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-beta 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-beta. Gene expression analysis using total RNA from embryonic murine microglia and BAM populations in the 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 embryonic microglia and BAM from E10.5 to E18.5 were sorted by FACS.中枢神经系统（Central nervous system, CNS）巨噬细胞包括脑实质小胶质细胞，以及驻留于脑膜、脉络丛和血管周围间隙的边界相关巨噬细胞（border-associated macrophages, BAMs）。除脉络丛巨噬细胞外，绝大多数中枢神经系统巨噬细胞起源于胚外卵黄囊（extra-embryonic yolk sac）的原始造血过程（primitive hematopoiesis）。然而目前仍未明确的是，小胶质细胞与BAMs究竟共享一套发育程序，还是源自各自独立的预先设定谱系。本研究鉴定出两类在表型、转录特征及局部微环境上均存在显著差异的脑巨噬细胞群体，二者分别发育为小胶质细胞与BAMs。在脑定植之前，卵黄囊中便已存在两类独立的巨噬细胞群体。借助命运图谱系统（fate-mapping system），我们证实：与小胶质细胞不同，脉络丛与脑膜中的胚胎BAMs库会逐渐被胚胎后期出现的前体细胞所替代。小胶质细胞的发育依赖转化生长因子-β（TGF-beta），而BAMs的发生与成熟则不依赖该细胞因子。综上，本研究数据表明，从个体发生、基因表达特征及TGF-β需求性来看，发育中的脑实质与非实质脑巨噬细胞属于两类独立的细胞实体。本研究通过提取小鼠胚胎小胶质细胞与BAMs群体的总RNA开展基因表达分析。实验采用经时间交配的C57Bl/6小鼠，观察到阴道栓的当日记为受孕后0.5天（E0.5）。通过荧光激活细胞分选（FACS）技术，分选出E10.5至E18.5时期的胚胎小胶质细胞与BAMs纯群体。