A conserved lamination pattern in the paleocortex and the neocortex revealed by single-cell RNA analyses

The piriform cortex, the largest paleocortical domain and a central olfactory cortex, is classically described as a three-layered structure, with layer II subdivided into semilunar and superficial pyramidal neurons. However, its cellular composition, developmental logic, and evolutionary relationship with the six-layered neocortex remain incompletely defined. Here, we aimed to resolve piriform cortical cell types and laminar organization and compare their molecular programs across mammalian cortical regions and the reptilian cortex. We performed single-nucleus RNA sequencing of the microdissected piriform cortex and integrated neuronal lineage data with published datasets, with spatial validation via the Allen Mouse Brain Atlas. Interspecies analyses identified piriform-dominant glutamatergic populations marked by Unc13c/Lmo3/Rora and an Rorb/Reln/Ntng1-enriched sensory-recipient subtype localized to the superficial layer IIa, consistent with semilunar neurons. Spatial transcriptomic mapping revealed that piriform layer IIb contains neocortical upper-layer-like corticocortical neurons, while piriform layer III segregates into layer V–like (IIIa), layer VI–like (IIIb), and VIb/VII (subplate)-equivalent populations, indicating a neocortex-like laminar framework with an inverted positioning of sensory recipients and corticocortical compartments relative to the neocortex. GABAergic neurons largely conformed to canonical MGE- and CGE-derived lineages but included a piriform-specific Rgs9+Pde7b+ subtype consistent with an LGE-related origin, suggesting region-specific diversification of GABAergic neurons. Disease ontology enrichment linked hippocampus-dominant glutamatergic programs to Alzheimer’s-related genes and piriform-dominant (and pan-cortical) excitatory and inhibitory programs to autism spectrum disorder and intellectual disability, implicating coordinated E/I circuit specializations in area-selective vulnerability. These findings support a revised view of the paleocortex as a divergent specialization of a conserved, multilayered cortical program and provide molecular markers and a comparative framework for studying cortical evolution and region-specific disease susceptibility.