Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map

While the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. Here, we developed a high-content pipeline, the Large-area Spatial Transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid, and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in early postnatal cortex, mostly persisted in adult mouse and human cortex. Single cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae. Gene expression analysis of purified astrocyte, non-astrocyte and bulk tissue from manually dissected upper and deep layers of the mouse cerebral cortex using RNA-sequencing. Astrocytes were purified from 14 day old transgenic animals harboring the Aldh1l1-GFP reporter via flow cytometry.尽管大脑皮层被组织为六层兴奋性神经元层，但目前尚不清楚神经胶质细胞是否也存在独特的分层模式。本研究开发了一种高内涵分析流程——大面积空间转录组（Large-area Spatial Transcriptomic, LaST）图谱，可实现原位单细胞基因表达定量。我们针对小鼠皮层中星形胶质细胞的多样性筛选了46个候选基因，鉴定出表层、中层与深层三类星形胶质细胞身份，其呈现的梯度层模式与神经元的层模式显著不同。星形胶质细胞的层特征在出生早期皮层中便已确立，并在成年小鼠和人类皮层中大多得以维持。单细胞RNA测序与空间重构分析进一步证实了成年皮层中星形胶质细胞层的存在。Satb2和Reeler突变可改变神经元的有丝分裂后发育，同时足以改变胶质细胞分层，这表明神经元信号对胶质细胞分层具有指导性作用。最后，小鼠不同皮层区域的星形胶质细胞层模式存在分化。上述研究结果表明，兴奋性神经元与星形胶质细胞各自形成了独特的谱系相关神经板层。本研究通过RNA测序，对手动分离的小鼠大脑皮层表层与深层组织中的纯化星形胶质细胞、非星形胶质细胞以及整体组织开展了基因表达分析。星形胶质细胞通过流式细胞术，从携带Aldh1l1-GFP报告基因的14日龄转基因动物中纯化获得。