Chemical Genetics: Drug Screens in Zebrafish

High throughput chemical genetic screens for compounds with specific biological activity in a whole organism are feasible using zebrafish embryos. At least two medium to large scale drug screens have been carried out to date, leading to the identification of compounds that disturb zebrafish development. Chemical genetics using zebrafish embryos may become an important step in the discovery of drugs and their targets.     

Small molecule screening in the zebrafish

The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.     

Making waves in cancer research: new models in the zebrafish

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.     

The hitchhiker's guide to Xenopus genetics

A decade after the human genome sequence, most vertebrate gene functions remain poorly understood, limiting benefits to human health from rapidly advancing genomic technologies. Systematic in vivo functional analysis is ideally suited to the experimentally accessible Xenopus embryo, which combines embryological accessibility with a broad range of transgenic, biochemical, and gain-of-function assays. The diploid X. tropicalis adds loss-of-function genetics and enhanced genomics to this repertoire. In the last decade, diverse phenotypes have been recovered from genetic screens, mutations have been cloned, and reverse genetics in the form of TILLING and targeted gene editing have been established. Simple haploid genetics and gynogenesis and the very large number of embryos produced streamline screening and mapping. Improved genomic resources and the revolution in high-throughput sequencing are transforming mutation cloning and reverse genetic approaches. The combination of loss-of-function mutant backgrounds with the diverse array of conventional Xenopus assays offers a uniquely flexible platform for analysis of gene function in vertebrate development.     

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

Behavioral screening for neuroactive drugs in zebrafish

The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.     

Identification of a novel retinoid by small molecule screening with zebrafish embryos

Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A-P) patterning to identify the target of DTAB, a compound that caused A-P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors gamma and beta. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules.     

Identification of Selected Gamma-Ray Induced Deficiencies in Zebrafish Using Multiplex Polymerase Chain Reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

Genetic screens for mutations affecting development of Xenopus tropicalis

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.     

Zebrafish: a model system for the study of human disease

The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which resemble human clinical disorders. The creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease.     

Bioenergetic profiling of zebrafish embryonic development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.     

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.     

Zebrafish genetic models for arrhythmia

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 Center, 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.     

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.     

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.     

斑马鱼模型在药物筛选中的应用

斑马鱼具有子代数量多、体外受精、胚胎透明、可以做大规模遗传突变筛选等生物学特性, 因此成为一种良好的脊椎动物模式生物。随着研究的深入, 斑马鱼不仅应用于遗传学和发育生物学研究, 而且拓展和延伸到疾病模型和药物筛选领域。作为一种整体动物模型, 斑马鱼能够全面地检测评估化合物的活性和副作用, 实现高内涵筛选。近年来, 科学家们不断地发展出新的斑马鱼疾病模型和新的筛选技术, 并找到了一批活性化合物。这些化合物大多数在哺乳动物模型中也有相似的效果, 其中前列腺素E2(dmPGE2)和来氟米特(Leflunomide)已经进入临床实验, 分别用来促进脐带血细胞移植后的增殖和治疗黑素瘤。这些成果显示了斑马鱼模型很适合用于药物筛选。文章概括介绍了斑马鱼模型的特点和近年来在疾病模型和药物筛选方面的进展, 希望能够帮助人们了解斑马鱼在新药研发中的应用, 并开展基于斑马鱼模型的药物筛选。     

Regulation of vertebrate eye development by Rx genes

The paired-like homeobox-containing gene Rx has a critical role in the eye development of several vertebrate species including Xenopus, mouse, chicken, medaka, zebrafish and human. Rx is initially expressed in the anterior neural region of developing embryos, and later in the retina and ventral hypothalamus. Abnormal regulation or function of Rx results in severe abnormalities of eye formation. Overexpression of Rx in Xenopus and zebrafish embryos leads to overproliferation of retinal cells. A targeted elimination of Rx in mice results in a lack of eye formation. Mutations in Rx genes are the cause of the mouse mutation eyeless (ey1), the medaka temperature sensitive mutation eyeless (el) and the zebrafish mutation chokh. In humans, mutations in Rx lead to anophthalmia. All of these studies indicate that Rx genes are key factors in vertebrate eye formation. Because these results cannot be easily reconciled with the most popular dogmas of the field, we offer our interpretation of eye development and evolution.     

A call to fins! Zebrafish as a gerontological model

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.     

In vivo imaging of embryonic vascular development using transgenic zebrafish

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.