Spatial atlas of the mouse central nervous system at molecular resolution

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain^1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS^3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm³, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. 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 analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas¹, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB^5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.