Reutilization of ancestral gill regulatory programs in outer ear evolution

How novel structures arise during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones1. In contrast, the evolutionary origins of the outer ear, another mammalian innovation, remain a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear. Overall design: From the adult male horseshoe crab, book gill tissue was dissected and subjected to mechanical dissociation of nuclei. We used FANS to perform nuclei sorting and then prepared 10X multiome libraries. These samples were pooled, sequenced using the Illumina Nextseq and Hiseq 4000 platforms, and de-multiplexed using sample-specific barcodes for further analysis.