Hematopoietic stem cell (HSC) biology impacts public health in that patients with blood disorders undergo irradiation to deplete malignant cells followed by transplantation of healthy HSCs to reconstitute their blood. Although much is known about HSC cellular and functional properties in mammals, relatively little is understood about the genetic pathways regulating their induction. The development of zebrafish as a model system has allowed for the unrivaled ability to discover genes in vertebrates through unbiased forward genetic screening approaches. Recent transgenic technology combined with the external fertilization and transparency of zebrafish embryos allows events in HSC specification and stem cell niche formation to be observed. This will address many long-standing questions regarding the molecular pathways regulating stem cell ontogeny. We propose to further characterize the hematopoietic phenotype in the Notch signaling mutant, mindbomb, pioneer transplantation technology in fish, and build a hierarchical network in the Notch pathway leading to HSC specification. To discover new genes required for HSC induction, we will use a mutagenesis strategy that has already uncovered 7 new mutants. The overall goal of this K01 application is to use the advantages of zebrafish to genetically dissect the role of the Notch pathway in HSC induction and to discover new genes governing HSC specification. These studies may elucidate new clinically relevant genes that translate from the fish into human patients. The research will be conducted in the laboratory of Leonard Zon, M.D. at Children's Hospital where the candidate, Caroline Erter Burns, Ph.D. is a fellow. Dr. Zon is a preeminent leader in the hematopoiesis, stem cell, and zebrafish fields and will provide mentorship and resources for the aims described. The candidate has institutional commitment from the Department of Medicine. A K01 award will aid Dr. Burns in achieving her goal of becoming an independent researcher in an academic setting studying the genetics of stem cell ontogeny.