(A) Six-day-old mice (n = 6, each genotype) had no middle ear effusions. Mesenchymal cells (black arrows) are found in the middle ear cavity, but the middle ear space (red arrows) is visible. Co, cochlea. Scale bars: 200 μm. (B) At days 11 and 12 (n = 3 mice of each genotype), mesenchymal cells (black arrows) have largely disappeared from the middle ear cavity of wild-type mice, but a few cells remain around the ossicles. M, malleus. Many more mesenchymal cells and collagen (asterisks), a by-product of mesenchyme regression, are present in the MECs of Eya4^–/– mice than in wild-type mice. See magnified inset (scale bars: 200 μm) showing mucoperiosteum and mesenchyme regression. The marked inward bulging of tympanic membrane in Eya4^–/– mice correlated with tympanic membrane retraction that was visible through the external auditory canal (EAC). Scale bar: 40 μm. (C) At days 14–16 (n = 5 for each genotype), some collagen (black asterisks) remains in both genotypes, but Eya4^–/– mice also show a hyperplastic mucosa (red asterisk) with inflammation. The opening of the eustachian tube (ET) is indicated (arrowheads). Inset shows a polyp (red arrowhead) and numerous inflammatory cells (black arrow) found in some Eya4^–/– mice.