Project Summary/Abstract: Hematopoietic stem cells (HSCs) give rise to all terminally differentiated cells in the blood. The ability of HSCs to reconstitute these blood cell lineages for life underlies the efficacy of bone marrow transplantation therapy for treatment of various blood disorders, including leukemias, anemia, and autoimmunity. Although this is an established and effective treatment, two-thirds of patients in need of a transplant lack a matched donor. Therefore, alternative sources of therapeutic HSCs would be a boon to the field. Human pluripotent stem cells (hPSCs) represent a potential source for cell-based therapies, including the derivation of patient-specific transplantable HSCs, which would additionally circumvent immune rejection and alloreactivity, both major issues in the clinic. The proposed collaborative research leverages the expertise of two Principal Investigators with complementary research interests and skills in stem cell biology, zebrafish genetics and development, murine HSC biology, hematopoietic development, and Wnt biology and biochemistry. Using zebrafish and hPSCs as model systems, they seek to identify and characterize the molecular cues that direct hematopoietic development during early embryonic stages, with an emphasis on the role of Wnt signaling. The proposed studies will build on their finding that a signaling axis regulated by Wnt9a/Frizzled9/EGFR is specifically required for HSC emergence and expansion across vertebrate phyla. This proposal will leverage lineage tracing methods in zebrafish and in vitro differentiation protocols of hPSCs combined with single-cell sequencing approaches to determine the molecular mechanisms of this requirement, and to provide a new level of understanding of how posterior lateral mesoderm is instructed to generate HSCs. The long-term goal of these studies is to gain a better understanding of how HSCs develop in the embryo in order to translate this information to hPSCs. Successful completion of this research will have a profound impact on HSC derivation and expansion, and thereby will be instrumental in overcoming current obstacles to the effective treatment of diseases requiring bone marrow transplant therapy.