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 zebrafish (Danio rerio) is a small tropical fish that has become a powerful animal model system for understanding the genetic basis of vertebrate development. The attributes of the zebrafish include its small size, fecundity, and production of optically clear embryos that undergo exceptionally rapid development ex utero. These qualities make the zebrafish amenable to forward genetic studies to dissect the molecular basis of developmental pathways as well as the phenotypic analysis of embryo-genesis and organogenesis in vivo. In the decades since George Streisinger and his dedicated colleagues pioneered the genetically and experimentally tractable zebrafish system, it has proven its value as a model organism and contributed to our understanding of developmental processes (1,2,3,4). Furthermore, as the sequencing of the zebrafish genome reaches completion, it is also clear that there is a high degree of genetic conservation between man and other vertebrates despite millions of years of divergent evolution (5,6). This is reflected by the reiteration of developmental gene pathways and regulatory mechanisms. However, the zebrafish is only beginning to be exploited as a model for human disease and biomedical research. The same attributes that have led to the emergence of the zebrafish as a preeminent vertebrate embryological model are now contributing to its strong potential for clinical applications (7,8,9,10,11). Unbiased forward genetic analyses of zebrafish models of human disease provide the means to identify novel genes in pathologic pathways, as well as an unprecedented capacity for the in vivo analysis of disease processes and progression. This review focuses on the current technology that takes advantage of the zebrafish to further our understanding of the genetic basis of cancer and its treatment.
Cancer is a genetically complex disease that results from the multistep accumulation of somatic, and occasionally inherited, mutations that lead to clonal neoplastic cell transformation (12). The genetic lesions associated with cancer include the activation of dominant oncogenes and the inactivation of tumor suppressor genes through mutation and loss of heterozygosity (LOH). Mouse models have provided important insights into these collaborating genetic events that lead to cancer formation. For example, retroviral insertion screens and mating strategies using genetically manipulated mice have identified numerous genes involved in enhancing and suppressing the onset of leukemia and lymphoma (13,14,15,16,17,18,19). However, a limitation of these methodologies is that knockout strategies require the a priori knowledge of genes to inactivate, and while retroviral insertion studies uncovered the overexpression of critical oncogenes, they have largely failed to identify inactivated tumor suppressor genes. Forward genetic screens using model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, can overcome these limitations as indicated by their enormous contribution to our understanding of developmental and signal transduction pathways. However, invertebrate models are generally unable to recapitulate the pathogenesis of many human diseases. By contrast, the zebrafish system allows forward genetic approaches in a vertebrate that can manifest the pathologies of human diseases, bridging the gap between mammalian and invertebrate model systems. In addition, the establishment of transgenic lines expressing fluoro-chromes, such as green fluorescent protein (GFP), in specific developing tissues (20,21,22,23,24,25) makes the transparent developing zebrafish particularly amenable to in vivo studies of neoplastic progression, metastasis, and remission.
Cancer encompasses a wide range of heterogeneous tumor types that arise in different tissues, each with different molecular genetic signatures that often change as the disease progresses through different stages of malignancy. Furthermore, cancers exhibit lesions in many other genes that affect diverse cellular processes regulating cell cycle controlled growth and proliferation, genome stability and repair, telomerase activity, apoptosis, and tissue-specific differentiation. Exploiting unbiased forward genetic approaches will help to address the increasingly multifaceted genetic etiology of cancer. It is likely that the use of zebrafish cancer models in modifier screens will uncover a variety of novel genes and chemicals affecting diverse pathways that enhance or suppress features of the tumorigenic phenotype. These screens will lead to the identification of elusive tumor suppressor genes and pathways that are essential to pathogenesis in man. Once identified, such modifiers can serve as new entry points for the development of more efficient anticancer drugs and therapies.