We synthesize and release more than 2 million red blood cells per second from our bone marrow to maintain circulating steady-state levels. This massive output of the erythropoietic system makes it a process that is essential for normal physiological function but also one that is exquisitely sensitive to direct injury such as radiation. Radiation injury provides a unique model for study of erythroid lineage injury, recovery, and regulation mechanisms and allows for a more thorough understanding of erythropoiesis as a dynamic process. The overall aim of this proposal is to better understand the mechanisms of injury and recovery of the erythroid lineage following xenotoxic exposure as modeled by sublethal radiation. Preliminary data obtained through traditional colony assays in combination with a novel multispectral imaging flow cytometry (MIFC) analysis indicate that erythroid progenitors and precursors are severely depleted at 48 hours post 4 Grey irradiation followed by a rapid increase beginning at 5 days post- irradiation. Aim 1 studies will further define the time course of differential radiosensitivity and recovery of erythroid progenitor and precursor populations as well as explore potential apoptotic mechanisms underlying erythroid loss following sublethal radiation injury. Interestingly, preliminary findings suggest that late-stage erythroid progenitors (CFU-E) may recover independent of more immature (BFU-E) progenitors. Based on these data, late-stage erythroid progenitors are hypothesized to proliferate in a modified stress erythropoiesis response that is responsible for the wave of erythroid recovery post-radiation. Therefore, Aim 2 will investigate the role of endogenous compounds important in the traditional stress erythroid response, such as erythropoietin (EPO), stem cell factor (SCF), and glucocorticoids, and their respective signaling pathways on the stimulation and subsequent proliferation of CFU-E cells during the recovery of the erythroid lineage from radiation. Finally, Aim 3 will combine proposed mechanisms underlying injury in Aim 1 and recovery in Aim 2 by determining if exogenous administration of cytokines such as EPO and SCF can reduce injury and enhance recovery of the erythroid lineage. Overall, these experiments will provide a more thorough understanding of normal hematopoiesis by investigating both its underlying sensitivity to radiation and the mechanisms of its recovery response following injury. These studies will ultimately lead to new treatments to protect and mitigate the hematopoietic system from clastogenic agents such as radiation and chemotherapy.