Phenotype based chemical screening in zebrafish. Male and female pairs are bred to produce hundreds of single cell embryos that are fertilized ex vivo. For high throughput screening, groups of males and females can be bred within in a larger tank (group breeding), producing high numbers of embryos for screening. Breeding is synchronized by the light/dark cycles, and the fish tend to breed within the first two hours of light in the morning. In this example, embryos are arrayed in 96-well plates, each with a different chemical compound, and observed for a specific phenotype. The chemical in well A2 causes a loss of melanocytes, like MoTP [18]. MoTP can specifically kill differentiated melanocytes, and has become a valuable chemical tool to explore melanocyte stem cell biology [42-44]. Other small molecule screens in zebrafish have also identified pigmentation phenotypes [85,86,96]. In another example of a small molecule screen, compounds are screened for inhibition of tail fin regeneration. The tail fin is clipped and grows back within a few days. A zebrafish embryo treated with the compound from well B3, a glucocorticoid, does not correctly regenerate its tail fin [97].

Creative examples of chemical biology in zebrafish. The zebrafish system can be used in a wide range of chemical biology experiments and screens. A. Zebrafish as young as four dpf have active and sleep-like states. Continuous tracking of movement behaviours of the embryos during rest and wake states, established by light and dark cycles, can be recorded by a camera and computer. High throughput screening for behavioural changes has identified new uses for poorly characterized compounds [80,82]. B. Genetic polymorphisms may underlie differences in sensitivity to poor nutrition. In this example, ENU mutagenized zebrafish (parental (P) generation) were screened for genetic mutations that showed sensitivity to sub-optimal copper nutrient conditions. Zebrafish embryos fertilized with UV-inactivated sperm can live as haploid embryos until about 3 dpf [2]. Haploid embryos of the heterozygous mother (F1 generation) were screened for loss of pigmentation, but only in the presence of the small molecule copper chelator, neocuproine [41]. C. Intensive efforts by the pharmaceutical industry to develop drugs that target the MAPK pathway to treat cancer patients may also be useful for the management of developmental diseases caused by mutations in the MAPK pathway. In the zebrafish model, expression of BRAF or MEK cardio-facio-cutaneous (CFC) mutant alleles interferes with early development. A one-hour treatment within a specific developmental time window with a MEK inhibitor is sufficient to allow normal development for a CFC zebrafish embryo [75]. D. The cardio-vasculature system is conserved in fish, mice and humans. A small molecule screen for changes in hematopoietic stem cells (HSC) development identified the prostaglandin pathway as critical for HSC establishment [59]. A long-acting compound, called dmPGE2, can stimulate HSC development in the embryo and adult zebrafish, as well as in the mouse. dmPGE2 can be safely administered to people, and a clinical trial is underway to see if dmPGE2 treatment of umbilical cord blood prior to transplant can benefit transplant patients (L.I. Zon, personal communication). E. The acute myelogenous leukemia oncogenic fusion AML1-ETO (AE) promotes a change from an erythrocytic fate to a granulocytic cell fate. Erythrocytes express the gata1 gene in the posterior blood island of the developing zebrafish (red dotted line). Heat-shock inducible expression of AE causes a cell fate change that can be visualized by loss of gata1 expression. A chemical screen identified that COX-2 inhibitors can suppress the AE cell fate change, and the embryos maintain gata1 expression in the presence of AE [72]. F. Zebrafish can play a valuable role in testing for drug toxicity and teratogenisity, as well as for testing direct chemical targets in vivo. Thalidomide is a valuable drug for multiple myeloma and leprosy, but caused severe developmental birth defects when taken by pregnant mothers in the late 1950s and early 1960s. Zebrafish are also sensitive to thalidomide, and treatment in early development prevents the proper development of embryonic fins [88]. Thalidomide binds CRBN, and knockdown of crbn in the developing zebrafish also causes a loss of fin phenotype. This suggests that the binding of thalidomide to CRBN in vivo may underlie its teratogenisty.