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PubMed2004

Identification of a zebrafish model of muscular dystrophy.

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ has been postulated previously and MTJ structural abnormalities have been identified in the dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.

Animals↗

PubMed2004

Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Dopaminergic deficiency in the brain of zebrafish was produced by systemic administration of two catecholaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the neurochemical and behavioural changes were characterized. The levels of dopamine and noradrenaline decreased significantly after the injection of MPTP and 6-OHDA. Corresponding to these changes, fish exhibited characteristic changes in locomotor behaviour, i.e. the total distance moved and velocity decreased after both neurotoxins. Tyrosine hydroxylase and caspase 3 protein levels were not altered after MPTP or 6-OHDA injections, as studied by immunohistochemistry and western blotting. The catecholaminergic cell clusters suggested to correspond to the mammalian nigrostriatal cell group displayed normal tyrosine hydroxylase immunoreactivity after the toxin treatment and did not show signs of DNA fragmentation that would indicate activation of cascades that lead to cell death. The results show that single systemic injections of MPTP and 6-OHDA induce both biochemical and behavioural changes in zebrafish, albeit failing to produce any significant morphological alteration in catecholaminergic cell clusters at the tested doses. This approach may be used for the screening of chemicals affecting the dopaminergic system. The model may be especially useful for evaluation of the role of novel genes in neurotoxicity, as a large number of zebrafish mutants are becoming available.

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

PubMedDate not supplied

Twenty ways to lose your bladder: common natural mutants in zebrafish and widespread convergence of swim bladder loss among teleost fishes.

Convergence is an important evolutionary phenomenon often attributed solely to natural selection acting in similar environments. The frequency of mutation and number of ways a phenotypic trait can be generated genetically, however, may also affect the probability of convergence. Here we report both a high frequency of loss of gas bladder (swim bladder) mutations in zebrafish and widespread convergent loss of gas bladders among teleost fishes. The phenotypes of 22 of 27 recessive lethal mutations, carried by a sample of 26 wild-caught zebrafish, involve loss or noninflation of the gas bladder. Nine of these bladderless mutations showed no other obvious phenotypic abnormalities other than the lack of an inflated gas bladder. At least 19 of the 22 bladderless mutations are genetically distinct, as shown by unique morphology or complementation. Although we were not able to obtain eggs for all 21 required crosses to demonstrate definitively that the remaining three mutations are different from all other bladderless mutations, all available evidence suggests that these mutants are also distinct. At least 79 of 425 families of extant teleosts include one or more species lacking a gas bladder as adults. Analysis of the trait's phylogenetic distribution shows that the gas bladder has been lost at least 30-32 times independently. Although adaptive explanations for gas bladder loss are convincing, a developmental bias toward bladderless phenotypes may also have contributed to the widespread convergence of this trait among teleosts. If gas bladder development in teleosts is as vulnerable to genetic perturbation as it is in zebrafish, then perhaps a supply of bladderless phenotypes has been readily available to natural selection under conditions for which it is advantageous not to have a gas bladder. In this way, developmental bias and selection can work together to produce widespread convergence.

Air Sacs↗

PubMed2006

A chemical genetic screen for cell cycle inhibitors in zebrafish embryos.

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

Animals↗

PubMed2004

Neuroepithelial oxygen chemoreceptors of the zebrafish gill.

In aquatic vertebrates, hypoxia induces physiological changes that arise principally from O(2) chemoreceptors of the gill. Neuroepithelial cells (NECs) of the zebrafish gill are morphologically similar to mammalian O(2) chemoreceptors (e.g. carotid body), suggesting that they may play a role in initiating the hypoxia response in fish. We describe morphological changes of zebrafish gill NECs following in vivo exposure to chronic hypoxia, and characterize the cellular mechanisms of O(2) sensing in isolated NECs using patch-clamp electrophysiology. Confocal immunofluorescence studies indicated that chronic hypoxia (P(O(2)) = 35 mmHg, 60 days) induced hypertrophy, proliferation and process extension in NECs immunoreactive for serotonin or synaptic vesicle protein (SV2). Under voltage clamp, NECs responded to hypoxia (P(O(2)) = 25-140 mmHg) with a dose-dependent decrease in K(+) current. The current-voltage relationship of the O(2)-sensitive current (I(KO(2))) reversed near E(K) and displayed open rectification. Pharmacological characterization indicated that I(KO(2)) was resistant to 20 mM tetraethylammonium (TEA) and 5 mM 4-aminopyridine (4-AP), but was sensitive to 1 mm quinidine. In current-clamp recordings, hypoxia produced membrane depolarization associated with a conductance decrease; this depolarization was blocked by quinidine, but was insensitive to TEA and 4-AP. These biophysical and pharmacological characteristics suggest that hypoxia sensing in zebrafish gill NECs is mediated by inhibition of a background K(+) conductance, which generates a receptor potential necessary for neurosecretion and activation of sensory pathways in the gill. This appears to be a fundamental mechanism of O(2) sensing that arose early in vertebrate evolution, and was adopted later in mammalian O(2) chemoreceptors.

Animals↗

PubMed2003

Development of form and function in peripheral auditory structures of the zebrafish (Danio rerio).

Investigations of the development of auditory form and function have, with a few exceptions, thus far been largely restricted to birds and mammals, making it difficult to postulate evolutionary hypotheses. Teleost fishes represent useful models for developmental investigations of the auditory system due to their often extensive period of posthatching development and the diversity of auditory specializations in this group. Using the auditory brainstem response and morphological techniques we investigated the development of auditory form and function in zebrafish (Danio rerio) ranging in size from 10 to 45 mm total length. We found no difference in auditory sensitivity, response latency, or response amplitude with development, but we did find an expansion of maximum detectable frequency from 200 Hz at 10 mm to 4000 Hz at 45 mm TL. The expansion of frequency range coincided with the development of Weberian ossicles in zebrafish, suggesting that changes in hearing ability in this species are driven more by development of auxiliary specializations than by the ear itself. We propose a model for the development of zebrafish hearing wherein the Weberian ossicles gradually increase the range of frequencies available to the inner ear, much as middle ear development increases frequency range in mammals.

Age Factors↗

PubMed2003

Myc-induced T cell leukemia in transgenic zebrafish.

The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c-myc under control of the zebrafish Rag2 promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c-myc- induced carcinogenesis.

Animals↗

PubMed2006

Conservation of RET regulatory function from human to zebrafish without sequence similarity.

Evolutionary sequence conservation is an accepted criterion to identify noncoding regulatory sequences. We have used a transposon-based transgenic assay in zebrafish to evaluate noncoding sequences at the zebrafish ret locus, conserved among teleosts, and at the human RET locus, conserved among mammals. Most teleost sequences directed ret-specific reporter gene expression, with many displaying overlapping regulatory control. The majority of human RET noncoding sequences also directed ret-specific expression in zebrafish. Thus, vast amounts of functional sequence information may exist that would not be detected by sequence similarity approaches.

Animals↗

PubMed1994

Contribution of early cells to the fate map of the zebrafish gastrula.

Previously, a tissue-specific fate map was compiled for the gastrula stage of the zebrafish embryo, indicating that development subsequent to this stage follows a reproducible pattern. Here it is shown that each early zebrafish blastomere normally contributes to a subset of the gastrula and thus gives rise to a limited array of tissues. However, the final contribution that any early blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed. Therefore, early cell divisions of the zebrafish embryo cannot reproducibly segregate determinants of tissue fates.

Animals↗

PubMed2001

The slow mo mutation reduces pacemaker current and heart rate in adult zebrafish.

Genetic studies in zebrafish have focused on embryonic mutations, but many physiological mechanisms continue to mature after embryogenesis. We report here that zebrafish homozygous for the mutation slow mo can be raised to adulthood. In the embryo, the slow mo gene is needed to regulate heart rate, and its mutation causes a reduction in pacemaker current (I(h)) and slowing of heart rate (bradycardia). The homozygous adult slow mo fish continues to manifest bradycardia, without other evident ill effects. Patch-clamp analysis of isolated adult cardiomyocytes reveals that I(h) has chamber-specific properties such that the atrial current density of I(h) is far greater than the ventricular current density of I(h). I(h) is markedly diminished in cardiomyocytes from both chambers of slow mo mutant fish. Thus I(h) continues to be a critical determinant of pacemaker rate even after adult neural and humoral influences have developed. It is clear that zebrafish may be used for genetic dissection of selected physiological mechanisms in the adult.

Animals↗

PubMed2002

Evidence for a widespread brain stem escape network in larval zebrafish.

Zebrafish escape behaviors, which typically consist of a C bend, a counter-turn, and a bout of rapid swimming, are initiated by firing of the Mauthner cell and two segmental homologs. However, after laser-ablation of the Mauthner cell and its homologs, escape-like behaviors still occur, albeit at a much longer latency. This might suggest that additional neurons contribute to this behavior. We therefore recorded the activity of other descending neurons in the brain stem using confocal imaging of cells retrogradely labeled with fluorescent calcium indicators. A large majority of identified descending neurons present in the larval zebrafish, including both ipsilaterally and contralaterally projecting reticulospinal neurons, as well as neurons from the nucleus of the medial longitudinal fasciculus, showed short-latency calcium responses after gentle taps to the head of the larva-a stimulus that reliably evokes an escape behavior. Previous studies had associated such in vivo calcium responses with the firing of action potentials, and because all responding cells have axons projecting into to spinal cord, this suggests that these cells are relaying escape-related information to spinal cord. Other identified neurons failed to show consistent calcium responses to escape-eliciting stimuli. In conjunction with previous lesion studies, these results indicate that the neural control systems for turning and swimming behaviors are widely distributed in the larval zebrafish brain stem. The degree of robustness or redundancy of this system has implications for the descending control of vertebrate locomotion.

Action Potentials↗

PubMed2004

Activation of ionotropic glutamate receptors on peripheral axons of primary motoneurons mediates transmitter release at the zebrafish NMJ.

The development and function of the vertebrate neuromuscular junction (NMJ) is continually being redefined. Previous studies have indicated that glutamate may play a role in the development or function of the NMJ by associating with presynaptic receptors. We have used larval zebrafish (Danio rerio) to investigate the presence of presynaptic ionotropic glutamate receptors (iGluRs) at the NMJ in vivo. In whole-mount zebrafish larvae, antibody staining directed to NR2A subunits colocalized with specific staining of motoneuron axon tracts. Whole cell voltage-clamp recordings of miniature endplate currents (mEPCs) from axial white muscle were performed during application of iGluR agonists and antagonists. Local perfusion of the NMJ with iGluR agonists resulted in significant increases in the frequency of spontaneous acetylcholine (ACh) release. These increases were blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (50 microM) and by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (50 microM). Further pharmacological investigation revealed no effect of the kainate receptor-specific antagonist (2S,4R)-4-methylglutamate (10 microM) on kainate-induced rises in the frequency of spontaneous ACh release. However, these were blocked with the AMPA receptor-specific antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (50 microM). Application of glutamate (1 mM) in the presence of the glutamate uptake inhibitor d-threo-beta-benzyloxyaspartate(200 microM) resulted in a significant increase in the frequency of mEPCs. These results suggest the presence of AMPA and NMDA receptors in association with motoneuron axons of larval zebrafish.

Acetylcholine↗

PubMed1995

Visualization of active neural circuitry in the spinal cord of intact zebrafish.

1. One of the major obstacles in studying vertebrate neural networks is the difficulty in simultaneously monitoring activity in a population of neurons. To take advantage of the transparency of larval zebrafish, we used confocal microscopy to look into the spinal cord of immobilized fish to monitor neural responses during an escape behavior. 2. Populations of identified neurons were labeled with a calcium indicator and neural activity was monitored on a millisecond time scale. The calcium dependent nature of the fluorescent signals was confirmed by monitoring the accumulation, diffusion, and removal of calcium that was introduced by electrical and sensory stimulation. 3. Zebrafish, like most swimming vertebrates, have two major classes of motoneurons: large primary motoneurons thought to be used primarily for rapid movements and smaller secondary motoneurons implicated in slower movements. Our optical approach allowed us to ask how these groups of primary and secondary motoneurons respond during the escape behavior--one of the fastest and most forceful motor behaviors produced by vertebrates. 4. We demonstrate a previously unknown synchrony in the response of populations of primary and secondary motoneurons. This synchrony can account for the massive activation of the axial musculature during powerful escapes. Detection of this synchrony depended on the rapid in vivo imaging of activity in this neuronal population. This optical approach will allow functional studies of neuronal populations in the brain and spinal cord of normal and mutant lines of zebrafish.

Animals↗

PubMed2000

Properties of miniature glutamatergic EPSCs in neurons of the locomotor regions of the developing zebrafish.

As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg(2+), a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.

2-Amino-5-phosphonovalerate↗

PubMed2004

Genomic characterization and expression analysis of the first nonmammalian renin genes from zebrafish and pufferfish.

Renin is a key enzyme in the renin-angiotensin system (RAS), a pathway which plays an important physiological role in blood pressure and electrolyte homeostasis. The origin of the RAS is believed to have accompanied early evolution of vertebrates. However, renin genes have so far only been unequivocally identified in mammals. Whether or not a bona fide renin gene exists in nonmammalian vertebrates has been an intriguing question of physiological and evolutionary interest. Using a genomic analytical approach, we identified renin genes in two nonmammalian vertebrates, zebrafish (Danio rerio) and pufferfish (Takifugu rubripes). Phylogenetic analysis demonstrates that the predicted fish renins cluster together with mammalian renins to form a distinct subclass of vertebrate aspartyl proteases. RT-PCR results confirm generation of the predicted zebrafish mRNA and its expression in association with the opisthonephric kidney of adult zebrafish. Comparative in situ hybridization analysis of wild-type and developmental mutants indicates that renin expression is first detected bilaterally in cells of the interrenal primordia at 24 h postfertilization, which subsequently migrate to lie adjacent to, but distinct from, the glomerulus of the developing pronephric kidney. Our report provides the first molecular evidence for the existence of renin genes in lower vertebrates. The observation that the earliest renin-expressing cells, arising during ontogeny of this teleost vertebrate, are of adrenocortical lineage raises an interesting hypothesis as regards the origin of renin-expressing cells in the metanephric kidney of higher vertebrates.

Adrenal Glands↗

PubMed2006

Transcriptome kinetics of arsenic-induced adaptive response in zebrafish liver.

Arsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8-96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver.

Adaptation, Physiological↗

PubMed2000

Reverse genetics in zebrafish.

The zebrafish has become a popular model system for the study of vertebrate developmental biology because of its numerous strengths as a molecular genetic and embryological system. To determine the requirement for specific genes during embryogenesis, it is necessary to generate organisms carrying loss-of-function mutations. This can be accomplished in zebrafish through a reverse genetic approach. This review discusses the current techniques for generating mutations in known genes in zebrafish. These techniques include the generation of chromosomal deletions and the subsequent identification of complementation groups within deletions through noncomplementation assays. In addition, this review will discuss methods currently being evaluated that may improve the methods for finding mutations in a known sequence, including screening for randomly induced small deletions within genes and screening for randomly induced point mutations within specific genes.

Animals↗

PubMed2006

Zebrafish as a "biosensor"? Effects of ionizing radiation and amifostine on embryonic viability and development.

The zebrafish (Danio rerio) has emerged as a popular vertebrate model system for cancer and treatment-related research. Benefits include ease of care, rapid development, optical clarity of embryos, which allows visualization of major organ systems, and opportunities for genetic manipulation. However, specific parameters of radiation sensitivity have not been systematically documented. We investigated the effects of radiation and a radiomodifier on zebrafish viability and embryonic development. Embryos were exposed to gamma-radiation (5, 10, or 20 Gy) at sequential times postfertilization and serially assessed for viability and morphologic abnormalities. As expected, lethality and morphologic perturbations were more pronounced earlier in embryogenesis and with higher radiation doses and were partially reversed by amifostine. The effects of radiation and concurrent treatment with amifostine on the developmental organization of the eye and brain were striking. Radiation resulted in hypocellularity and disorganization of the cellular layers of the retina, effects partially reversed by amifostine, as well as lens opacification. Radiation strikingly reduced the volume of brain, but the volume loss was substantially blocked by amifostine. Increased terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling signal was noted in both the irradiated eye and brain, but reduced by amifostine. Finally, irradiating embryos resulted in caspase activation detectable in 96-well microplates, which was proportional to the number of embryos and radiation dose; the degree of activation was markedly reduced by amifostine. These results together suggest the power and versatility of the zebrafish in assessing the effects of radiation and radiomodifiers on organ and tissue development.

Amifostine↗