SummaryMicroglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease.

Central nervous system (CNS) macrophages comprise microglia and border-associated macrophages (BAMs) residing in the meninges, the choroid plexus, and the perivascular spaces. Most CNS macrophages emerge during development, with the exception of choroid plexus and dural macrophages, which are replaced by monocytes in adulthood. Whether microglia and BAMs share a developmental program or arise from separate lineages remains unknown. Here, we identified two phenotypically, transcriptionally, and locally distinct brain macrophages throughout development, giving rise to either microglia or BAMs. Two macrophage populations were already present in the yolk sac suggesting an early segregation. Fate-mapping models revealed that BAMs mostly derived from early erythro-myeloid progenitors in the yolk sac. The development of microglia was dependent on TGF-b, whereas the genesis of BAMs occurred independently of this cytokine. Collectively, our data show that developing parenchymal and non-parenchymal brain macrophages are separate entities in terms of ontogeny, gene signature, and requirement for TGF-b. ll