The cerebral cortex shows species-specific variations in size and organization, which probably account for distinct behavioural abilities1. These structural differences may reflect evolutionary changes in the developmental expression of shared genes. Here, to investigate this possibility, we used machine vision to identify and compare cell-type-specific gene expression patterns in the developing mouse and human neocortex, and in human cortical organoids. Using this approach, we identified genes with evolutionarily conserved or divergent transcriptional regulation, revealing species-specific cyto-temporal gene expression patterns. Among such genes, the transcription factor gene JUNB showed mutually exclusive expression in human progenitors and mouse neurons. Through cell-type-specific gain- and loss-of-function experiments in mice and human cortical organoids, we show that JUNB bidirectionally controls human cortical features, including progenitor proliferation rates, neuronal production timing and total neuronal output. We identify IRF1 as a human radial glia-specific regulator that, when expressed in mouse radial glia, activates JUNB and recruits human-like gene regulatory networks, demonstrating cross-species activation of poised developmental programmes. Together, these findings reveal how cyto-temporal regulation of shared genes drives species-specific cortical features, and provide a molecular framework for understanding and manipulating these evolutionary programmes.
