PROJECT SUMMARY/ABSTRACT Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment for many blood diseases and cancers. However, these procedures still need to be optimized to improve patient outcomes and survival. HSPCs reside in a microenvironment surrounded by niche cells that help regulate their function. Our research proposal seeks to address fundamental questions regarding the cellular interactions between HSPCs and niche cells. During development in the embryo, HSPCs move through different tissues and have changing requirements for contact with the microenvironment. HSPCs first arise in the dorsal aorta, a large vessel in the embryo, and are then released into circulation. Next, HSPCs migrate to the fetal liver where the population of cells expands via symmetric divisions. Finally, HSPCs migrate again to colonize the bone marrow where they will remain throughout adulthood. As HSPCs are migrating between these different hematopoietic tissues, they are also becoming more mature and are programmed towards their adult quiescent state. We use zebrafish and mice as model systems that are highly conserved with humans and have many genetic tools for functional testing and live imaging of cellular behaviors. In zebrafish, we have found a novel role for vascular endothelial growth factor c (vegfc) in the release of HSPCs from the dorsal aorta. Vegfc also regulates a fate decision in pre-HSPCs that determines if they will become a stem cell or a different type of progenitor cell. At later stages, we found that integrin alpha 4 (itga4) in the caudal hematopoietic tissue (CHT), the zebrafish equivalent of the fetal liver, is required for proper epigenetic programming of HSPCs. We hypothesize that dissecting niche-dependent HSPC programming, using mutants with prolonged or shortened retention in developmental niches, will enable us to decode mechanisms governing HSPC fate decisions and track their long-term impact on hematopoiesis. We will address this hypothesis through the following Specific Aims: 1) Determine the role of vegfc in fate determination and release of HSPCs from the hemogenic endothelium of the dorsal aorta; 2) Establish how itga4-dependent contact with the fetal/CHT niche programs HSPCs as they transition from immature to mature states. We will perform in vivo genetic knockdown and small molecule treatments, together with live imaging, to understand the dynamic interactions between HSPCs and niche cells. We will use a multiomics approach to infer and functionally test the gene regulatory networks involved in HSPC programming. Together, our proposed aims will identify functionally critical signals and networks that integrate to program HSPCs. Our goal is for these novel insights and paradigms to improve HSPC-based therapies for patients.