Comparative transcriptomics reveals shifts in cortical architecture at the metatherian/eutherian transition

The neocortex, a layered structure unique to mammals, supports higher-order functions such as perception, learning, and decision-making. While its overall organization is broadly conserved, it remains unclear whether changes to the cell type-specific organization of the cortical column-the basic unit of cortical processing- occurred at the metatherian/eutherian split. 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 of a metatherian (Monodelphis domestica) and a eutherian mammal (Mus musculus). We find distinctions between supragranular (layer 2/3) and infragranular (layers 5 and 6) intratelencephalic (IT) neurons to be more pronounced in mice, with layer 2/3 neurons enriched for transcripts involved in synaptic transmission, adhesion, and dendritic development. Mouse cortex also exhibits expanded populations of disinhibitory interneurons, redistribution of perineuronal nets, and reduced oligodendrocyte density-features known to enhance cortical plasticity. These findings suggest the metatherian-eutherian split was accompanied by remodeling of the cortical column, highlighting potential substrates of neocortical evolution in early mammals.