Neanderthal-derived variants shape craniofacial enhancer activity at a human disease locus

Facial appearance is one of the most variable morphological traits in humans, influenced by both rare and common genetic variants that can impact facial form between individuals and in disease. Deletion of an enhancer cluster 1.45 megabases upstream of the SOX9 gene (EC1.45) results in Pierre Robin sequence, a human craniofacial disorder characterised by underdevelopment of the lower jaw and frequently associated with cleft palate. We reasoned that single nucleotide variants in EC1.45 may cause more subtle alterations to facial morphology. Here, we took advantage of recent human evolution, and the distinct morphology of the Neanderthal lower jaw, to investigate the impact of three Neanderthal-derived single nucleotide variants on EC1.45 function and jaw development. Utilising a dual enhancer-reporter system in zebrafish, we observed enhanced Neanderthal regulatory activity relative to the human orthologue during a specific developmental window. At this same stage, we show that EC1.45 appears to be selectively active in neural crest- derived progenitor cells which lie in close apposition with and are transcriptionally related to precartilaginous condensations that contribute to craniofacial skeletal development. To examine the potential consequences of increased SOX9 expression in this specific cellular population during jaw development, we overexpressed human SOX9 specifically in EC1.45-active cells and observed an increase in the volume of developing cartilaginous precursors. Taken together, our work implicates Neanderthal-derived variants in increased regulatory activity for a disease- associated enhancer with the potential to impact craniofacial skeletal development and jaw morphology across recent hominin evolution. To explore the relative activity and cell type specificity of the human versus Neanderthal EC1.45 Peak1-2 enhancers further, we performed single cell-RNA sequencing (scRNA-seq) for the two human-Neanderthal enhancer reporter lines at 2 dpf. Fluorescence-activated cell sorting (FACS) was used to isolate all cells that were eGFP-positive, mCherry-positive or double-positive from dissected embryonic cranial regions at 2 dpf. This resulted in 40,000 cells for the Tg(Hu:GFP;Ne:Ch) line and 10,000 cells for the Tg(Ne:GFP;Hu:Ch) line which were processed as two separate samples using the 10x Genomics Chromium single-cell 3′ gene expression technology (v3.1).