A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages

Genetic tools to target microglia specifically and efficiently from the early stages of the embryonic development are missing. Here we present Crybb1-Cre, a new constitutive Cre deleter producing a nearly complete recombination of embryonic brain macrophages (microglia and embryonic BAMs) by the perinatal period, with limited recombination leakage in peripheral myeloid cells. Using this tool, in combination with Flt3-Cre lineage tracer, scRNA-seq analysis and confocal imaging we were able to accurately fate-map embryonic derived versus monocyte derived BAMs in the mouse cortex and identified specific surface markers to resolve either population. Furthermore, we used Crybb1-Cre to delete the transcription factor SMAD4. Using multiomics analysis combining single-cell RNA-seq and ATAC-seq we showed that SMAD4-deficient microglia underwent an arrest of their homeostatic maturation and at the same time acquired a BAMs specification signature. By contrast, BAMs development was unaffected. Therefore, SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate. This data provides a valuable resource to study molecular underpinnings of microglia development. Immune cells were isolated from the brains of Smad4 F/F and Smad4 cKO mice and subjected to multi-omic profiling using 10X genomics reagents. Immune cells were isolated from the brains of WT and 5XFAD mice and subjected to scRNAseq using 10X genomics reagents.

目前尚缺乏能够在胚胎发育早期特异性、高效靶向小胶质细胞(microglia)的遗传工具。本研究报道了一种新型组成型Cre删除工具Crybb1-Cre，该工具可在围产期实现胚胎脑巨噬细胞（小胶质细胞与胚胎脑相关巨噬细胞(brain-associated macrophages, BAMs)）的近乎完全重组，且仅在外周髓系细胞中存在极少量重组渗漏。我们将该工具与Flt3-Cre谱系示踪剂、单细胞RNA测序(single-cell RNA-sequencing, scRNA-seq)分析及共聚焦成像相结合，实现了小鼠大脑皮层中胚胎来源与单核细胞来源脑相关巨噬细胞的精准命运图谱分析，并鉴定出可区分这两类细胞群的特异性表面标志物。此外，我们利用Crybb1-Cre工具敲除了转录因子SMAD4。通过结合单细胞RNA测序与转座酶可及性测序(assay for transposase-accessible chromatin using sequencing, ATAC-seq)的多组学分析，我们发现SMAD4缺陷型小胶质细胞出现了稳态成熟阻滞，同时获得了脑相关巨噬细胞的特化特征。与之相反，脑相关巨噬细胞的发育未受影响。由此可见，SMAD4调控一套转录与表观遗传程序，该程序对于脑巨噬细胞定向分化为小胶质细胞命运是不可或缺的。本数据集为研究小胶质细胞发育的分子基础提供了宝贵的研究资源。我们从Smad4 F/F与Smad4条件性敲除(cKO)小鼠的脑组织中分离免疫细胞，并利用10X基因组学(10X Genomics)试剂完成多组学图谱分析。我们从野生型(wild type, WT)与5XFAD小鼠的脑组织中分离免疫细胞，并利用10X基因组学试剂完成单细胞RNA测序分析。