The fruit fly Drosophila melanogaster was chosen by Thomas Hunt Morgan nearly 100 years ago as a model system for working out the basic mechanisms of inheritance, for which he was awarded the Nobel Prize in 1933. Relative to nematodes, there is a more comprehensive set of classical genetic tools available due to the long history of genetic research in flies, and sequence conservation between flies and humans is somewhat higher.86 Its small body size, short generation time (2 weeks), large generation size (~50 eggs per day), and the comprehensive set of genetic tools such as balancer chromosomes and a dense genetic and physical map have proven invaluable in conducting genetic analyses, especially forward genetic screens. The instigation of saturating screens for fly embryogenesis mutants in the early 1980s marked a milestone in genetic analysis in metazoans, providing much of the basis for our current understanding of cell signaling pathways.87
As with other model organisms, forward genetic screens have been used to hunt for potential drug targets that play a role in fly models of human diseases. Of particular usefulness in this regard is the fact that the signaling pathways involved in normal eye development are well studied, and can be manipulated to produce an easily scored phenotype. Such engineered eye screens have been used to identify suppressors of polyglutamine-induced neurodegeneration in fly models for Huntington's disease88 and spinocerebellar ataxia 1.89 The genetic analysis of Ras signaling in flies has been used as a model for cancer; receptor tyrosine kinase activation of Ras, leading to activation of a MAPK pathway, plays a role in normal eye development, and the fly equivalent of oncogenic, constitutively active Ras causes overproliferation of certain cells in the eye.90 Screens for suppressors or enhancers of this cancerlike phenotype have identified a number of key players in Ras signaling, including novel potential oncology targets.91'92
The fly genome sequence was completed in 2000; at approximately 14000 genes, it is somewhat smaller than the nematode genome.93 Matching a sample of 929 human disease genes to the fly genome revealed that 77% of the human genes had at least one fly homolog, indicating the utility of fly genomics to target discovery.94 Many of the typical genomics tools and datasets are available at flybase95 and the Berkeley Drosophila Genome Project.96 A project is underway to systematically disrupt every gene via transposon insertions.97 An ingenious early application of genomics technologies in combination with classical genetic tools for studying fly development used automated embryo sorting and gene expression profiling to study the effects of perturbations of the twist transcription factor on mesoderm development.98 As in nematodes, RNAi can be triggered by dsRNA, but in flies most genome-scale screening is done with cultured cells arrayed in microwell plates rather than in the whole organism. Some fly cell lines can naturally take up dsRNA, others require addition of a transfection reagent. Large-scale genome screens have been conducted to study hedgehog signaling pathways,99 cell proliferation and viability,99 cell morphology,100 cardiac development,101 and Wnt signaling.102 In the Wnt study, 238 potential regulators were identified, including both known pathway components and some genes without previously assigned function. Several regulators were validated by testing for effects on Wnt signaling in Drosophila embryos as well as in zebrafish embryos and cultured mammalian cells, indicating that these genes have evolutionarily conserved functions in Wnt signaling.
The potential power of whole-genome screening for novel drugs and simultaneously identifying the targeted pathway was demonstrated in a parallel screen for small-molecule and dsRNA inhibitors of cytokinesis.103 A dsRNA library covering more than 90% of the fly genome and a small-molecule library consisting of 51 000 compounds was screened in a cell line for the binucleate phenotype indicative of a cytokinesis block by automated fluorescent microscopy. Fifty compounds and 214 dsRNAs gave a detectable cytokinesis block. The 25 most potent compounds and 40 best dsRNA inhibitors were analyzed further by testing for the immunolocalization pattern of a set of 15 proteins involved in various aspects of cytokinesis. By comparing the patterns of localization phenotypes among the dsRNA and small-molecule cytokinesis inhibitors, a match was made between one compound and the Aurora B kinase, suggesting that this compound targets either Aurora B or an upstream member of the Aurora pathway. This type of pattern matching approach, utilizing a combination of small molecule and RNAi perturbations and assessing multiple phenotypic changes, should prove to be a powerful method for target identification in the future.
Although flies and worms allow for efficient functional genomics studies of disease-relevant genes and pathways in the context of a multicellular organism, the zebrafish Danio rerio is becoming the system of choice for identifying novel targets in the context of a whole vertebrate animal. The zebrafish was originally developed by George Streisinger as model system for doing forward genetic screens in vertebrates and has proven to be a powerful tool for understanding early development.104 Its relatively short generation time (3-4 months), large generation size (up to 200 eggs per clutch), optically clear embryos, and small body size (4cm) make it uniquely suited among vertebrates for large-scale genetic analysis, especially phenotypic screening.105,106 The genome sequence is nearly complete,107 and many of the standard genetics and genomics tools are available, including transcription profiling with DNA microarrays,108 a cDNA collection,109 and high-resolution physical maps to facilitate positional cloning.110,111 Antisense phosphorodiamidate morpholino oligonucleotides (morpholinos) can be used for efficient gene knockdown,112 possibly allowing for genome-wide, reverse-genetic functional screening in the future, although the current mode of delivery (microinjection) is rate-limiting. A reverse-genetics strategy allowing for efficient recovery of randomly generated mutations in specific genes, called targeting induced local lesions in genomes (TILLING)113 has been used successfully in zebrafish.114 High-throughput, whole-organism compound screens can be conducted as well by simply arraying embryos and compounds together in multiwell plates.115
The zebrafish system has proven useful for the identification of novel drug targets by the application of classical forward genetics (or knockdown by morpholino) to identify mutations that mimic human disease phenotypes. The obvious advantage of zebrafish over other model systems is that organ-level and vertebrate-specific diseases can be modeled. The first demonstration of the utility of zebrafish for identification of human disease genes was in the area of anemia; the iron transporter ferroportin 1 was originally discovered in zebrafish116 and subsequently found to be mutated in the hereditary iron overload disease hemochromatosis.117,118 Models for polycystic kidney disease,119-121 melanoma,122 and various leukemias123-125 have been developed. Complex nervous system and sensory defects have been modeled in zebrafish, including neurodegeneration caused by heterologous expression of mutant tau126 and various forms of blindness and deafness.127,128
The zebrafish system has been particularly useful in the area of heart disease. Mutations in TBX5 result in congenital defects in cardiac development in both humans and fish,129,130 and mutations in the muscle filament titin, which cause familial cardiomyopathies in humans,131 exhibit a similar phenotype in zebrafish.132 Mutations in the ether-a-go-go related gene hERG cause arrhythmias in both humans and zebrafish, and drugs targeting the potassium channel encoded by hERG can induce arrhythmia in both organisms.133,134 Since many commonly used drugs have the side effect of causing arrhythmia via this mechanism, the zebrafish system may prove useful in screening out cardiotoxic drug candidates.
A powerful strategy for identification of novel drugs in zebrafish uses a disease model to identify small molecules that suppress the disease phenotype. In this scenario, the target must still be identified by traditional means, so the advantage of using fish lies as much in the presumptive quality of leads as in target identification, since issues of toxicity and metabolism of the drug are reduced by whole organism screening. The utility of this approach has been demonstrated in a heart disease model.135 The hypomorphic gridlock allele of the zebrafish hey2 (hiairy/enhancer of split-related) transcription factor produces congenital aortic defects that are phenotypically similar to aortic coarctation in humans: constriction of the aorta upstream of the aortic branch leading to the trunk results in reduced circulation. In the zebrafish screen, mutant embryos arrayed in 96-well plates were treated with a set of 5000 compounds, and a pyridinyl-thioether compound was found that suppresses the effects of the hey2 mutation, leading to restoration of proper aortic development. In a first step toward addressing the mechanism of action, candidate genes were tested for expression levels, and vascular endothelial growth factor (VEGF) was found to be upregulated by treatment with the drug in both zebrafish and cultured human cells, leading to increased angiogenic potential in both systems. Although the target of this compound remains to be identified, it should prove useful for investigating angiogenic mechanisms. This disease-suppression approach should at least prove valuable for identifying novel pathways to target, and perhaps in the future the application of focused morpholino libraries or panels of candidate mutants produced by TILLING combined with a chemical genomics approach will speed target identification in zebrafish.
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