Our goal is to understand the molecular mechanisms that regulate the emergence and subsequent transitions of the hemato-vascular program during development. This should facilitate progress toward manipulating stem and progenitor cell populations, modulating differentiation of specific lineages, and impacting treatment strategies for certain leukemias. We identified the BMP signaling pathway as essential for the generation of the initial hematopoietic system, and we characterized the function of several interacting pathways. In the last funding cycle we discovered that Smad signaling promotes expansion of the earliest hemato-vascular progenitors (the hemangioblast), yet also restricts the proliferation of these cells in order to maintain a stem cell population and control the development of committed progenitors. Our results indicate that Smad-dependent pathways continue to regulate hematopoiesis in the definitive lineage and even in the adult. Due to compensation and/or embryonic lethality it is challenging to study in vivo the role of BMP signaling components. This renewal application proposes to investigate lineage and stage-specific functions for Smad signaling during hematopoietic developmental transitions, to understand the specificity of function among Smads, and to define molecular mechanisms by which Smad activity regulates the commitment of stem and progenitor cells to hemato-vascular fates. The proposal exploits complementary experimental advantages of the zebrafish model and murine ES cells to understand how stem and progenitor cell populations are normally regulated by conserved Smad-dependent molecular pathways, considering that these might ultimately be modulated for clinical benefit. The Aims are to: 1. Define the spatio-temporal requirements of Smad signaling for hemato-vascular development. We will generate novel transgenic and genetic models to define precisely when and where Smad-dependent signaling functions in different hemato-vascular progenitor populations. 2. Delineate the specificity of Smadl and SmadS for hematopoiesis. We will determine why the closely related BMP pathway mediators Smadl and SmadS have distinct regulatory functions with respect to hemato-vascular development. 3. Determine the molecular mechanisms by which Smad function regulates hematopoiesis. We propose to define the specificity, the pathways that are the targets for this activity, and the epigenetic changes that occur with respect to a key target gene, Gata2. Relevance to public health: Growth factors such as BMPs can regulate stem cell activity but their use in the clinic will not be feasible until we can accurately predict how they affect genetic programs in different progenitor cells. Our experiments should help to define the specificity of an important stem and progenitor cell regulatory pathway that is controlled in blood cells by BMPs.