Comparative transcriptomics reveals differences in cortical cell type organization between metatherian and eutherian mammals

The neocortex, a layered structure unique to mammals, supports higher-order functions including perception, learning, and decision-making. While its laminar architecture is broadly conserved, the cell type-specific organization of the cortical column has not been compared across species that diverged early in mammalian evolution. To address this, we used single-nucleus RNA sequencing and spatial transcriptomics to compare gene expression, cell types, and laminar architecture in the primary visual cortex (V1) of metatherian (Monodelphis domestica) and eutherian (Mus musculus) mammals. We show that spatio-transcriptomic distinctions between supragranular (layer 2/3) and infragranular (layer 5) intratelencephalic (IT) neurons are more pronounced in mice, consistent with lineage-specific specialization. Mouse cortex also exhibits a lower relative density of parvalbumin-positive (PV) GABAergic neurons and redistributed perineuronal nets, consistent with altered constraints on plasticity. Together, these findings demonstrate substantial variation in the cellular and spatial organization of the cortical column across deeply diverged mammals, challenging the view that local cortical architecture is uniformly conserved. Significance StatementThe neocortex supports perception and flexible behavior in mammals, yet how its cellular composition varies across early mammalian divergences has not been directly examined. By comparing transcriptomic cell types and their spatial organization in the primary visual cortex of a metatherian (opossum) and a eutherian (mouse), we show that major classes and laminar structure are broadly conserved, but intratelencephalic neurons differ substantially. These differences are accompanied by shifts in inhibitory circuitry and extracellular scaffolding, indicating divergent modes of cortical organization and plasticity across mammalian lineages. Our findings challenge the assumption that neocortical columns are uniformly conserved and identify specific cellular populations that vary across deeply diverged therian mammals.