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

Abstract

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.