Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Spatially charting molecular cell types at single-cell resolution across the three-dimensional (3D) volume of the brain is critical for illustrating the molecular basis of the brain anatomy and functions. Single-cell RNA sequencing (scRNA-seq) has profiled molecular cell types in the mouse brain, but cannot capture their spatial organization. Here, we employed an in situ sequencing technique, STARmap PLUS, to map 1.09 million high-quality cells across the whole adult mouse brain and the spinal cord, profiling 1,022 genes at subcellular resolution with a voxel size of 194 X 194 X 345 nm3 in 3D. We developed computational pipelines to segment, cluster, and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analyses of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell type nomenclature and allowed the identification of tissue architectures previously undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we further integrated the STARmap PLUS measurements with a published scRNA-seq atlas, imputing 11,844 genes at the single-cell level. Finally, we delineated the tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6, across the molecular cell types and tissue regions of the whole mouse brain. Together, this annotated dataset provides a comprehensive, high-resolution, single-cell resource that integrates a spatial molecular atlas, cell taxonomy, brain anatomy, and genetic manipulation accessibility of the mammalian central nervous system (CNS).在大脑的三维（3D）容积中以单细胞分辨率对分子细胞类型进行空间绘图，对于阐明大脑解剖结构与功能的分子基础至关重要。单细胞RNA测序（scRNA-seq）已对小鼠大脑中的分子细胞类型完成表达谱分析，但无法捕捉其空间组织特征。本研究采用原位测序技术STARmap PLUS，对整个成年小鼠大脑与脊髓内的109万个高质量细胞进行空间绘图，以194×194×345 nm³的体素尺寸，在三维空间中对1022个基因完成亚细胞分辨率的表达谱分析。我们开发了计算流程，基于单细胞基因表达特征对230种分子细胞类型进行分割、聚类与注释，并基于空间微环境基因表达特征划分出106个分子组织区域。通过对分子细胞类型与分子组织区域开展联合分析，我们构建了一套系统化的分子空间细胞类型命名体系，并识别出现有脑解剖学研究中尚未定义的组织架构。为构建全转录组空间图谱，我们进一步将STARmap PLUS测序数据与已发表的scRNA-seq图谱进行整合，在单细胞水平上对11844个基因完成表达量插补。最后，我们绘制了全脑转基因递送工具AAV-PHP.eB⁵,⁶在整个小鼠大脑的分子细胞类型与组织区域中的嗜性分布特征。综上，本注释数据集提供了一套全面、高分辨率的单细胞研究资源，整合了哺乳动物中枢神经系统（central nervous system, CNS）的空间分子图谱、细胞分类系统、大脑解剖结构与遗传操作可及性信息。