Hemoglobin switching is an evolutionarily conserved process that occurs during vertebrate erythropoiesis. The fetal gamma-globin gene in humans is silenced during the transition from the fetal to adult stage of development, yet the factors involved in this process remain to be defined. In the grant, we propose to study fetal globin silencing in zebrafish. We have previously utilized mutant zebrafish as animal models of human disease and through mutagenesis screens, we have isolated novel genes involved in erythropoiesis. The zebrafish exhibit globin switching from embryonic to larval life, and from larval to adult stages. The zebrafish beta-e1 globin gene has a similar developmental pattern of expression as the human gamma-globin gene. It is expressed during larval life and at 25 days of age, expression is silenced. We have shown that larval beta-e1 globin expression is induced in adults treated with hydroxyurea and butyrate. These two drugs have been shown in mammalian erythroid cells to prevent gamma-globin gene silencing. We plan to take a classical genetic and chemical genetic approach to understanding factors that regulate beta-e1 globin silencing. First, we will undertake a temperature sensitive screen for mutants with defects in beta-e1 globin silencing using gynogenetic diploid zebrafish. A transgenic line will be created with green fluorescent protein expressed under the beta-e1 globin regulatory elements, allowing in vivo visualization of beta-e1 globin gene expression in adults. We will also create a stable transgenic zebrafish line with a BAC containing the entire human globin locus. Zebrafish mutants obtained in the genetic screen can then be tested for human gamma-globin silencing. Finally, we plan to undertake a chemical genetic screen for drugs that will prevent globin gene silencing in the early zebrafish adulthood. The genes and compounds found by this proposal can be further studied in mammalian systems. These studies should improve our understanding of the basic molecular biology of globin gene regulation and could be therapeutically useful for patients with sickle anemia and thalassemia.