The outer ear is unique to mammals, but its evolutionary origin has remained a mystery. According to a new study published in Nature from the USC Stem Cell lab of Gage Crump, this intricate coil of cartilage has a surprisingly ancient origin in the gills of fishes and marine invertebrates.
“When we started the project, the evolutionary origin of the outer ear was a complete black box,” said corresponding author Crump, professor of stem cell biology and regenerative medicine at the Keck School of Medicine of USC. “We had been studying the development and regeneration of the jawbones of fishes, and an inspiration for us was Stephen Jay Gould’s famous essay ‘An earful of jaw,’ which laid out how fish jawbones transformed into the middle ear bones of mammals. This made us wonder whether the cartilaginous outer ear may also have arisen from some ancestral fish structure.”
The first clue toward cracking this mystery was the team’s discovery that gills and outer ears are both composed of a relatively rare tissue type: elastic cartilage. “When we started the study, there was very little out there about whether elastic cartilage existed outside of mammals,” said Crump. “So it wasn’t really known if fish had elastic cartilage or not. It turns out that they do.”
Gills and outer ears look and function quite differently from one another. They also do not mineralize, which means they are rarely recovered in the fossil record. Therefore, a new type of approach was needed to determine if they were evolutionarily related. The study’s first author Mathi Thiruppathy, a PhD student in the Crump lab, focused on gene control elements called enhancers. While the genes that these enhancers control are often involved in the development of many unrelated tissues and organs, enhancers tend to be much more tissue specific.
The scientists were able to incorporate enhancers that help form the elastic cartilage of the human outer ear into the genomes of zebrafish . Remarkably, the human outer ear enhancers were active specifically in the gills of these transgenic zebrafish. The scientists also succeeded in doing the experiment in reverse, creating transgenic mice with genomes incorporating zebrafish enhancers typically involved in the formation of the gills, and found them active in the outer ears of the mice. These enhancers were key in connecting structures that at first glance do not appear to be very similar.
With collaborators, the researchers then investigated whether the human outer ear and fish gill enhancers could be used to follow the evolution of gills into outer ears across intermediate species, such as amphibians and reptiles. They found that when either human ear or fish gill enhancers were incorporated into the genomes of tadpoles, the enhancers showed activity in their gills. However, when reptiles came on the scene, the elastic cartilage of gills moved to the ear canal, which the scientists demonstrated in a series of experiments with green anole lizards. This cartilage eventually became further elaborated to form the prominent outer ears of early mammals.
An additional surprise was that the elastic cartilage of gills may have arisen much earlier than previously thought. Older reports had characterized cartilage-like tissue in the gills and tentacles of several marine invertebrates, including horseshoe crabs, which have changed very little since emerging close to 400 million years ago. The researchers performed DNA sequencing on individual cells of the horseshoe crab gills and discovered a crab enhancer that, when placed in the genome of zebrafish, had gill activity. This suggests that the very first elastic cartilage, similar to what is in our outer ears, may have first arisen in ancient marine invertebrates.
“This work provides a new chapter to the evolution of the mammalian ear,” said Crump. “While the middle ear arose from fish jawbones, the outer ear arose from cartilaginous gills. By comparing how the same gene control elements can drive development of gills and outer ears, the scientists introduce a new method of revealing how structures can dramatically change during evolution to perform new and unexpected functions.”
About the study
Additional authors are Lauren Teubner, Ryan R. Roberts, Seth Ruffins, Arijita Sarkar, Jade Tassey, Denis Evseenko, and Thomas P. Lozito from USC; Micaela Lasser and Helen Rankin Willsey from the University of California, San Francisco; Alessandra Moscatello and Ya-Wen Chen from the the Icahn School of Medicine at Mount Sinai; Christian Hochstim from Children’s Hospital Los Angeles and USC; and J. Andrew Gillis from the Marine Biological Laboratory at Woods Hole.
Funding was provided by NIDCR (grant numbers R35DE027550 and F31DE030706). Lizard experiments were funded by NIH/NIGMS (grant number R01GM115444). Human tissue experiments were funded by USC Stem Cell Challenge Grants. Helen Rankin Willsey is a Chan Zuckerberg Biohub Investigator.