Title: Development of the resident macrophage lineage in mouse and human Summary/abstract Our long-range goal in our previous funded RO1 application was to elucidate mechanisms that underlie the development and specification of embryo-derived tissue-resident macrophages in mice. Our program of work aimed to understand how the identities and functions of resident macrophages are specified and to provide tools and concepts to identify pathophysiological mechanisms underlying their roles in developmental, inflammatory, degenerative and tumoral diseases. This work allowed us to identify key novel functions of resident macrophages and mechanisms that underly these functions and to develop new genetically tractable models which pave the way for our current project. Yet, a comprehensive understanding of the developmental origin and functions of mammalian resident macrophages, which has essential implications for genetic and pharmacological investigations of their specialized functions, still faces several important challenges, which are identified and addressed in this renewal proposal. 1) The development of resident macrophage in mice cannot be directly extrapolated to humans because the earlier steps of extraembryonic hematopoiesis and macrophage development present with differences among vertebrates, for example between Zebrafish and mice, and are overall poorly understood. Notably, 2) the early hemato-endothelial progenitor(s) that give rise to EMPs and HSCs are still elusive, which impairs our ability to fully characterize resident macrophages. In addition, 3) it is important to elucidate the roles of individual LDTFs and their subset-specific combinations for the control of tissue-specific functions of macrophages, because, in addition to identify molecular mechanisms that underly macrophage functions, these studies have the potential to unveil therapeutic strategies for leveraging macrophage functions in human. We propose to take advantage of novel genetic tools to identify the hemato- endothelial progenitors that generate the macrophage lineage in mice (AIM1), to identify conserved macrophages progenitors in human using a genetically tractable model for hematopoietic differentiation in human induced pluripotent stem cell (hiPSC)-derived embryoid bodies, and to take advantage of this versatile in vitro model to characterize the role of macrophage LDTFs in the control of macrophage tissue-specific functions (AIM2). We expect that our results will provide a robust experimental basis to transform and improve our understanding of the cellular, genetic, and molecular determinants of resident-macrophage development in mice and, importantly, in human, and provide important genetic tools for the molecular understanding and manipulation of their functions in physiology and diseases.