Redeployment of an ancestral gill program in outer ear evolution

The outer ear is a mammalian innovation whose evolutionary origins have remained a mystery. It is formed through outgrowth of neural crest-derived cells and contains a unique type of elastic cartilage. Here we find that the gills of fishes and amphibia share common gene regulatory programs with the outer ear for both their developmental outgrowth and later formation of elastic cartilage. Comparative single-nuclei multiomics of the human fetal outer ear and zebrafish gill cartilages reveals common gene expression and enrichment of transcription factor binding motifs in differentially accessible regions (DARs) of chromatin. We also identify multiple DARs at gill outgrowth stages that are sequence conserved and differentially accessible in the human outer ear. This conserved gene regulatory architecture is reflected by specific transgenic activity of human outer ear enhancers in zebrafish and tadpole gills, and reciprocal activity of zebrafish gill enhancers in tadpole gills and the mouse outer ear. These include a conserved Dlx5/6 enhancer, consistent with Dlx5 expression in the forming gills and outer ear and enrichment of a conserved DLX-binding motif in gill and outer ear DARs. Further, this DLX-mediated gill program may be conserved in the book gills of the Atlantic horseshoe crab, which are supported by an elastic-like cartilage. Single-cell multiomics of horseshoe crabs reveals a gill mesenchyme population expressing the DLX homolog distalless and with DARs enriched for the conserved DLX-binding motif. Remarkably, a horseshoe crab distalless gill enhancer drives specific expression in zebrafish gills. We propose that a DLX-mediated gill outgrowth program, commonly associated with later development of elastic-like cartilage, first arose in invertebrates and has been redeployed in vertebrates to generate structures as diverse as gills and outer ears.