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

Abstract

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications. Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown. HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development. Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

Resolving the three-dimensional interactome of Human Accelerated Regions during human and chimpanzee neurodevelopment

“…Determining the specific traits that HARs and HGEs influenced ultimately requires genetic models, such as humanized mice or cerebral organoids, in which their effects on neurodevelopment and function can be measured. A small number of HARs and HGEs have been shown to drive changes in gene expression and regulation in humanized mouse models, as well as developmental phenotypes including changes in radial glial self-renewal that lead to an increased number of neurons in the cortex 12,15,24 . These studies all share two advantages: first, strong evidence that the HAR or HGE encoded human-specific regulatory activity in a particular tissue, developmental time point or cell type; and second, a clear understanding of the identity and biological function of the likely gene targets.…”

“…Multiple studies support that HARs include transcriptional enhancers with human-specific activity 10,11 . HARs have been shown to drive changes in gene expression in genetically modified mouse models [12][13][14][15] , while massively parallel reporter assays (MPRAs) comparing HARs with their non-human primate orthologs have identified HARs that exhibit species-specific changes in enhancer activity [16][17][18] . Targeted perturbations and large-scale CRISPR proliferation screens have also been used to link HARs to changes in gene expression and neural stem cell phenotypes 19,20 .…”

“…HARs and HGEs may drive changes in gene expression via two non-exclusive mechanisms. First, HARs, HGEs and their chimpanzee orthologs may target conserved sets of orthologous genes in each species, with HARs and HGEs acting to alter expression of those genes in human [12][13][14][15]24 . Alternatively, it has been suggested HARs may have gained new gene targets in human neurodevelopment, a phenomenon referred to as enhancer hijacking 7 .…”