Zebrafish: swimming into the development mainstream.
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Animals↗
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Laser-induced thrombosis in zebrafish larvae: a novel genetic screening method for thrombosis.
Classical genetic approaches to study hemostasis and thrombosis have not been available until our recent introduction of the teleost, Danio rerio (the zebrafish), as an effective genetic model for in vivo coagulation assays. The genetic screen for this model is carried out using the genome saturation mutagenesis approach. The resulting mutants are screened for hemostatic or thrombotic defects. We developed a global physiological screening method for thrombosis by utilizing a laser to induce thrombosis in a specifically targeted area of the major artery and vein. Using this assay, we have screened many fish for abnormal hemostasis, and have isolated a number of mutants with abnormal coagulation parameters. These mutants can be grown, bred, and further evaluated for the genetic etiology of their abnormal hemostatic pathways.
Animals↗
tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.
TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53(N168K) and tp53(M214K)). Although homozygous tp53(N168K) mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37 degrees C, cells in the tp53(M214K) embryos failed to undergo apoptosis in response to gamma radiation at both 28 and 37 degrees C. Unlike wild-type control embryos, irradiated tp53(M214K) embryos also failed to up-regulate p21 and did not arrest at the G(1)/S checkpoint. Beginning at 8.5 months of age, 28% of tp53(M214K) mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.
Alleles↗
Chlorpyrifos exposure of developing zebrafish: effects on survival and long-term effects on response latency and spatial discrimination.
Chlorpyrifos (CPF) is a widely used insecticide, which has been shown to interfere with neurobehavioral development. Rat models have been key in demonstrating that prenatal CPF exposure causes choice accuracy deficits and motor alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the molecular mechanisms underlying the persisting behavioral effects of developmental CPF exposure. Zebrafish with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant impacted neurodevelopment. To facilitate the use of the zebrafish model and to compare it to the more typical rodent models, the behavioral phenotype of CPF toxicity in zebrafish must be well characterized. Our laboratory has developed methods for assessing spatial discrimination learning in zebrafish, which can differentiate response latency from choice accuracy in a three chambered fish tank. Low and high doses of CPF (10 and 100 ng/ml on days 1-5 postfertilization) both had significant persisting effects on both spatial discrimination and response latency over 18 weeks of testing. The high, but not the low dose, significantly accelerated mortality rates of the fish during the study from 20-38 weeks of age. Developmental exposure to either 10 or 100 ng/ml of CPF caused significant spatial discrimination impairments in zebrafish when they were adults. The impairment caused by 10 ng/ml was seen during early but not later testing, while the impairment caused by 100 ng/ml became more pronounced with continued testing. The higher dose caused a more pervasive impairment. The 10 and 100 ng/ml doses had opposite effects on response latency. The low 10 ng/ml dose significantly slowed response latency, while the high 100 ng/ml dose significant increased response latency. Both of these effects diminished with continued testing. CPF exposure during early development caused clear behavioral impairments, which lasted throughout adulthood in zebrafish. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.
Animals↗
Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development.
Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C(18), we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C(18) labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C(18) into the circulation of zebrafish embryos and identified the presence of DiI-C(18)-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.
Animals↗
A quantitative analysis of pharyngeal tooth shape in the zebrafish (Danio rerio, Teleostei, Cyprinidae).
To test whether successive replacement cycles in the pharyngeal dentition of the zebrafish, a polyphyodont vertebrate model organism, entail overall shape changes in the teeth, a qualitative and quantitative analysis was made of size and shape variables in the five ventral teeth. The following measurements were defined: tooth length, tooth height, neck-crown angle, cusp depth, and crown curvature. Ontogenetic changes in fish, ranging between 6 and 29 mm standard length (SL), were analysed by linear regressions on to SL. The teeth became significantly larger with growth of the fish, through successive replacements and cusp depth also increased over time. Neck--crown angle and crown curvature did not change over time. Position-dependent differences were analysed by Friedman ANOVA and Kendall concordance tests. Measurements differed significantly according to tooth position in the pharyngeal jaws. Tooth 1V was always the smallest, 3V the largest. The neck--crown angle and curvature of the crown increased from 1V to 5V. Cusp depth increased from 1V to 3V, and then decreased again. These results indicate that successive replacement cycles entail a size increase accompanied by shape changes apparently restricted to the crown. These quantitative data lay the basis for further descriptive and experimental studies of tooth shape in this model-species.
Analysis of Variance↗
Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors.
To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kilobase (kbp) zebrafish rod opsin genomic clone, which consists of 18 kbp of 5'-flanking region, the entire coding region, and 0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, Southern blotting, and DNA sequencing revealed the rod opsin gene lacks introns. The transcription start site was localized 94 nucleotides upstream of the translation initiation site. Sequence alignment with orthologous promoters revealed conserved cis-elements including glass, NRE, OTX/Bat-1, Ret-1/PCE-1, Ret-4, and TATA box. A 1.2-kbp promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1- to 2-cell stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by polymerase chain reaction amplification of fin genomic DNA, exhibited EGFP expression in the retina, confirming the germline transmission of the transgene. Frozen eye sections demonstrated the EGFP expression was rod-specific and exhibited a similar developmental expression profile as the rod opsin protein. This stable transgenic line provides a novel tool for identification of genes regulating development and maintenance of rod photoreceptors.
Amino Acid Sequence↗
Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria.
Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.
Animals↗
The zebrafish. An overview of its early development.
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Animals↗
Nicotinic involvement in memory function in zebrafish.
Zebrafish are an emerging model for the study of the molecular mechanisms of brain function. To conduct studies of the neural bases of behavior in zebrafish, we must understand the behavioral function of zebrafish and how it is altered by perturbations of brain function. This study determined nicotine actions on memory function in zebrafish. With the methods that we have developed to assess memory in zebrafish using delayed spatial alternation (DSA), we determined the dose effect function of acute nicotine on memory function in zebrafish. As in rodents and primates, low nicotine doses improve memory in zebrafish, while high nicotine doses have diminished effect and can impair memory. This study shows that nicotine affects memory function in zebrafish much like in rats, mice, monkeys and humans. Now, zebrafish can be used to help understand the molecular mechanisms crucial to nicotine effects on memory.
Acetylcholine↗
The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos.
Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.
Amino Acid Sequence↗
Depletion of zebrafish titin reduces cardiac contractility by disrupting the assembly of Z-discs and A-bands.
The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb, and annotated the full-length genomic sequences for both genes. We found that ttna, but not ttnb, is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon-containing and N2A exon-containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon-containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon-containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in "titinopathies" may be attributable to the disruption of different titin isoforms.
Alternative Splicing↗
Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish.
As many as 10% of humans suffer chronic sleep disturbances, yet the genetic mechanisms that regulate sleep remain essentially unknown. It is therefore crucial to develop simple and cost-effective vertebrate models to study the genetic regulation of sleep. The best characterized mammalian sleep/wake regulator is hypocretin/orexin (Hcrt), whose loss results in the sleep disorder narcolepsy and that has also been implicated in feeding behavior, energy homeostasis, thermoregulation, reward seeking, addiction, and maternal behavior. Here we report that the expression pattern and axonal projections of embryonic and larval zebrafish Hcrt neurons are strikingly similar to those in mammals. We show that zebrafish larvae exhibit robust locomotive sleep/wake behaviors as early as the fifth day of development and that Hcrt overexpression promotes and consolidates wakefulness and inhibits rest. Similar to humans with insomnia, Hcrt-overexpressing larvae are hyperaroused and have dramatically reduced abilities to initiate and maintain rest at night. Remarkably, Hcrt function is modulated by but does not require normal circadian oscillations in locomotor activity. Our zebrafish model of Hcrt overexpression indicates that the ancestral function of Hcrt is to promote locomotion and inhibit rest and will facilitate the discovery of neural circuits, genes, and drugs that regulate Hcrt function and sleep.
Animals↗
Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes.
Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.
Animals↗
Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation.
The vertebrate neural tube develops by two distinct mechanisms. Anteriorly, in the brain and future trunk (cervicothoracic) region, 'primary neurulation' occurs, where an epithelial sheet rolls or bends into a tube. Posteriorly, in the future lumbar and tail region, the neural tube forms by 'secondary neurulation', where a mesenchymal cell population condenses to form a solid rod that undergoes transformation to an epithelial tube. Teleost neurulation has been described as different from that of other vertebrates. This is principally because the teleost trunk neural tube initially forms a solid rod (the neural keel) that later develops a lumen. This process has also been termed secondary neurulation. However, this description is not accurate since the teleost neural tube derives from an epithelial sheet that folds. This best fits the description of primary neurulation. It has also been suggested that teleost neurulation is primitive, however, both primary and secondary neurulation are found in groups with a more ancient origin than the teleosts. The similarity between neurulation in teleosts and other vertebrates indicates that this group includes viable models (such as the zebrafish) for understanding human neural tube development.
Animals↗
Using the adult zebrafish visual system to study cadherin-2 expression during central nervous system regeneration.
Zebrafish is a good model organism for the study of vertebrate development due to numerous experimental advantages. Here we show that zebrafish are also suitable for the study of cellular and molecular mechanisms underlying tissue regeneration in the vertebrate central nervous system. In this report we describe simple protocols to introduce injuries to the adult zebrafish visual structures and analyze cadherin-2 expression in the regenerating visual system using immunocytochemistry and immunoblotting.
Animals↗
Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish.
The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.
Animals↗
Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor.
The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.
Amino Acid Sequence↗