Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.增强子（enhancer）可整合转录因子信号通路，驱动发育中大脑的细胞命运特化过程。本研究将增强子标记技术与单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）相结合，对胚胎发育第11.5天（E11.5）小鼠内侧神经节隆起（medial ganglionic eminences, MGE）、外侧神经节隆起（lateral ganglionic eminences, LGE）及尾侧神经节隆起（caudal ganglionic eminences, CGE）内的神经元谱系特化进行解析与区分。研究证实，基于转录因子的单细胞RNA测序聚类分析可提升区域群体与发育群体的分辨率，而增强子活性可用于识别神经节隆起（ganglionic eminences, GE）来源的特异性及重叠神经元群体。首先，我们在体内以单细胞分辨率绘制了7个进化保守的脑增强子的活性图谱，发现所选增强子在神经节隆起各区域的特定祖细胞与神经元群体中展现出多样化的活性模式。随后，我们结合基于增强子的标记技术、单细胞RNA测序及原位杂交数据分析，成功区分了MGE来源的γ-氨基丁酸能（GABAergic）、胆碱能投射神经元与中间神经元的转录特征迥异且空间定位明确的亚型。本研究结果明确了胚胎基底神经节神经发生背后的发育起源与特化路径，并展现了单细胞RNA测序与增强子标记技术联用的强大能力，可用于解析小鼠大脑发育过程中神经元特化的复杂路径。