PROJECT SUMMARY Tissue resident macrophage function has emerged as a critical component controlling the balance between organ health and disease. In the mammary gland, macrophages are closely associated with the ductal epithelium and have important phagocytotic roles to maintain tissue homeostasis. Recent studies from our laboratory and others revealed distinct ductal- and stromal-associated tissue resident macrophages that contribute to maintaining the developing ductal structures. However, the important macrophage-derived factors that regulate mammary gland development and the mechanisms by which these distinct macrophage populations maintain the homeostatic state are poorly understood. The overall goal of these studies is to define the functions and mechanisms by which tissue resident macrophages contribute to ductal remodeling throughout distinct stages of mammary gland development. Our preliminary studies identify transcriptional profiles of distinct macrophage populations in the mammary glands of adult mice. We show that two key factors, Cebpb and Gas6, are highly expressed in ductal- and stromal-associated macrophages, respectively, providing potential mechanisms for macrophage function in mammary gland development. Genetic ablation of these factors results in alterations in the mammary gland during key developmental windows. We hypothesize that C/EBPb and Gas6 are crucial effector molecules that drive distinct functions of ductal and stromal macrophages, respectively, during key stages of mammary gland development. In aim 1, we will characterize macrophage heterogeneity throughout postnatal development and determine the functions of distinct macrophage populations in mammary gland development. In Aim 2, the mechanisms of how C/EBPb-induced factors in ductal macrophages alter stem cell expansion will be determined. In Aim 3, Gas6-dependent mechanisms of collagen homeostasis by stromal macrophages will be determined. Impact: Throughout mammary gland development, there are critical windows that are highly susceptible to mutagenic events. Although macrophages have been well-studied during tumor progression and metastasis, the mechanisms driving tissue resident macrophage function during these key stages of development are not known. Understanding these mechanisms will advance our understanding of how tissue resident macrophages impact mammary tissue homeostasis, and provide insight into how disrupted tissue homeostasis leads to disease, such as tumorigenesis.