Early fate defines microglia and non-parenchymal brain macrophage development

Central nervous system (CNS) macrophages comprise parenchymal microglia and border-associated macrophages (BAMs) residing in the meninges, the choroid plexus and the perivascular spaces. With the exception of choroid plexus macrophages, most CNS macrophages emerge during primitive hematopoiesis in the extra-embryonic yolk sac. What remains unknown however, is whether microglia and BAMs share a developmental program or arise from separate predefined lineages. Here, we identified two phenotypically, transcriptionally and locally distinct brain macrophage populations throughout development, giving rise to microglia and BAMs. Two independent macrophage populations were already present in the yolk sac prior to their seeding of the brain. Using fate-mapping system, we demonstrate that in contrast to microglia, the pool of embryonic BAMs in the choroid plexus and the meninges was gradually replaced by precursors emerging later in embryogenesis. The development of microglia was dependent on TGF-ß whereas the genesis and maturation 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 expression signatures and requirement for TGF-ß. Overall design: Single cell sequencing of embryonic macrophages from E16.5 C57Bl/6 brain. C57Bl/6 mice were time-mated and the day of vaginal plug observation was denoted as 0.5 day after conception (E0.5). Pure populations of CD45+ F4/80+ CD11b+ Ly6C- cells from E16.5 brain were sorted by FACS, and applied onto 5-10 uM-diameter C1 Integrated Fluidic Circuit (IFC; Fluidigm) to capture single cells. Please note that the sample characteristics are identical across all samples as we discovered the two distinct macrophage populations only after the data processing steps and this is addressed in the supplementary file.