A human-specific enhancer fine-tunes radial glia potency and corticogenesis

Humans have evolved an extraordinarily expanded and complex cerebral cortex associated with developmental and gene regulatory modifications^1-3. Human accelerated regions (HARs) are highly conserved DNA sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to species-specific cortical development remains largely unknown^4,5. HARE5 is a HAR transcriptional enhancer of the WNT signalling receptor Frizzled8 that is active during brain development⁶. Here, using genome-edited mouse (Mus musculus, Mm) and primate models, we demonstrated that human (Homo sapiens, Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacities of neural progenitor cells. Hs-HARE5 knock-in mice have significantly enlarged neocortices, containing more excitatory neurons. By measuring neural dynamics in vivo, we showed that these anatomical features result in increased functional independence between cortical regions. We assessed underlying developmental mechanisms using fixed and live imaging, lineage analysis and single-cell RNA sequencing. We discovered that Hs-HARE5 modifies radial glial cell behaviour, with increased self-renewal at early developmental stages, followed by expanded neurogenic potential. Using genome-edited human and chimpanzee (Pan troglodytes, Pt) neural progenitor cells and cortical organoids, we showed that four human-specific variants of Hs-HARE5 drive increased enhancer activity that promotes progenitor proliferation. Finally, we showed that Hs-HARE5 increased progenitor proliferation by amplifying canonical WNT signalling. These findings illustrate how small changes in regulatory DNA can directly affect critical signalling pathways to modulate brain development. Our study uncovered new functions of HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.