Mammals have evolved a more complex brain, exemplified by the transformation of the single-layer dorsal cortex of excitatory projection neurons (ExNs) in ancestors into a multilayered cerebral neocortex1–4 enriched with diverse intratelencephalic and extratelencephalic ExN subtypes5–7, thereby establishing specialized projection systems that enhance brain connectivity and functionality5–8. This is in contrast to modern reptiles and birds with single-layered or pseudolayered columnar organization of ExNs4,9–12. However, the mechanisms underlying these mammalian-specific adaptations remain elusive. By comparing the landscape of gene expression and putative cis-regulatory elements (CREs) in mouse ExN subtypes and through cross-species examination, we identified mammalian-specific CREs, including a subset bound by the transcription factor ZBTB18 (also RP58, ZFP238 or ZNF238) and associated with genes defining intratelencephalic and extratelencephalic subtypes and connectivity, which have been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished cortico-spinal and callosal projections and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, thereby resembling non-mammalian brain. ZBTB18 binding motifs are highly enriched in callosally projecting intratelencephalic-biased putative CREs and show higher conservation specifically in mammals. This study uncovers critical components and mammalian-specific evolutionary adaptations within a regulatory node essential for neocortical ExN identity and connectivity.
