The Human Accelerated Region HACNS1 modifies developmental gene expression in humanized mice

Morphological innovations that arose during human evolution are ultimately encoded in genetic changes that altered development. Human Accelerated Regions (HARs), which include developmental enhancers that harbor a significant excess of human-specific sequence changes, are leading candidates for driving novel physical modifications in humans. Here we examine the role of the HAR HACNS1 (also known as HAR2) in human limb evolution by directly interrogating its cellular and developmental functions in a humanized mouse model. HACNS1 encodes an enhancer with human-specific activity in the developing limb in transgenic mouse reporter assays, and exhibits increased epigenetic signatures of enhancer activity in the human embryonic limb compared to its orthologs in rhesus macaque and mouse. Here we find that HACNS1 maintains its human-specific enhancer activity compared to its chimpanzee ortholog in the mouse embryonic limb, and that it alters expression of the transcription factor gene Gbx2 during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in humanized limb bud chondrogenic mesenchyme, implicating HACNS1-mediated Gbx2 expression in early skeletal patterning. Our findings establish that HARs direct changes in the level and distribution of gene expression during development, and illustrate how humanized mouse models provide insight into regulatory pathways modified in human evolution.