Project Summary/Abstract: Investigating the origin of cell types is key to understanding basic processes in developmental biology and to enable in vitro production of cells and tissues for therapeutic benefit. While major advances in our understanding the ontogeny of hematopoietic cells have been made, significant technical limitations have precluded us from having a full picture of the lineage relationships of blood cells during development. For instance, the prevailing view in the field is that hematopoietic stem cells (HSCs) that emerge in the embryo are the cells responsible for lifelong blood production in the mammal. However, limited data exist that analyse the long-term fate of the earliest hematopoietic progenitors entirely in situ. Furthermore, the field still lacks a conclusive understanding of the true anatomical site of origin of the cells that will become the lifelong HSCs/progenitors in the adult. The Camargo and Hormoz laboratories have pioneered the use of in situ mammalian barcoding strategies to perform lineage tracing at the single cell level. Our proposal here aims to utilize these systems investigate the developmental origins of hematopoiesis. Our application is based on our identification of a population of non-transplantable embryonic multipotent progenitors (eMPPs) that contribute long-term to post-natal hematopoiesis. These findings imply that progenitor populations in addition to traditional HSCs have an active and substantial contribution to adult multilineage blood production. Our first goal in this application is to further characterize eMPPs molecularly and functionally. We will also explore the idea that functional differences in hematopoietic lineages can arise based on their eMPP or HSC ontogeny. Finally, we will extend the use of our barcoding approaches to broadly and unbiasedly characterize developmental waves of blood production and their exact site of origin. Our work has the potential to uncover basic mechanisms of blood development that could be useful for therapeutic manipulation.