Specification of tissue resident macrophages during organogenesis
Venue: Science

Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also serve tissue specific purposes: For example, microglia in the central nervous system support neuronal circuit development, Kupffer cells scavenge blood particles and dying red blood cells in the liver, and alveolar macrophages uptake surfactant and remove airborne pollutants and microbes from the airways. Resident macrophage diversity in adult mice is reflected in tissue specific gene expression profiles, which may be due to responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types. Altogether, the mechanisms responsible for the generation of tissue resident macrophage diversity remain unclear.  Tissue resident macrophages originate, at least in part, from mesodermal erythro myeloid progenitors (EMPs) from the yolk sac, which invade the embryo proper at the onset of organogenesis. These tissue resident macrophages are also self maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore hypothesized that resident macrophages represent a founding cell type within most organ anlagen. In this model, the generation of macrophage diversity, as observed in the tissues of postnatal mice, may be integral to organogenesis.  To test this hypothesis and explore the molecular basis of macrophage diversity in mammals, we performed a spatiotemporal analysis of macrophage development in mice, from embryonic day 9 (E9) to 3 weeks after birth. Unbiased single cell RNA sequencing (RNA seq) analysis of CD45+ cells, combined with RNA seq analyses of sorted cell populations, genetic fate mapping, and in situ analyses, revealed that EMPs give rise to a population of premacrophages (pMacs) that colonize the whole embryo from E9.5, as they acquire a core macrophage differentiation program that includes pattern recognition, scavengers, and cytokine receptors. The chemokine receptor Cx3cr1 is up regulated in pMacs and is important for embryo colonization, which is delayed in Cx3cr1 deficient embryos. Fate mapping of pMacs using a Tnfrsf11a Cre reporter labels homogeneously fetal and adult tissue resident macrophages but not HSCs and their progeny. Transcriptional regulators that identify postnatal tissue resident macrophages in the brain, liver, kidney, skin, and lung were specifically up regulated immediately after colonization. These dynamic changes mark the onset of diversification into adult macrophages. We identified Id3 as a Kupffer cell specific transcriptional regulator. Deletion of Id3 in pMacs resulted in Kupffer cell deficiency but did not affect development of microglia and kidney macrophages.  Our study shows that EMP derived precursors colonize embryonic tissues and simultaneously acquire a full core macrophage program. This is followed by their diversification into tissue specific macrophages during organogenesis, likely via the expression of distinct sets of transcriptional regulators. These results indicate that differentiation of tissue resident macrophages is an integral part of organogenesis and identify a spatiotemporal molecular road map for the generation of macrophage diversity in vivo. Our findings provide a conceptual framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to development, homeostasis, and disease pathogenesis in different tissues and will support efforts to differentiate specialized macrophages in vitro. Specification of tissue resident macrophages. Erythro myeloid progenitors (EMPs) from the yolk sac colonize the fetal liver and give rise to macrophage precursors (pMacs) that acquire a core macrophage transcriptional program and colonize the embryo from E9.5 in a Cx3cr1 dependent manner (green arrows) Specification of F4/80+ resident macrophages (brown arrows) starting from E10.25, is initiated by the expression of tissue specific transcriptional regulators. Id3 (red) is important for Kupffer cell development. Transcription factors noted in blue have been shown to be important for the differentiation or the maintenance of the corresponding macrophage subsets. MPh, macrophage. Tissue resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine receptor dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.