Project Summary/Abstract In humans, aging is associated with chronic inflammation and a decline in adaptive immunity, leading to higher rates of infection and cardiovascular disease, two of the leading causes of death in adults over 65. These hematologic defects are due in part to deficiencies in aged hematopoiesis, which is biased toward myeloid lineages. Many studies on aged hematopoiesis rely on transplantation assays or isolation of specific hematopoietic stem cell (HSC) populations for molecular analysis. While these studies highlight important features of aged HSCs, their approaches focus on long-term (LT) HSCs, a small subset of hematopoietic cells, often to the exclusion of other progenitor populations. Recent studies employ inducible genetic tags to track unperturbed native hematopoiesis. Many of these studies suggest that a large number of progenitor clones, rather than LT-HSCs, drive the bulk of adult hematopoiesis. However, the precise origin of these progenitors remains unclear. Our preliminary work using the transposon tagging technique developed in our lab to tag cells throughout embryonic development indicates that a large portion of adult blood bears tags associated with MPPs, without a corresponding tag in the HSC population. Thus, these MPPs are not clonally related to HSCs, but rather were tagged as a previously unappreciated population of embryonic MPPs (eMPPs). Mature blood contributions from eMPPs were detected when tagging was induced as early as E9.5. However, it remains unclear at which stage of development eMPPs first emerge, and whether earlier hematopoietic cells, such as hemogenic endothelium, are primed to generate eMPPs. By E11.5, increased expression of several lymphoid- associated transcripts appears to separate eMPPs from HSCs. We hypothesize that unique transcriptomic and epigenetic features can predict eMPP divergence during development. To test this hypothesis, we will perform single-cell ATAC-seq and RNA-seq on hematopoietic cells from embryos at several developmental stages. We anticipate that tracking transcriptomic and epigenetic state in embryonic hematopoietic populations will allow us to identify the emergence of eMPPs and, potentially, populations which are primed to generate them. Preliminary data also indicate that eMPPs’ large contributions to hematopoiesis decline with age, and that eMPPs generate the majority of mature lymphoid cells throughout all adult life. We hypothesize that eMPPs are vital to lymphoid production, and their age-dependent decline underlies myeloid bias in aged hematopoiesis. To test this hypothesis, we will genetically ablate eMPPs during fetal development and detect the effects of the loss of eMPPs on the lineage balance of mature blood and expansion of HSCs in the bone marrow. Conversely, we will attempt to rescue the aged phenotype via overexpression of Hoxb4 and Meis1, two genes associated with self- renewal in HSCs, in MPPs. Our findings will not only elucidate the orig...