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PubMed2003

The dystrophin associated protein complex in zebrafish.

Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins.

Amino Acid Sequence↗
PubMed2006

Human laterality disorders.

Heterotaxia is a group of congenital disorders characterized by a misplacement of one or more organs according to the left-right axis. Bilateral asymmetry of internal organs is conserved among all vertebrate species. Analyses in animal models such as mouse, chicken, frog and zebrafish allowed for a remarkable progress of knowledge on the embryonic and genetic mechanisms underlying internal left-right asymmetry. In this review we focus on the insights from these model organisms that are useful for a better understanding of the etiology and pathogenesis of human heterotaxia. The known causes of human heterotaxia are reviewed and situated within the conceptual framework that originates from vertebrate model organisms. Furthermore, we attempt to apply the rapidly increasing insights gained from both animal models and human genetics to clinical practice in order to contribute to a more accurate conceptual classification, genetic diagnosis and counseling.

Animals↗
PubMed2006

In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.

The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.

Animals↗
PubMed2005

The mechanical scaling of coasting in zebrafish (Danio rerio).

Many fish species span two or three orders of magnitude in length during the growth from larvae to adults, and this change may have dramatic consequences for locomotor performance. We measured how the performance of coasting changes over the life history of zebrafish (Danio rerio) and examined the scaling of mechanics underlying this change. Adult zebrafish coast disproportionately further and faster and maintain their speed for a longer duration than do larvae and juveniles. Measurements of drag on tethered dead fish suggest that adult fish operate in an inertial regime by coasting at relatively high Reynolds numbers (Re > 1000), and in vivo drag measurements showed adults to operate with a drag coefficient (Cinert approximately 0.024) that was consistent with previously published estimates. However, drag scaled differently at lower Re values than those assumed in previous studies. We found a viscous regime at Re < 300, which corresponds to the routine coasting of larvae and juveniles. Despite these changes in hydrodynamics over growth, a mathematical model of coasting mechanics suggests that the disproportionately longer coasting of adults is caused primarily by their large body mass and high speed at the beginning of coasting. We therefore propose that changes in coasting performance with growth are dictated primarily by the scaling of momentum rather than resulting from hydrodynamic changes. These results provide an opportunity for new interpretations of function in the growth and evolution of fish.

Animals↗
PubMed2004

Genetics in zebrafish, mice, and humans to dissect congenital heart disease: insights in the role of VEGF.

Heart development and the establishment of a functional circulatory circuit are complex biological processes in which subtle perturbations may result in catastrophic consequences of cardiovascular birth defects. Studies in model organisms, most notably the mouse and the zebrafish, have identified genes that also cause these life-threatening defects when mutated in humans. Gradually, a framework for the genetic pathway controlling these events is now beginning to emerge. However, the puzzling phenotypic variability of the cardiovascular disease phenotype in humans and the recent identification of phenotypic modifiers using model organisms indicates that other genetic loci might interact to modify the disease phenotype. To illustrate this, we review the role of vascular endothelial growth factor (VEGF) during vascular and cardiac development and stress how zebrafish and mouse genetic studies have helped us to understand the role this growth factor has in human disease, in particular in the Di-George syndrome.

Animals↗
PubMed2006

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

Amino Acid Sequence↗
PubMed2006

Zebrafish DJ-1 is evolutionarily conserved and expressed in dopaminergic neurons.

Loss-of-function mutations in the human PARK7 gene, encoding DJ-1, are a rare cause of autosomal recessive Parkinson's disease (ARPD). To facilitate generation of a novel vertebrate model, in which to examine the biochemical functions of DJ-1 in vivo, we cloned and characterized the zebrafish orthologue of DJ-1 (zDJ-1). The 0.95 kb zDJ-1 mRNA is expressed in adult zebrafish brain, muscle and gut, and in the embryo from 24 h post-fertilization. The zDJ-1 transcript encodes a 19.8 kDa, 189 amino acid protein, which is 83% identical to human DJ-1. Residues thought to be functionally important sites of post-translational modification in human DJ-1, and critical positions affected by pathogenic missense mutations in ARPD patients, are conserved in zDJ-1. The 14 kb zDJ-1 gene contains six exons and is located on zebrafish chromosome 8; the structure of the gene is highly homologous to human DJ-1, except that there are no alternatively spliced non-coding 5' exons. The single zDJ-1 first exon shows 5' end heterogeneity, reflecting multiple transcription start sites. In the adult zebrafish brain, zDJ-1 immunoreactivity was prominent in the cytoplasm of most neurons, and in the neuropil, but was less evident within white matter tracts, consistent with neuronal somatic and dendritic localization. Dopaminergic neurons in each of the major forebrain and diencephalic TH-positive cell groups expressed zDJ-1. These studies show that zDJ-1 is very similar to human DJ-1 and delineate essential resources, allowing further examination of the function and regulation of DJ-1, using the zebrafish as a model.

Animals↗
PubMed2004

Screen for genes differentially expressed during regeneration of the zebrafish caudal fin.

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up-regulated during "initiation" (1 day) and "outgrowth and differentiation" (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT-PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration.

Amputation, Surgical↗
PubMed2001

Investigations of photoreceptor synaptic transmission and light adaptation in the zebrafish visual mutant nrc.

PURPOSE: To characterize the retinal physiology of the zebrafish visual mutant no optokinetic response c (nrc) and to identify the genetic map position of the nrc mutation. METHODS: Electroretinograms were recorded from wild-type and nrc zebrafish larvae between 5 to 6 days postfertilization. Responses to flash stimuli, On and Off responses to prolonged light stimuli, and responses to flash stimuli with constant background illumination were characterized. The glutamate agonist, 2-amino-4-phosphonobutyric acid (APB) was used to examine the photoreceptor specific a-wave component of the electroretinogram. Amplified fragment length polymorphism methodology was used to place the nrc mutation on the zebrafish genomic map. RESULTS: nrc and wild-type zebrafish larvae 5 to 6 days postfertilization have similar threshold responses to light, but the b-wave of the nrc electroretinogram is significantly delayed and reduced in amplitude. On and Off responses of nrc larvae to prolonged light have multiple oscillations that do not occur in normal zebrafish larvae after 5 days postfertilization. Analysis of the b-wave demonstrated a light adaptation defect in nrc that causes saturation at background light levels approximately 1 order of magnitude less than those with wild-type larvae. Application of the glutamate analog, APB, uncovered the photoreceptor component of the electroretinogram and revealed a light adaptation defect in nrc photoreceptors. The nrc mutation was placed approximately 0.2 cM from sequence length polymorphism marker Z7504 on linkage group 10. CONCLUSIONS: The zebrafish mutant nrc is a possible model for human retinal disease. nrc has defects in photoreceptor synaptic transmission and light adaptation. The nrc mutant phenotype shows striking similarities with phenotypes of dystrophin glycoprotein complex mutants, including patients with Duchenne/Becker muscular dystrophy. Localization of the nrc mutation now makes it possible to evaluate candidate genes and clone the nrc gene.

Adaptation, Ocular↗
PubMed2006

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens.

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.

Amino Acid Sequence↗
PubMed2005

Analysis of hematopoietic development in the zebrafish.

The zebrafish (Danio rerio) has emerged as a powerful vertebrate genetic and developmental model that is particularly amenable to the study of hematopoiesis. The zebrafish embryo develops externally and its optical clarity allows the number and morphology of circulating blood cells to be visualized using a dissecting microscope. Both the morphology of the blood lineages and the expression of critical blood genes are highly conserved between zebrafish and mammals. The high fecundity and short generation time of zebrafish facilitate genetic analysis, and a number of large-scale mutagenesis screens have identified mutations in genes affecting blood development. The discovery of novel hematopoietic genes, as well as the cloning of zebrafish homologs of known hematopoietic genes, necessitates the use of efficacious and reliable methods for complete gene characterization. In this chapter, we illustrate frequently used techniques that are essential for evaluating hematopoiesis in the zebrafish, including whole-mount in situ hybridization, the detection of erythrocytes by o-dianisidine staining, and a description of the microinjection procedure, which has various applications, including overexpression of messenger ribonucleic acid, gene "knockdown" by antisense technology, and the creation of transgenic zebrafish. Also included is an explanation of the use of flow cytometry to separate hematopoietic lineages from the adult kidney and to isolate relatively pure populations of cell types from transgenic embryos based on the expression of fluorescent markers.

Animals↗
PubMed2005

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.

Animals↗
PubMed2005

Resolving mechanisms of toxicity while pursuing ecotoxicological relevance?

In this age of modern biology, aquatic toxicological research has pursued mechanisms of action of toxicants. This has provided potential tools for ecotoxicologic investigations. However, problems of biocomplexity and issues at higher levels of biological organization remain a challenge. In the 1980s and 1990s and continuing to a lesser extent today, organisms residing in highly contaminated field sites or exposed in the laboratory to calibrated concentrations of individual compounds were carefully analyzed for their responses to priority pollutants. Correlation of biochemical and structural analyses in cultured cells and tissues, as well as the in vivo exposures led to the production and application of biomarkers of exposure and effect and to our awareness of genotoxicity and its chronic manifestations, such as neoplasms, in wild fishes. To gain acceptance of these findings in the greater environmental toxicology community, "validation of the model" versus other, better-established often rodent models, was necessary and became a major focus. Resultant biomarkers were applied to heavily contaminated and reference field sites as part of effects assessment and with investigations following large-scale disasters such as oil spills or industrial accidents. Over the past 15 years, in the laboratory, small aquarium fish models such as medaka (Oryzias latipes), zebrafish (Danio rerio), platyfish (Xiphophorus species), fathead minnow (Pimephales promelas), and sheepshead minnow (Cyprinodon variegatus) were increasingly used establishing mechanisms of toxicants. Today, the same organisms provide reliable information at higher levels of biological organization relevant to ecotoxicology. We review studies resolving mechanisms of toxicity and discuss ways to address biocomplexity, mixtures of contaminants, and the need to relate individual level responses to populations and communities.

Animals↗
PubMed1995

Distribution of tissue progenitors within the shield region of the zebrafish gastrula.

The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish.

Animals↗
PubMed2006

Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals.

Zebrafish has emerged as a valuable model for immunological studies. However, little is known about the overall picture of its immune response to infectious pathogens. Here we present the first systematic study of its immune response to Aeromonas salmonicida and Staphylococcus aureus, a Gram-negative and a Gram-positive bacteria, respectively. Genes induced upon infection were identified with suppression subtractive hybridization, with many of them encoding acute phase proteins (APPs). When compared with mammals, striking similarities and obvious differences have been observed. Both similar APPs (SAA, hepcidin and haptoglobin, etc.) and a similar system for the induction of APPs (which involves the TLRs, pro-inflammatory cytokines and C/EBPs) were identified, implying evolutionary conserved mechanisms among fish and mammals. Some novel APPs were also discovered, suggesting different immune strategies adopted by fish species. Among which, LECT2 was induced by up to 1000-fold upon infection, shedding new lights on the function of this gene. Our results constitute the first demonstration of a similar while different immune response in zebrafish and open new avenues for the investigation of evolutionary conserved and fish specific mechanisms of innate immunity.

Acute-Phase Reaction↗
PubMed2001

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.

Animals↗
PubMed2005

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.

Abnormalities, Drug-Induced↗
PubMed2000

Vertebrate ancient-long opsin: a green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells.

Nonretinal/nonpineal photosensitivity has been found in the brain of vertebrates, but the molecular basis for such a "deep brain" photoreception system remains unclear. We conducted an extensive search for brain opsin cDNAs of the zebrafish (Danio rerio), a useful animal model for genetic studies, and we have isolated a partial cDNA clone encoding an ortholog of vertebrate ancient (VA) opsin, the function of which is unknown. Subsequent characterization revealed the occurrence of two kinds of mRNAs encoding putative splicing variants, VA and VA-Long (VAL) opsin, the latter of which is a novel variant of the former. Both opsins shared a common core sequence in the membrane-spanning domains, but VAL-opsin had a C-terminal tail much longer than that of VA-opsin. Functional reconstitution experiments on the recombinant proteins showed that VAL-opsin with bound 11-cis-retinal is a green-sensitive pigment (lambdamax approximately 500 nm), whereas VA-opsin exhibited no photosensitivity even in the presence of 11-cis-retinal. Immunoreactivity specific to this functionally active VAL-opsin was localized at a limited number of cells surrounding the diencephalic ventricle of central thalamus, and these cells were distributed over approximately 200 micrometer along the rostrocaudal axis. Taken together with the previous study on the locus of the teleost brain photosensitivity (von Frisch K, 1911), it is strongly suggested that the VAL-positive cells in the zebrafish brain represent the deep brain photoreceptors. The VAL-specific immunoreactivity was also detected in a subset of non-GABAergic horizontal cells in the zebrafish retina. The existence of VAL-opsin, a new member of the rhodopsin superfamily, in these tissues may indicate its multiple roles in visual and nonvisual photosensory physiology.

Animals↗