Adaptive Evolution of Gene Regulatory Networks in Mammalian Neocortical Neurons

Mammals have evolved a more complex brain, exemplified by the transformation of single layer dorsal cortex of excitatory projection neurons (ExNs) in ancestors to multilayered cerebral neocortex enriched with diverse intratelencephalic (IT) and extratelencephalic (ET) ExN subtypes, establishing specialized projection systems to enhance brain connectivity and functionality. This is in stark contrast to modern reptiles and birds with single- or pseudo-layered columnar organization of ExNs. 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 by cross-species examination, we identified mammalian-specific CREs, including a subset bound by transcription factor (TF), ZBTB18 (also RP58, ZFP238, or ZNF238) and associated with genes defining IT and ET subtypes and connectivity, and have previously been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished corticospinal and callosal projections, and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, resembling the non-mammalian brain. ZBTB18 binding motifs are highly enriched in callosally projecting IT-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.