PROJECT SUMMARY/ABSTRACT Elucidation of the genetic and molecular mechanisms that underlie normal blood cell development is an important prerequisite for understanding and treating hematopoietic disorders such as leukemias. Drosophila has proven to be an extremely powerful system for genetic dissection of such mechanisms, which are remarkably conserved in mammals. This study uses the excellent genetic tools available in Drosophila to address specific questions related to how the balance between progenitors and differentiated cells is achieved. Preliminary data suggests that the tight coupling between cell-cycle regulation and differentiation pathways in progenitors plays an important role in determining the balance of progenitors and differentiated cells. In addition, two signals that originate in distinct transitory populations define parallel paths of hematopoietic development, and we hypothesize that they subsequently affect the balance between progenitor maintenance and differentiation that is crucial for homeostasis. These mechanisms will be explored in three aims. In Aim 1, a thorough genetic analysis will be aimed towards obtaining evidence for the spatiotemporal developmental mechanisms that generate parallel paths in hematopoiesis. The genetic data will be fully integrated with transcriptomic analysis to determine how the system generates lineage biases in cells with related, but not identical transcriptomes. In Aim 2, the signals that distinguish the two transitory populations will be explored as will the downstream effects of these signals on the balance between progenitor maintenance and differentiation. This will involve the genetic dissection of a JNK-related and a PVR-related signal that are unique attributes of each transitory population. Lastly, in Aim 3, a genetic/functional analysis of integrated Hh- and Wnt6-dependent pathways that control the balance between cell proliferation and differentiation will be investigated. The role of Wnt6 in controlling cell growth and oxidative status of the progenitors will be tested to obtain a mechanistic understanding of how and why the control of cell cycle and differentiation are closely and directly linked to each other. Overall, the proposed research will yield important insights into the fundamental mechanisms that govern the balance between the opposing forces of progenitor maintenance and differentiation in blood cell development. As these pathways are conserved in humans, this analysis will help bridge the gap between broadly available descriptions of genetic control during hematopoiesis with the less well-defined in vivo functional relevance of such genetic networks to the controlled process of hematopoiesis during homeostatic conditions and the genetic perturbations that lead to blood disorders. These experiments, done in the Drosophila model, will further our understanding of, and provide guidance for, future analysis of blood development and disorders in humans.