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The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed.

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes.

In the past two decades, zebrafish genetic screens have identified a wealth of mutations that have been essential to the understanding of development and disease biology. More recently, chemical screens in zebrafish have identified small molecules that can modulate specific developmental and behavioural processes. Zebrafish are a unique vertebrate system in which to study chemical genetic systems, identify drug leads, and explore new applications for known drugs. Here, we discuss some of the advantages of using zebrafish in chemical biology, and describe some important and creative examples of small molecule screening, drug discovery and target identification.

Over the last decade the zebrafish has emerged as a major genetic model organism. While stimulated originally by the utility of its transparent embryos for the study of vertebrate organogenesis, the success of the zebrafish was consolidated through multiple genetic screens, sequencing of the fish genome by the Sanger Centre, and the advent of extensive genomic resources. In the last few years the potential of the zebrafish for in vivo cell biology, physiology, disease modeling and drug discovery has begun to be realized. This review will highlight work on cardiac electrophysiology, emphasizing the arenas in which the zebrafish complements other in vivo and in vitro models; developmental physiology, large scale screens, high-throughput disease modeling and drug discovery. Much of this work is at an early stage, and so the focus will be on the general principles, the specific advantages of the zebrafish and on future potential.

Background

Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.

Results

Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.

Conclusions

Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

Zebrafish studies in the past two decades have made major contributions to our understanding of hematopoiesis and its associated disorders. The zebrafish has proven to be a powerful organism for studies in this area owing to its amenability to large-scale genetic and chemical screening. In addition, the externally fertilized and transparent embryos allow convenient genetic manipulation and in vivo imaging of normal and aberrant hematopoiesis. This review discusses available methods for studying hematopoiesis in zebrafish, summarizes key recent advances in this area, and highlights the current and potential contributions of zebrafish to the discovery and development of drugs to treat human blood disorders.

The zebrafish model is rapidly gaining prominence in the study of development, hematopoiesis, and disease. The zebrafish provides distinct advantages over other vertebrate models during early embryonic development by producing transparent, externally fertilized embryos. Embryonic zebrafish are easily visualized and manipulated through microinjection, chemical treatment, and mutagenesis. These procedures have contributed to large-scale chemical, suppressor, and genetic screens to identify hematopoietic gene mutations. Genomic conservation and local synteny between the human and zebrafish genomes make genome-scale and epigenetic analysis of these mutations (by microarray, chromatin immunoprecipitation sequencing, and RNA sequencing procedures) powerful methods for translational research and medical discovery. In addition, large-scale screening techniques have resulted in the identification of several small molecules capable of rescuing hematopoietic defects and inhibiting disease. Here, we discuss the contributions of the zebrafish model to the understanding of hematopoiesis, hematopoietic stem cell development, and disease-related discovery. We also highlight the recent discovery of small molecules with clinical promise, such as dimethyl prostaglandin E2, 3F8, and thiazole-carboxamide 10A.

The discovery of truly efficacious treatments that lead to full recovery is a daunting task in psychiatric illness. A systems-based orientation to in vivo pharmacology has been suggested as a way to transform psychiatric drug discovery and development. A critical catalyst in the success of recent systems biology efforts has been the incorporation of data mining strategies. Our approach to the drug discovery problem has been to utilize the whole animal to provide a systems response that is subsequently mined for predictive attributes with known psychopharmacological value. Our in vivo data mining approach, termed Pattern Array, establishes a framework for screening novel chemical entities based upon a response that represents the net pharmacological effect on the system of interest, namely the central nervous system (CNS). Large scale screening of small molecules by non-conventional approaches such as this at a systems level may improve the identification of novel chemical entities with psychiatric utility. This type of approach will compliment the more labor-intensive models based upon construct validity. It will take the collective effort of many disciplines and numerous strategies in close association with clinical colleagues to address quality of life issues and breakthrough treatment barriers in psychiatric illness.

In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts.

In vivo chemical screening is a broadly applicable approach not only for dissecting genetic pathways governing hematopoiesis and hematological diseases, but also for finding critical components in those pathways that may be pharmacologically modulated. Both high-throughput chemical screening and facile detection of blood-cell-related phenotypes are feasible in embryonic/larval zebrafish. Two recent studies utilizing phenotypic chemical screens in zebrafish have identified several compounds that promote hematopoietic stem cell formation and reverse the hematopoietic phenotypes of a leukemia oncogene, respectively. These studies illustrate efficient drug discovery processes in zebrafish and reveal novel biological roles of prostaglandin E2 in hematopoietic and leukemia stem cells. Furthermore, the compounds discovered in zebrafish screens have become promising therapeutic candidates against leukemia and included in a clinical trial for enhancing hematopoietic stem cells during hematopoietic cell transplantation.

Many of today’s drug discovery programs utilize high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or linewidth to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR-based screens that use 1D 1H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.

Chemical genetic screening can be described as a discovery approach in which chemicals are assayed for their effects on a defined biological system. The zebrafish, Danio rerio, is a well-characterized and genetically tractable vertebrate model organism that produces large numbers of rapidly developing embryos that develop externally. These characteristics allow for flexible, rapid, and scalable chemical screen design using the zebrafish. We describe a protocol for screening compounds from a chemical library for effects on early zebrafish development using an automated in situ based read-out. Because screens are performed in the context of a complete, developing organism, this approach allows for a more comprehensive analysis of the range of a chemical’s effects than that provided by, for example, a cell culture-based or in vitro biochemical assay. Using a twenty-four hour chemical treatment, one can complete a round of screening in six days.

During the past decade, single gene disruption in mice and large-scale mutagenesis screens in zebrafish have elucidated many fundamental genetic pathways that govern early heart patterning and differentiation. Specifically, a number of genes have been revealed serendipitously to play important and selective roles in cardiac valve development. These initially surprising results have now converged on a finite number of signaling pathways that regulate endothelial proliferation and differentiation in developing and postnatal heart valves. This review highlights the roles of the most well-established ligands and signaling pathways, including VEGF, NFATc1, Notch, Wnt/β-catenin, BMP/TGF-β, ErbB, and NF1/Ras. Based on the interactions among and relative timing of these pathways, a signaling network model for heart valve development is proposed.

The highly successful zebrafish workshop on infectious disease and cancer in zebrafish in Leiden (Netherlands) in 20071 strongly motivated the authors of this report to organize a follow-up meeting.Prompted by the prediction that similarities between defense mechanisms against microbes and cancer cells can reveal new insights into specific determinants of innate immune responses, this workshop focused on cancer models and infection studies. Due to the amenability of zebrafish to large scale forward and reverse genetic screens this model organism is ideal for discovery of novel gene functions in disease processes at a throughput level that can not be matched by rodent models. Furthermore, owing to its small size and optical transparency, disease manifestations and resulting immune responses can be studied at the whole organism level. Particularly advantageous in this context are fluorescence multicolor labeling techniques that allow tagging of the players in disease processes (e.g. cancer cells, immune cells, and microbes) for easy detection in vivo. In the following report the oral presentations at the meeting are summarized chronologically.

Neuroactive small molecules are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high—throughput (HT) behavior—based approach to neuroactive small molecule discovery in the zebrafish. We use automated screening assays to evaluate thousands of chemical compounds and find that diverse classes of neuroactive molecules cause distinct patterns of behavior. These `behavioral barcodes' can be used to rapidly identify novel psychotropic chemicals and to predict their molecular targets. For example, we identify novel acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining HT screening technologies with behavioral phenotyping in vivo, behavior—based chemical screens may accelerate the pace of neuroactive drug discovery and provide small—molecule tools for understanding vertebrate behavior.

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Zebrafish are a widely utilised animal model in developmental genetics, and owing to recent advances in our understanding of zebrafish behaviour, their utility as a comparative model in behavioural neuroscience is beginning to be realised. One widely reported behavioural measure is the novel tank-diving assay, which has been often cited as a test of anxiety and stress reactivity. Despite its wide utilisation, and various validations against anxiolytic drugs, reporting of pre-test housing has been sparse in the literature. As zebrafish are a shoaling species, we predicted that housing environment would affect their stress reactivity and, as such, their response in the tank-diving procedure. In our first experiment, we tested various aspects of housing (large groups, large groups with no contact, paired, visual contact only, olfactory contact only) and found that the tank diving response was mediated by visual contact with conspecifics. We also tested the basal cortisol levels of group and individually housed fish, and found that individually housed individuals have lower basal cortisol levels. In our second experiment we found ethanol appeared to have an anxiolytic effect with individually housed fish but not those that were group housed. In our final experiment, we examined the effects of changing the fishes' water prior to tank diving as an additional acclimation procedure. We found that this had no effect on individually housed fish, but appeared to affect the typical tank diving responses of the group housed individuals. In conclusion, we demonstrate that housing represents an important factor in obtaining reliable data from this methodology, and should be considered by researchers interested in comparative models of anxiety in zebrafish in order to refine their approach and to increase the power in their experiments.

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Histological characterization is used in clinical and research contexts as a highly sensitive method for detecting the morphological features of disease and abnormal gene function. Histology has recently been accepted as a phenotyping method for the forthcoming Zebrafish Phenome Project, a large-scale community effort to characterize the morphological, physiological, and behavioral phenotypes resulting from the mutations in all known genes in the zebrafish genome. In support of this project, we present a novel content-based image retrieval system for the automated annotation of images containing histological abnormalities in the developing eye of the larval zebrafish.

The zebrafish offers a scalable vertebrate model for many areas of biologic investigation. There is substantial conservation of genetic and genomic features and, at a higher order, conservation of intermolecular networks, as well as physiologic systems and phenotypes. We highlight recent work demonstrating the extent of this homology, and efforts to develop high-throughput phenotyping strategies suited to genetic or chemical screening on a scale compatible with in vivo validation for systems biology. We discuss the implications of these approaches for functional annotation of the genome, elucidation of multicellular processes in vivo, and mechanistic exploration of hypotheses generated by a broad range of ‘unbiased’ ‘omic technologies such as expression profiling and genome-wide association. Finally, we outline potential strategies for the application of the zebrafish to the systematic study of phenotypic architecture, disease heterogeneity and drug responses.

Background

Zebrafish is becoming an increasingly attractive model organism for understanding biology and developing therapeutics, because as a vertebrate, it shares considerable similarity with mammals in both genetic compositions and tissue/organ structures, and yet remains accessible to high throughput phenotype-based genetic and small molecule compound screening.

Objective/method

The focus of this review is on the nervous system, which is arguably the most complex organ and known to be afflicted by more than six hundred disorders in humans. I discuss the past, present, and future of using zebrafish to assess the impact of small molecule drugs on neural development and function, in light of understanding and treating neurodevelopmental disorders such as autism, neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Hungtington’s disease, and neural system dysfunctions such as anxiety/depression and addiction.

Conclusion

These studies hold promise to reveal fundamental mechanisms governing nervous system development and function, and to facilitate small molecule drug discovery for the many types of neurological disorders.

There is a remarkable array of new chemical entities in the current antiepileptic drug (AED) development pipeline. In some cases, the compounds were synthesized in an attempt improve upon the activity of marketed AEDs. In other cases, the discovery of antiepileptic potential was largely serendipitous. Entry into the pipeline begins with the demonstration of activity in one or more animal screening models. Results from testing in a panel of such models provide a basis to differentiate agents and may offer clues as to the mechanism. Target activity may then be defined through cell-based studies, often years after the initial identification of activity. Some pipeline compounds are believed to act through conventional targets, whereas others are structurally novel and may act by novel mechanisms. Follow-on agents include the levetiracetam analogs brivaracetam and seletracetam that act as SV2A-ligands; the valproate-like agents valrocemide, valnoctamide, propylisopropyl acetamide, and isovaleramide; the felbamate analog flurofelbamate, a dicarbamate, and the unrelated carbamate RWJ-333369; the oxcarbazepine analog licarbazepine, which probably acts as a use-dependent sodium channel blockers, and its prodrug acetate BIA 2-093; and various selective partial benzodiazepine receptor agonists, including ELB139, which is a positive allosteric modulator of α3-containing GABAA receptors. A variety of AEDs that may act through novel targets are also in clinical development: lacosamide, a functionalized amino acid; talampanel, a 2,3-benzodiazepine selective noncompetitive AMPA receptor antagonist; NS1209, a competitive AMPA receptor antagonist; ganaxolone, a neuroactive steroid that acts as a positive modulator of GABAA receptors; retigabine, a KCNQ potassium channel opener with activity as a GABAA receptor positive modulator; the benzanilide KCNQ potassium channel opener ICA-27243 that is more selective than retigabine; and rufinamide, a triazole of unknown mechanism.

The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo’s developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo’s innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model, that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening.

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.