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 (<i>Monodelphis domestica</i>) and eutherian (<i>Mus musculus</i>) mammals. We show that spatio-transcriptomic distinctions between supragranular (layer 2/3) and infragranular (layer 5) intratelencephalic neurons are more pronounced in mice, consistent with lineage-specific specialization. Mouse cortex also exhibits a lower relative density of parvalbumin-positive 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.