Specification of CNS macrophage subsets occurs postnatally in defined anatomical niches

All tissue resident macrophages of the central nervous system (CNS), including parenchymal microglia as well as CNS-associated macrophages (CAMs) such as meningeal (mMF) and perivascular macrophages (pvMF) are part of the CNS endogenous innate immune system that acts as the first line of defense during infections or trauma. It has been suggested that microglia and all CAM subsets are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAMs subsets in situ are poorly understood. Using novel fate mapping systems, single-cell profiling and cell-specific mutants, we show that only mMF and microglia share a common prenatal progenitor. In contrast, pvMF originate from perinatal mMF only after birth in an integrin-dependent manner. Furthermore, the establishment of pvMF critically requires the presence of vascular smooth muscle cells. In summary, our data reveal a novel precisely timed process in distinct anatomical niches for the establishment of CNS macrophage subsets. A1/A2 cells were FACS-sorted, from extraembryonic YS at E9.5 into a 384-well (Bio-Rad Laboratories, Munich, Germany) plate containing lysis buffer and barcodes. The gating strategies for FACS-sorting, related to the datasets shown in Fig. 2a-d, Extended Data Fig. 5b-d, were shown in Extended Data Fig. 5a.中枢神经系统（central nervous system, CNS）的所有组织驻留巨噬细胞，包括脑实质小胶质细胞，以及脑膜巨噬细胞（meningeal macrophage, mMF）、血管周围巨噬细胞（perivascular macrophage, pvMF）这类中枢神经系统相关巨噬细胞（CNS-associated macrophages, CAMs），均属于中枢神经系统内源性先天免疫系统，是机体应对感染或创伤时的第一道防线。 此前已有研究提出，小胶质细胞与所有CAM亚群均起源于卵黄囊中的产前细胞类群——早期红髓系祖细胞。然而，目前学界对于不同CAM亚群在原位发育的精确谱系关系、潜在转录调控程序以及驱动其发育的分子信号仍知之甚少。 本研究借助新型命运图谱系统、单细胞分析（single-cell profiling）与细胞特异性突变模型开展实验，结果显示仅脑膜巨噬细胞与小胶质细胞共享共同的产前祖细胞。与之相反，血管周围巨噬细胞仅在出生后以整合素依赖的方式由围产期脑膜巨噬细胞分化而来。此外，血管周围巨噬细胞的建立严格依赖血管平滑肌细胞的存在。 综上，本研究数据揭示了在不同解剖微环境中，中枢神经系统巨噬细胞亚群建立的新型精确时序调控过程。 A1/A2细胞经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）从胚胎第9.5天（E9.5）的胚外卵黄囊中分离，接种至含裂解液与条形码的384孔板（Bio-Rad Laboratories，德国慕尼黑）中。与图2a-d、扩展数据图5b-d所示数据集相关的荧光激活细胞分选门控策略，已在扩展数据图5a中予以展示。