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

Abstract

During organogenesis, patterns and gradients of gene expression underlie organization and diversified cell specification to generate complex tissue architecture. While the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells are diversified to mimic neuronal laminae or show distinct layering. To determine the molecular architecture of the mammalian cortex, we developed a high content pipeline that can quantify single-cell gene expression in situ. The Large-area Spatial Transcriptomic (LaST) map confirmed expected cortical neuron layer organization and also revealed a novel neuronal identity signature. Screening 46 candidate genes for astrocyte diversity across the cortex, we identified gray matter superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from 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 identity or organization, respectively, were sufficient to alter glial layering, indicating an instructive role for neuronal cues. In normal mouse cortex, astrocyte layer patterns diverged between functionally distinct cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae, which together show higher order neuroglial complexity of cortical architecture.