Project Summary Erythropoiesis is the tightly regulated process by which red blood cells are formed from hematopoietic stem cells. Clinically, the only erythropoiesis-stimulating agents available for the treatment of anemia are the hormone erythropoietin (Epo), and glucocorticoids, leaving a gap where they are ineffective or contraindicated. In this inter-disciplinary project, we will use in vivo mouse models of erythropoietic stress, mouse genetics, and cutting-edge scRNA-Seq approaches to explore a largely uncharted area of non-Epo growth factor signaling and regulatory mechanisms in early erythroid progenitors. We will specifically focus on a potent new regulator that we have discovered: the cytokine IL-17, which we found stimulates both human and mouse erythroid progenitors, acting specifically through its receptor IL-17RA. We first identified IL17 as a novel erythropoietic regulator by overcoming an important technical challenge in studying erythropoiesis. Until recently, a major challenge in identifying novel erythropoietic regulators was a lack of information about the earliest stages of erythroid differentiation from stem cells. Early progenitors could only be defined retrospectively by their erythroid colony forming potential, namely “burst” forming units and colony forming units [BFU-e(rythroid) and CFU-e respectively]. FACS-based schemes could enrich for BFU-e/ CFU-e, but not purify them. We have now, for the first time, prospectively isolated distinct early stages of erythropoiesis, guided by novel single cell RNA sequencing (scRNA-Seq) technology and using FACS. We found that these newly defined progenitors express multiple growth factor receptors that were not previously recognized as erythroid regulators, and we identified an erythropoietic regulatory role for the first three such growth-factor receptors that we tested, including IL-17. Our project is structured around three aims: we will determine IL-17 pathway components involved in erythropoietic control, the cell biological and transcriptional responses to IL-17 in erythroid and non-erythroid blood progenitors, and finally, we will investigate the ability of IL-17RA activation to modulate erythropoiesis in mice in stress and disease, including anemic conditions. In the process, we will apply scRNA-seq technology to reveal the dynamic structure of the hematopoietic/erythropoietic hierarchies in physiology, disease and during stress, and the sensitivity of these stem and progenitor cell hierarchies to IL-17 signaling.