Orthogonal Modes of Gene Expression Evolution Shape Human Neocortical Development and Disease Vulnerability

The neocortex is responsible for higher-order cognitive abilities such as language, abstract reasoning, and executive function—capacities that are particularly advanced in humans. To elucidate the molecular foundations of neocortical evolution in the human lineage, it is essential to examine how conserved gene repertoires have undergone expression changes relative to other mammals. The expression changes in conserved genes are widely regarded as key drivers of the phenotypic evolution of human-specific traits. In this study, we performed a comprehensive comparative single-cell transcriptomic analysis of fetal neocortical development across four mammalian species: human, macaque, mouse, and ferret. To validate human-specific expression changes, we further analyzed brain organoids derived from both human and chimpanzee stem cells. Genes exhibiting human-specific expression shifts were systematically classified along three orthogonal dimensions: overall expression level (Human Level Distinctive; HLD), temporal expression trend (Human Trend Distinctive; HTD), and differentiation lineage specificity (Human Differentiation trajectory Distinctive; HDD). HLD genes were frequently enriched for long introns and located near Human Accelerated Regions (HARs), and showed pronounced upregulation in humans. These genes were strongly associated with neurodevelopmental disorders such as autism spectrum disorder and developmental delay, as well as with megalencephaly and glioblastoma. HTD genes, in contrast, exhibited a unique pattern in humans, peaking early in development and subsequently declining—opposite to the steadily increasing trends observed in other species. These genes were significantly enriched for oxidative phosphorylation and ribosomal functions, pointing to a temporally restricted elevation in biosynthetic activity in early human corticogenesis. HDD genes displayed a marked shift in lineage-specific expression: cilia-related genes that are typically expressed in apical progenitors in non-human species were instead highly expressed in outer radial glia (oRGs) in humans. This spatial reorganization of ciliary gene activity suggests an oRG-specific adaptation in signaling architecture. Together, these results highlight the diversity of regulatory changes that have shaped human cortical development. Distinct classes of gene expression evolution—mediated in part by HARs—appear to have contributed not only to the expansion and increased complexity of the human neocortex, but also to its heightened vulnerability to neurodevelopmental and oncogenic pathologies. The identified human distinctive genes will be the target of future experimental verification to elucidate the precise molecular mechanisms regulating human-specific aspects of cortical development.