ABSTRACT Millions of individuals worldwide are affected by hypoproliferative anemias, such as anemia of inflammation or myeloodysplastic syndrome (MDS). Erythropoietin (Epo) is a central regulator of erythropoiesis and is known to act directly on erythroid progenitors and precursors. Non-erythroid cells have been shown to be responsive to Epo via expression of the erythropoietin receptor (EpoR), which may be important in the context of erythroid progenitor and precursor niches. Hematopoietic progenitors are known to reside in the vascular endothelial niche. We have recently found that endothelial cells of EpoR-Cre tdTomato mice show robust tdTomato signal, indicating that Epo signaling may play a role in this niche. Conflicting reports exist about the presence of EpoR within the erythroblastic island (EBI) macrophage (Mφ), which is the erythroid precursor niche. We have recently published that EBI Mφs show a heterogeneous phenotype, indicating that more investigation is needed to determine the expression pattern of EpoR in this niche. Knowing that non-erythroid cells have the capacity to respond to Epo, it will be essential to understand how specifically the erythroid niches respond to Epo treatment. The long-term goal of this project is to provide insight to the structure and function of erythroid progenitor and precursor niches to improve the outcome for patients with hypoproliferative anemia, such as anemia of inflammation or myelodysplastic syndrome, where erythroid-extrinsic factors compromise erythropoiesis. The objectives of this proposal are to determine how EpoR expression in the vascular endothelial cells impacts the erythroid progenitor production and determine the expression pattern of EpoR in the EBI Mφ population and its functional consequence to the erythroid precursors in the EBIs. To meet these objectives, the progenitor and precursor niches will be evaluated in each aim. We will validate the active presence of EpoR, by quantifying downstream signaling readouts and we will evaluate the significance of EpoR signaling in the bone marrow stroma utilizing mouse models of tissue specific deletion of EpoR, breeding the EpoRflox/flox mouse with Tie2Cre mice and Csf1RCre mice to delete EpoR in endothelial cells and Mφs respectively. To evaluate the effect of EpoR in the progenitor niche, erythroid progenitors will be quantified in Tie2Cre;EpoR∆/∆ mice by colony assays and flow cytometry. Downstream readouts of EpoR signaling will also be evaluated to determine the presence of EpoR in EBI Mφs. Tissue-specific (Csf1R-Cre driven) deletion in Mφs will determine the functional significance of EpoR signaling within EBI Mφs in terms of erythroid precursor population. Because EBI Mφs are heterogeneous, it will be necessary to characterize transcriptomic and proteomic changes in the presence and absence of Epo with single cell resolution. Our studies will evaluate the BM stromal cells serving as niches for erythroid precursors and progenitors and...