Having three ossicles in the middle ear is one of the defining features of mammals. All reptiles and birds have only one middle ear ossicle, the stapes or columella. How these two additional ossicles came to reside and function in the middle ear of mammals has been studied for the last 200 years and represents one of the classic example of how structures can change during evolution to function in new and novel ways. From fossil data, comparative anatomy and developmental biology it is now clear that the two new bones in the mammalian middle ear, the malleus and incus, are homologous to the quadrate and articular, which form the articulation for the upper and lower jaws in non-mammalian jawed vertebrates. The incorporation of the primary jaw joint into the mammalian middle ear was only possible due to the evolution of a new way to articulate the upper and lower jaws, with the formation of the dentary-squamosal joint, or TMJ in humans. The evolution of the three-ossicle ear in mammals is thus intricately connected with the evolution of a novel jaw joint, the two structures evolving together to create the distinctive mammalian skull.

High incidence of chronic otitis media is associated with human craniofacial syndromes, suggesting that defects in the formation of the middle ear and associated structures can have a knock-on effect on the susceptibility to middle ear inflammation. Patients with branchio-oto-renal (BOR) syndrome have several defects in the ear leading to both sensorineural and conductive hearing loss, including otitis media. 40% of BOR syndrome cases are due to Eya1 haploinsufficiency, with mouse models affecting Eya1, mimicking many of the defects found in patients. Here, we characterize the onset, consequences, and underlying causes of chronic otitis media in Eya1 heterozygous mice. Cavitation defects were evident in these mice from postnatal day (P)11 onwards, with mesenchyme around the promontory and attic regions of the middle ear space. This mesenchyme was still prominent in adult Eya1 heterozygous mice, while the wild-type littermates had fully aerated ears from P14 onwards. MicroCT analysis highlighted a significantly smaller bulla, confirming the link between bulla size defects and the ability of the mesenchyme to retract successfully. Otitis media was observed from P14, often presenting unilaterally, resulting in hyperplasia of the middle ear mucosa, expansion of secretory cells, defects in the motile cilia, and changes in basal epithelial cell markers. A high incidence of otitis media was identified in older mice but only associated with ears with retained mesenchyme. To understand the impact of the environment, the mouse line was rederived onto a super-clean environment. Cavitation defects were still evident at early stages, but these generally resolved over time, and importantly, no signs of otitis media were observed at 6 weeks. In conclusion, we show that a small bulla size is closely linked to defects in cavitation and the presence of retained mesenchyme. A delay in retraction of the mesenchyme predates the onset of otitis media, making the ears susceptible to its development. Early exposure to OM appears to exacerbate the cavitation defect, with mesenchyme evident in the middle ear throughout the animal’s life. This highlights that permanent damage to the middle ear can arise as a consequence of the early onset of OM.