Project Summary/Abstract Hematopoietic stem cells (HSCs) are self-renewing, transplantable cells that have the capacity to reconstitute all blood lineages for life. Consequently, HSC transplantation (HSCT) is used in treatment of leukemia and other blood disorders. In principle, induced pluripotent stem cell (iPSC)-derived HSCs could provide an unlimited source of HSCs for transplant, patient-specific in vitro studies, drug studies and as a vector for delivery of gene therapy. However, to date, it has not been possible to generate bona fide HSCs from iPSCs with high engraftment potential and multilineage reconstitution. One means of generating HSCs from iPSCs would be to reproduce the normal embryonic specification of HSCs in vitro. HSCs arise from hemogenic endothelial (HE) cells in the embryonic dorsal aorta (DA). The extracellular matrix (ECM) provides the physical environment in which HSC precursors receive signals that induce their commitment to the hematopoietic lineage. Despite the likely importance of the ECM, we do not yet understand how or whether the ECM influences HSC specification. The lysyl oxidase (Lox) family of ECM-modifying enzymes controls ECM stiffness, display of growth factor signals, and other properties by catalyzing the cross-linking of ECM proteins including collagen and fibronectin. Using the zebrafish model of hematopoietic development, I have shown that Lox activity is required for specification of the earliest HSCs. My preliminary studies indicate that at least three Lox family members are required for proper specification and maturation of the definitive hematopoietic system. In Aim 1, I will systematically examine Lox family contribution to HSC specification. In Aim 2, I will determine the mechanism of Lox regulation of HSC specification to distinguish between the most likely possibilities: Lox-induced ECM stiffness regulates HSCs directly through integrin-ECM interactions, indirectly through modulating existing growth factor signals, or both. The research and training proposed in this F32 application will take place under the guidance of my mentor, Dr. Wilson Clements and my mentorship team at St. Jude Children’s Research Hospital. The proposal describes research that will provide exceptional training opportunities and generate original and meaningful scientific findings to launch my career as an independent scientist. The aims build on my prior experience while simultaneously providing training in the field of HSC biology. I will gain expertise in genome editing and cutting- edge techniques for visualization and analysis of ECM, including second harmonic generation (SHG) and polarized light microscopy. In addition, I will take advantage of numerous career development resources.