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Small molecule modifier screen for kit-dependent functions in zebrafish embryonic melanocytes.

ABSTRACT: Zebrafish is gaining popularity as a vertebrate model for screening small molecules that affect specific phenotypes or genetic pathways. In this study, we present a targeted drug screen to identify drug modifiers of the melanocyte migration defect of a temperature-sensitive allele of the Kit receptor tyrosine kinase, kit(ts). We first test two candidate drugs, the phosphatidylinositol-3-kinase kinase inhibitor (LY294002) and the Erk/MAP kinase inhibitor (PD98059), for their effect on melanocyte migration and survival. We find that LY294002 enhances the migration defect of kit(ts), implicating the phosphatidylinositol-3-kinase kinase pathway in promoting kit-dependent melanocyte migration, but not survival. We then used the kit(ts)-sensitized genetic background to screen a panel of 1280 pharmacologically active drugs to identify drug enhancers and suppressors of the kit(ts) melanocyte migration defect. We identified three drug enhancers of migration, two of which, Papaverine and Isoliquiritigenin, specifically enhance the kit(ts) migration defect, while 8-DPAT affected both melanocyte migration and survival. These drugs now provide additional experimental tools for investigating the mechanisms of kit-promoted melanocyte migration and survival in the zebrafish embryo.

SUBMITTER: Hultman KA

PROVIDER: S-EPMC2757780 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The retention of particular genes after the whole genome duplication in zebrafish has given insights into how genes may evolve through partitioning of ancestral functions. We examine the partitioning of expression patterns and functions of two zebrafish kit ligands, kit ligand a (kitla) and kit ligand b (kitlb), and discuss their possible coevolution with the duplicated zebrafish kit receptors (kita and kitb). In situ hybridizations show that kitla mRNA is expressed in the trunk adjacent to the notochord in the middle of each somite during stages of melanocyte migration and later expressed in the skin, when the receptor is required for melanocyte survival. kitla is also expressed in other regions complementary to kita receptor expression, including the pineal gland, tail bud, and ear. In contrast, kitlb mRNA is expressed in brain ventricles, ear, and cardinal vein plexus, in regions generally not complementary to either zebrafish kit receptor ortholog. However, like kitla, kitlb is expressed in the skin during stages consistent with melanocyte survival. Thus, it appears that kita and kitla have maintained congruent expression patterns, while kitb and kitlb have evolved divergent expression patterns. We demonstrate the interaction of kita and kitla by morpholino knockdown analysis. kitla morphants, but not kitlb morphants, phenocopy the null allele of kita, with defects for both melanocyte migration and survival. Furthermore, kitla morpholino, but not kitlb morpholino, interacts genetically with a sensitized allele of kita, confirming that kitla is the functional ligand to kita. Last, we examine kitla overexpression in embryos, which results in hyperpigmentation caused by an increase in the number and size of melanocytes. This hyperpigmentation is dependent on kita function. We conclude that following genome duplication, kita and kitla have maintained their receptor-ligand relationship, coevolved complementary expression patterns, and that functional analysis reveals that most or all of the kita receptor's function in the embryo are promoted by its interaction with kitla.

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Small molecule modifier screen for kit-dependent functions in zebrafish embryonic melanocytes.

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