morpholinos also protected neurons from MPTP damage. Thus, the mechanism for MPTP-induced dopaminergic neuron toxicity in mammals is conserved in zebrafish larvae. Effects on swimming behavior and touch response that result from MPTP damage are partially ameliorated by both l-deprenyl and DAT knockdown.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Initiation of V(D)J recombination in zebrafish (Danio rerio) ovaries.

The assembly of mammalian antigen receptor genes is a lymphoid-specific process. However, rearranged immunoglobulin genes can also be recovered from non-lymphoid tissues of cartilaginous fish. This event, known as germline rearrangement, has been speculated to arise from recombination-activating gene (RAG)-mediated recombination in germ cells. In this report, we demonstrate that zebrafish (Danio rerio) oocytes expressing high levels of RAG-RNA can readily initiate recombination cleavage at immunoglobulin gene loci, providing direct evidence for an ongoing process of attempted germline rearrangement in zebrafish ovaries. This attempted rearrangement is largely unproductive, yielding no accumulation of germline-joined immunoglobulin genes in zebrafish, which is consistent with their general absence in this species. Our data, therefore, substantiate the speculation that RAG might have been derived from a transposase, invading germ cells of ancient species, and later become a dedicated recombinase only expressed in developing lymphocytes.

Animals↗

PubMed2006

Embryonic zebrafish neuronal growth is not affected by an applied electric field in vitro.

Naturally occurring electric fields (EFs) have been implicated in cell guidance during embryonic development and adult wound healing. Embryonic Xenopus laevis neurons sprout preferentially towards the cathode, turn towards the cathode, and migrate faster towards the cathode in the presence of an external EF in vitro. A recent Phase 1 clinical trial has investigated the effects of oscillating EFs on human spinal cord regeneration. The purpose of this study was to investigate whether embryonic zebrafish neurons respond to an applied EF, and thus extend this research into another vertebrate system. Neural tubes of zebrafish embryos (16-17 somites) were dissected and dissociated neuroblasts were plated onto laminin-coated glass. A 100 mV/mm EF was applied to cell cultures for 4 or 20 h and the responses of neurons to the applied EFs were investigated. After 4h in an EF neurites were significantly shorter than control neurites. No other statistically significant effects were observed. After 20 h, control and EF-exposed neurites were no different in length. No length difference was seen between cathodally- and anodally-sprouted neurites. Application of an EF did not affect the average number of neurons in a chamber. Growth cones did not migrate preferentially towards either pole of the EF and no asymmetry was seen in neurite sprout sites. We conclude that zebrafish neurons do not respond to a 100 mV/mm applied EF in vitro. This suggests that neurons of other vertebrate species may not respond to applied EFs in the same ways as Xenopus laevis neurons.

Animals↗

PubMed2006

Cloning and characterization of zebrafish (Danio rerio) cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by neuron-specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in primary neurogenesis remains unexplored. Here, we have cloned and characterized the zebrafish cdk5 ortholog. Zebrafish cdk5 is 96% identical to its human counterpart. In situ hybridization analyses demonstrated that zebrafish cdk5 transcripts are ubiquitously expressed as early as the blastula stage. At 11.5h of development, cdk5 transcripts were present in the neural plate at the domains where primary neurons begin to be specified. RT-PCR analyses showed equal levels of cdk5 transcripts up to 72 h of development. SiRNA-mediated cdk5 knockdown resulted in a reduction in primary sensory neurons of the trigeminal ganglia of the peripheral nervous system, suggesting that cdk5 plays a crucial role in the development of the peripheral nervous system.

Animals↗

PubMed2004

The too few mutant selectively affects subgroups of monoaminergic neurons in the zebrafish forebrain.

Monoaminergic neurons are present in small numbers and in multiple distinct locations of the vertebrate CNS. They are involved in important functions such as movement coordination, motivation, and the response to environmental stress. However, the mechanisms involved in their subtype specification are not well understood. In this study, we examined the states of forebrain dopaminergic (DA) and serotonergic (5HT) neurons in larval and adult zebrafish of wild type and the too few mutant. The majority of DA and 5HT neuronal subgroups that were found in adults were established in the 6-day old larval zebrafish. Rather than affecting all monoaminergic neurons in the forebrain, selective subgroups of these neurons are reduced in the too few mutant, starting from the larval stage. Taken together, our study establishes that similar to DA neurons, distinct subtypes of 5HT neurons exist in larval as well as adult zebrafish. The development of a subset of these monoaminergic neurons is dependent on the too few gene product. Thus, this mutant is potentially important for understanding the development as well as the function of forebrain DA and 5HT neurons.

Animals↗

PubMedDate not supplied

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC(50)) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.

Abnormalities, Multiple↗

PubMedDate not supplied

Zebrafish as a neurotoxicological model.

At a time when common regulatory pathways are being identified in several different species and genomics is beginning to allow comparisons of genes, how they are arranged on chromosomes and how they are regulated, zebrafish has emerged as a valuable and complementary vertebrate model. Some of the characteristics that prove of value are described and illustrated. Fluorescent transgenic lines of zebrafish embryos are presented for time-line studies with neurotoxicants. While genetic knockout technology has yet to be developed for the model, the anti-sense, morpholino approach allows for knockdown of expression of genes for the 3 day, embryonic period. This can provide for phenocopies of mutant genes for those genes essential to embryonic development or it can provide for a limited inhibition of gene expression that allows subsequent development of the fish. With the zebrafish genomic sequencing effort, microarray technology is now developing for the model system. These resources and technologies allow one to challenge the system with toxicants, and to view the immediate effects of the toxicants with transgenic embryos that fluoresce in part or all of the nervous system. Behavioral and learning protocols have been developed for the organism so that early exposures can be assayed for effects upon adult fish. Microarray technology should allow for one to identify specific genes and pathways affected by a neurotoxicant. In the future, these approaches should provide a working protocol for exploring molecular mechanisms of neurotoxicants. This type of complementary approach should then allow for more efficient examination and testing of mechanisms in mammalian models.

Animals↗

PubMed2006

Uptake and elimination of ethanol by young zebrafish embryos.

Among animal models being explored to understand ethanol-induced teratogenesis, the zebrafish (Danio rerio) is attracting attention because its embryonic development is well characterized and readily visualized. Despite the potential of the zebrafish embryo in research on developmental anomalies produced by ethanol exposure, little is known about the relationship between embryonic ethanol content and the nature/severity of ethanol-mediated deficits. Here, using gas chromatography and radiometry of labeled ethanol carbon, we examine accumulation and clearance of ethanol by dechorionated zebrafish embryos during blastulation/gastrulation. Our data indicate that: (a) rates of uptake and loss of ethanol are directly proportional to the extra-/intra-embryonic ethanol concentration gradient and (b) ethanol in the water fraction of embryos reaches near equimolarity with ethanol in the exposure medium. It appears that, within a wide range of exposure concentrations, embryonic ethanol content can be predicted accurately according to exposure time. Furthermore, it appears that embryonic ethanol can be adjusted rapidly to and maintained at a targeted concentration.

Analysis of Variance↗

PubMed2006

Developmental plasticity of ventilatory control in zebrafish, Danio rerio.

To determine whether development of ventilatory control in zebrafish (Danio rerio) exhibits plasticity, embryos were exposed to hypoxia, hyperoxia or hypercapnia for the first 7 days post-fertilization. Their acute reflex breathing responses to ventilatory stimuli (hypoxia, hypercapnia and external cyanide) were assessed when they had reached maturity (3 months or older). Zebrafish reared under hyperoxic conditions exhibited significantly higher breathing frequencies at rest (283+/-27min(-1) versus 212+/-16min(-1) in control fish); breathing frequency was unaffected in adult fish subjected to hyperoxia for 7 days. The respiratory responses of fish reared in hyperoxic water to acute hypoxia, hypercapnia or external cyanide were blunted (hypoxia, cyanide) or eliminated (hypercapnia). Adult fish exposed for 7 days to hyperoxia showed no change in acute responses to these stimuli. The respiratory responses to acute hypoxia, hypercapnia or external cyanide of fish reared under hypoxic or hypercapnic conditions were similar to those in fish reared under normal conditions. A subset of all fish examined exhibited episodic breathing; an analysis of breathing patterns demonstrated that fish reared under hypercapnic conditions had an increased tendency to display episodic breathing. The results of this study reveal that there is flexibility in the design and functioning of the embryonic or larval respiratory system in zebrafish.

Aging↗

PubMed2005

Modulation of peroxisome proliferator-activated receptors (PPARs) by PPAR(alpha)- and PPAR(gamma)-specific ligands and by 17beta-estradiol in isolated zebrafish hepatocytes.

Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPAR(alpha) include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPAR(beta) activators include fatty acids, prostaglandin A2 (PGA2) and prostacyclin (PGI2). PPAR(gamma) is the most selective receptor and, among others, 15-deoxy-Delta(12,14) prostaglandin J2 (PGJ2) has been described to be a PPAR(gamma)-specific ligand. The aim of the present study was to determine if known PPAR(alpha) and PPAR(gamma) ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPAR(alpha) specific ligand (8S-HETE), a PPARgamma specific ligand (PGJ) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17beta-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPAR(alpha) and PPAR(gamma) by clofibrate (at 0.5 mM for PPAR(alpha) and at 1 and 2 mM for PPAR(gamma)), by HETE (1 microM), and by PGJ2 (0.3 and 1 microM for PPAR(alpha) and 0.3 microM for PPAR(gamma)). Expression of PPARgamma was also induced at 10 microM by 17beta-estradiol. The percentage of PPAR(alpha) positive nuclei increased significantly at 1 microM HETE and the percentage of PPAR(gamma) positive cells decreased at 10 microM 17beta-estradiol. As a conclusion, clofibrate, HETE and PGJ2 are able to induce expression of both PPAR(alpha) and PPAR(gamma) in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.

Animals↗

PubMed2005

Carbofuran and malathion inhibit nucleotide hydrolysis in zebrafish (Danio rerio) brain membranes.

Carbofuran and malathion are broad spectrum pesticides widely used in agricultural practice throughout the world. Toxicity of these pesticides has been correlated with their inhibitory effects on acetylcholinesterase activity. Nucleotides are extracellular signaling molecules, which trigger multiple biological effects. Studies have demonstrated the co-transmission of acetylcholine and ATP at the nerve endings. The control of neurotransmitter ATP levels is promoted by enzymes named ectonucleotidases, which include nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. Since acetylcholine and ATP are co-released at the synapse and the acetylcholinesterase inhibition is an important target for pesticide action, here we verified the effect of exposure in vitro and in vivo to carbofuran and malathion on ectonucleotidase activities from brain membranes of zebrafish. To verify if carbofuran and malathion have a direct inhibitory effect on NTPDase and 5'-nucleotidase activities in brain membranes of zebrafish, we have tested in vitro concentrations of pesticides varying from 0.25 to 5 mM. Carbofuran, in vitro, inhibited ATP and ADP hydrolysis in an uncompetitive manner, but no effect was observed on AMP hydrolysis. Malathion decreased ATP and ADP hydrolysis in competitive and an uncompetitive manner, respectively, but not altered AMP hydrolysis. After exposure to carbofuran (50 and 500 microg/L) during 7 days, ADP hydrolysis was significantly decreased in both concentrations tested (by 19 and 24.5%, respectively). Malathion, at 500 microg/L, was able to inhibit ADP and AMP hydrolysis (by 28 and 58.5%, respectively). This study has shown that ectonucleotidases from brain membranes of zebrafish can be a potential target for pesticide neurotoxicity.

5'-Nucleotidase↗

PubMedDate not supplied

Neurotoxicity assessment using zebrafish.

INTRODUCTION: Transparency is a unique attribute of zebrafish that permits direct assessment of drug effects on the nervous system using whole mount antibody immunostaining and histochemistry. METHODS: To assess pharmacological effects of drugs on the optic nerves, motor neurons, and dopaminergic neurons, we performed whole mount immunostaining and visualized different neuronal cell types in vivo. In addition, we assessed neuronal apoptosis, proliferation, oxidation and the integrity of the myelin sheath using TUNEL staining, immunostaining and in situ hybridization. The number of dopaminergic neurons was examined and morphometric analysis was performed to quantify the staining signals for myelin basic protein and apoptosis. RESULTS: We showed that compounds that induce neurotoxicity in humans caused similar neurotoxicity in zebrafish. For example, ethanol induced defects in optic nerves and motor neurons and affected neuronal proliferation; 6-hydroxydopamine caused neuronal oxidation and dopaminergic neuron loss; acrylamide induced demyelination; taxol, neomycin, TCDD and retinoic acid induced neuronal apoptosis. DISCUSSION: Effects of drug treatment on different neurons can easily be visually assessed and quantified in intact animals. These results support the use of zebrafish as a predictive model for assessing neurotoxicity.

Acrylamide↗

PubMed2006

Use of PCR-restriction fragment length polymorphism for the identification of zoonotic mycobacteriosis in zebrafish caused by Mycobacterium abscessus and Mycobacterium chelonae.

Skin ulcers, scoliosis, and dropsy-like scale edema were observed in laboratory-maintained zebrafish. Affected fish had multifocal granulomas not only in internal organs such as the liver, intestine, genital organs, kidney, muscle, and spleen but also in the fin, epithelium, gills, and sclera of the eyes. Large numbers of acid-fast-rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas with Gram's stain and Ziehl-Neelson's stain. The size of the Mycobacterium spp. was 1-2 microm x 2-3 microm with a double-layered cell wall, based upon electron-microscopical features. Definitive diagnosis of these outbreaks was obtained by culture on selective media followed by PCR-restriction fragment length polymorphism analysis (PRA) of the rpoB gene for species identification. The amplified 360-bp products of the rpoB gene of mycobacteria isolated from zebrafish were digested with MspI restriction enzyme, which revealed unique band patterns matching those of Mycobacterium abscessus and Mycobacterium chelonae which are responsible for skin and soft tissue infection caused by rapidly growing mycobacteria in humans. This is the first documentation of the precise identification of zoonotic non-tuberculous mycobacteria isolated from laboratory-maintained zebrafish by the PRA of the rpoB gene; this study thus provides a great deal of useful epidemiological information and reduces the likelihood that epizootics will occur.

Animals↗

PubMed2006

Photodecomposition of bisphenol A on nanometer-sized TiO2 thin film and the associated biological toxicity to zebrafish (Danio rerio) during and after photocatalysis.

We investigated the relationship between the TiO2 photocatalytic decomposition of bisphenol A and biological toxicity to zebrafish (Danio rerio). TiO2 particles, which prepared using a solvothermal method, were applied to produce a nanometer-sized TiO2 thin film. An alcoholic solution containing the TiO2 particles and an inorganic binder was directly coated on the UV-lamp substrate. It was equipped in a photoreactor that was manufactured in our laboratory. The attachment of the thin TiO2 film to the UV-lamp substrate resulted in a stable and transparent coating. The TiO2 particles on the thin film were approximately 20-30 nm in size, and the resulting film thickness was approximately 200 nm after a single coat. The bisphenol A, which was eluted from epoxy resin in a drinking water tank, was completely degraded by the TiO2 photocatalysis. We initially detected approximately 7.8 ng/ml of bisphenol A in the epoxy-resin tank, but its concentration was undetectable after a 48-h photocatalytic reaction over TiO2. We observed a decreased survival rate in zebrafish that were reared in water exposed to the leaching process of the epoxy resin. After the photocatalysis, however, no toxic effects on the hatching rates or morphogenesis of the zebrafish were observed. In summary, toxicity during the TiO2 photocatalysis was observed; however, toxicity was no longer observed once the bisphenol A was completely decomposed by the TiO2 photocatalysis. On the basis of these experimental observations, we suggest that TiO2 photocatalysis can be adopted as a treatment method to purify an epoxy-resin tank.

Animals↗