Maintaining hematopoiesis is critical for life. Generating the proper numbers of functional blood cells across all lineages requires cell-intrinsic developmental programs, but these programs require guidance from cell-extrinsic mechanisms that correctly convey the physiologic needs for these cells. Glycans on the cell surface and in the extracellular milieu reside at the interphase through which the cell-extrinsic cues are conveyed. The overarching Program Hypothesis is that cell-intrinsic and extrinsic glycan-mediated mechanisms regulate maintenance, differentiation, and function of hematopoietic cells. Glycosyltransferases such as the sialyltransferase ST6GAL1 are also residents of the extracellular milieu. Our preliminary data point to a role for the extracellular, or extrinsic ST6GAL1, and possibly other glycosyltransferases in the marrow to influence hematopoietic decisions on multiple levels of blood cell development. Outside of the marrow, platelets upon activation release the sialic acid donor substrate required to drive extrinsic sialyltransferase catalysis. Together with Project 1, we will explore the idea that megakaryocytes, the precursors of platelets, control marrow extrinsic sialylation by a similar mechanism. Together with Project 3, we have uncovered a totally unexpected interaction between glycosaminoglycan components of the extracellular matrix with ST6GAL1, and we have identified a number of GAG-mimetics with striking ability to modulate extrinsic ST6GAL1 sialylation on target cells. Disturbed hematopoiesis with highly heterogenous presentation and varied underlying driver mutations is the defining hallmark of clonal myeloid diseases such as myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). However, a key common feature of MPN and MDS is dysplastic megakaryocytes with altered circulating platelet numbers and function. Our preliminary data point to distinct glycosylation signatures associated with platelets from patients of these diseases, suggesting fundamental alterations to the bone marrow environment and the megakaryocytes that produce the platelets. We hypothesize that glycosylation, especially extrinsic glycosylation, regulates blood cell homeostasis, ultimately impacting the number and function of circulating blood cells in health and in disease. We will test this hypothesis in three Specific Aims to 1) understand how extrinsic ST6GAL1 regulates HSPC maintenance; 2) understand how extrinsic sialylation in the marrow is regulated; and 3) initiate a first-time study on how glycosylation is modified in clinical MDS and MPN and in mouse models. The proposed study is expected to yield transformative understanding of extrinsic glycosylation in cell-niche interactions critical to maintaining blood cell production. This is also a pioneering investigation into the glycoscience of clinical MDS and MPN, with potential for novel therapeutics and diagnostics.