Zebrafish as a model system for biomedical studies

Zebrafish (Danio rerio) is now firmly recognized as a powerful research model for many areas of biology and medicine. Here, we review some achievements of zebrafish-based assays for modeling human diseases and for drug discovery and development. For drug discovery, zebrafish is especially valuable during the earlier stages of research as its represents a model organism to demonstrate a new treatment’s efficacy and toxicity before more costly mammalian models are used. This review considers some examples of known compounds which exhibit both physiological activity and toxicity in humans and zebrafish. The major advantages of zebrafish embryos consist in their permeability to small molecules added to their incubation medium and chorion transparency that enables the easy observation of the development. Assay of acute toxicity (LC₅₀ estimation) in embryos can also include the screening for developmental disorders as an indicator of teratogenic effects. We have used the zebrafish model for toxicity testing of new drugs based on phospholipid nanoparticles (e.g. doxorubicin). Genome organization and the pathways involved into control of signal transduction appear to be highly conserved between zebrafish and humans and therefore zebrafish may be used for modeling of human diseases. The review provides some examples of zebrafish application in this field.     

Modeling human muscle disease in zebrafish

Zebrafish reproduce in large quantities, grow rapidly, and are transparent early in development. For these reasons, zebrafish have been used extensively to model vertebrate development and disease. Like mammals, zebrafish express dystrophin and many of its associated proteins early in development and these proteins have been shown to be vital for zebrafish muscle stability. In dystrophin-null zebrafish, muscle degeneration becomes apparent as early as 3 days post-fertilization (dpf) making the zebrafish an excellent organism for large-scale screens to identify other genes involved in the disease process or drugs capable of correcting the disease phenotype. Being transparent, developing zebrafish are also an ideal experimental model for monitoring the fate of labeled transplanted cells. Although zebrafish dystrophy models are not meant to replace existing mammalian models of disease, experiments requiring large numbers of animals may be best performed in zebrafish. Results garnered from using this model could lead to a better understanding of the pathogenesis of the muscular dystrophies and the development of future therapies.     

Zebrafish: a new model on the pharmaceutical catwalk

Zebrafish is recognized as one of the most important vertebrate model organisms; however, its value in pharmacological studies has not been extensively explored and exploited. In this review, I summarize significant findings about the effects of drugs and medicines on important physiological processes in zebrafish. Our experiments have shown that cardiovascular, anti-angiogenic and anti-cancer drugs elicit comparable responses in zebrafish embryos to those in mammalian systems. Similar observations have been reported by other laboratories, exposing zebrafish to a variety of pharmaceutical active compounds affecting a range of different processes. All the data summarized indicate that zebrafish represents a very valuable organism for different kinds of pharmacological studies, such as screenings of chemical libraries, lead validation and optimization, mode-of-action studies, analysis of gene function, predictive toxicology and teratogenicity, pharmacogenomics and toxicogenomics. Zebrafish pharmacological assays have specific advantages compared to in vitro cell culture studies and in vivo experiments using mice, complementing these assays to give valuable guides for future tests of new drugs for human therapy.     

The use of mature zebrafish (Danio rerio) as a model for human aging and disease

Zebrafish (Danio rerio) have been extensively utilized for understanding mechanisms of development. These studies have led to a wealth of resources including genetic tools, informational databases, and husbandry methods. In spite of all these resources, zebrafish have been underutilized for exploring pathophysiology of disease and the aging process. Zebrafish offer several advantages over mammalian models for these studies, including the ability to perform saturation mutagenesis and the capability to contain thousands of animals in a small space. In this review, we will discuss the use of mature zebrafish as an animal model and provide specific examples to support this novel use of zebrafish. Examples include demonstrating that clinical pathology can be performed in mature zebrafish and that age-associated changes in heat shock response can be observed in zebrafish. These highlights demonstrate the utility of zebrafish as a model for disease and aging.     

Developmental toxicity screening in zebrafish

Given the ever-increasing toxic exposure ubiquitously present in our environment as well as emerging evidence that these exposures are hazardous to human health, the current rodent-based regulations are proving inadequate. In the process of overhauling risk assessment methodology, a nonrodent test organism, the zebrafish, is emerging as tractable for medium- and high-throughput assessments, which may help to accelerate the restructuring of standards. Zebrafish have high developmental similarity to mammals in most aspects of embryo development, including early embryonic processes, and on cardiovascular, somite, muscular, skeletal, and neuronal systems. Here, we briefly describe the development of these systems and then chronicle the toxic impacts assessed following chemical exposure. We also compare the available data in zebrafish toxicity assays with two databases containing mammalian toxicity data. Finally, we identify gaps in our collective knowledge that are ripe for future studies.     

Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin,Fgf, and sonic hedgehog function

BACKGROUND: Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS: Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS: Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol‐induced decreases in GAD1 and Atonal1a gene expression. CONCLUSIONS: These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.     

Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish

Altered neurological function will generally be behaviourally apparent. Many of the behavioural models pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that software solutions are readily available from multiple vendors to automatically test behavioural responses in 96 larvae simultaneously, zebrafish are a potent and rapid model for screening neurological impairments. Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio)

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor γ and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research.     

Zebrafish Models of p53 Functions

Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease. The growing number of genetic tools available in zebrafish research has resulted in the identification of many genes involved in developmental and disease processes. In particular, studies in the zebrafish have clarified roles of the p53 tumor suppressor in the formation of specific tumor types, as well as roles of p53 family members during embryonic development. The zebrafish has also been instrumental in identifying novel mechanisms of p53 regulation and highlighting the importance of these mechanisms in vivo. This article will summarize how zebrafish models have been used to reveal numerous, important aspects of p53 function.The zebrafish, Danio rerio, is a small model organism that has long been used to study vertebrate development. Zebrafish embryos are optically clear and develop externally to the mother, facilitating the study of early developmental processes. In addition, zebrafish have increasingly been used in modeling human diseases, including a number of cancers. The availability of forward and reverse genetic tools in the zebrafish has resulted in the identification and characterization of many genes involved in development and disease. One gene that has been extensively studied is the p53 tumor suppressor gene, which is structurally and functionally conserved in the zebrafish. This article will discuss how studies in the zebrafish have increased our understanding of how p53 contributes to the formation of specific tumor types, resulted in the identification of novel mechanisms of p53 regulation, and showed how p53 and p53 family members are involved in embryonic development.     

Zebrafish models for the functional genomics of neurogenetic disorders

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Influences of Domoic Acid Exposure on Cardiac Development and the Expression of Cardiovascular Relative Genes in Zebrafish (Daniorerio) Embryos

Domoic acid (DA) is a highly toxic phycotoxin that is generated from marine diatoms Pseudonitzschia spp. It has been found that bivalves or cephalopods can accumulate DA to a high level through their feeding activities and cause illness or death in consumers. Zebrafish have been used as a model to investigate and characterize the developmental toxicity of DA. However, there is no report about the relationship between DA and cardiac development in zebrafish. Here, zebrafish embryos were exposed to DA with at the dose of 1, 10, 100, and 1000 ng/L. High mortality and some developmental toxicity including pericardial and yolk sac edema, dorsal curvature, and cardiac defects were observed in the DA‐treated larvae. We found that DA exposure not only disrupted normal cardiac development but also altered the expression of some cardiac development correlated genes and calcium ion channels, such as Anf, Bnp, Atp2a2a, Atp2a2b, Ncx1h, Ryr2b, and Tbx5.     

Development of zebrafish epidermis

Zebrafish epidermal ionocytes are analogous to mammalian kidney cells in terms of expression and function of ion transporters. In this review, we summarize current findings about the development of the zebrafish epidermis and demonstrate how the zebrafish regulate stress acclimation through induction of cell differentiation. In addition, cellular homologies between zebrafish epidermal ionocytes and mammalian kidney cells are presented to show the potential of zebrafish epidermis as an in vivo model to study the development and function of mammalian cells.     

Zebrafish models to study hypoxia-induced pathological angiogenesis in malignant and nonmalignant diseases

Most in vivo preclinical disease models are based on mouse and other mammalian systems. However, these rodent-based model systems have considerable limitations to recapitulate clinical situations in human patients. Zebrafish have been widely used to study embryonic development, behavior, tissue regeneration, and genetic defects. Additionally, zebrafish also provides an opportunity to screen chemical compounds that target a specific cell population for drug development. Owing to the availability of various genetically manipulated strains of zebrafish, immune privilege during early embryonic development, transparency of the embryos, and easy and precise setup of hypoxia equipment, we have developed several disease models in both embryonic and adult zebrafish, focusing on studying the role of angiogenesis in pathological settings. These zebrafish disease models are complementary to the existing mouse models, allowing us to study clinically relevant processes in cancer and nonmalignant diseases, which otherwise would be difficult to study in mice. For example, dissemination and invasion of single human or mouse tumor cells from the primary site in association with tumor angiogenesis can be studied under normoxia or hypoxia in zebrafish embryos. Hypoxia-induced retinopathy in the adult zebrafish recapitulates the clinical situation of retinopathy development in diabetic patients or age-related macular degeneration. These zebrafish disease models offer exciting opportunities to understand the mechanisms of disease development, progression, and development of more effective drugs for therapeutic intervention.     

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1 Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Development of sensory systems in zebrafish (Danio rerio)

Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.     

Large-scale analysis of acute ethanol exposure in zebrafish development: a critical time window and resilience

Background

In humans, ethanol exposure during pregnancy causes a spectrum of developmental defects (fetal alcohol syndrome or FAS). Individuals vary in phenotypic expression. Zebrafish embryos develop FAS-like features after ethanol exposure. In this study, we ask whether stage-specific effects of ethanol can be identified in the zebrafish, and if so, whether they allow the pinpointing of sensitive developmental mechanisms. We have therefore conducted the first large-scale (>1500 embryos) analysis of acute, stage-specific drug effects on zebrafish development, with a large panel of readouts.

Methodology/Principal Findings

Zebrafish embryos were raised in 96-well plates. Range-finding indicated that 10% ethanol for 1 h was suitable for an acute exposure regime. High-resolution magic-angle spinning proton magnetic resonance spectroscopy showed that this produced a transient pulse of 0.86% concentration of ethanol in the embryo within the chorion. Survivors at 5 days postfertilisation were analysed. Phenotypes ranged from normal (resilient) to severely malformed. Ethanol exposure at early stages caused high mortality (≥88%). At later stages of exposure, mortality declined and malformations developed. Pharyngeal arch hypoplasia and behavioral impairment were most common after prim-6 and prim-16 exposure. By contrast, microphthalmia and growth retardation were stage-independent.

Conclusions

Our findings show that some ethanol effects are strongly stage-dependent. The phenotypes mimic key aspects of FAS including craniofacial abnormality, microphthalmia, growth retardation and behavioral impairment. We also identify a critical time window (prim-6 and prim-16) for ethanol sensitivity. Finally, our identification of a wide phenotypic spectrum is reminiscent of human FAS, and may provide a useful model for studying disease resilience.     

Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

Background

The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines.

Methodology/Principal Findings

In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification.

Conclusion

Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.     

Developmental stages of zebrafish (Danio rerio) embryos and toxicological studies using foldscope microscope

Zebrafish (Danio rerio), is a well‐established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the zebrafish embryos. Briefly, the developmental stages, such as zygote, cleavage, blastula, gastrula, segmentation, and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar‐turmerone along with rhodamine B (bio‐coupler) on zebrafish embryos are analyzed upto 72 hr using foldscopes in live condition. The lethal endpoints, such as coagulation, lack of somite formation, non‐detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio‐evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C. longa, ar‐turmerone and rhodamine B exposed embryos remains healthy up to 96, 48, and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared with the control embryos. It is confirmed that the test compounds are not highly toxic to zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural‐regeneration and its neuronal development in zebrafish embryos.     

Antibiotic Toxicity and Absorption in Zebrafish Using Liquid Chromatography-Tandem Mass Spectrometry

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10–1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD₅₀ values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.