Regression of bulla mesenchyme in wild-type mice. (A,C,D) E15.5 Fbxo11^+/+ (mixed C57BL/6J C3H background) and (B) Fbxo11^Jf/+ mice have a first pharyngeal pouch lined with endoderm-derived epithelium (unlabelled arrowhead in A), E-cadherin and (D) wide-spectrum cytokeratin IHC. (E,G-L) E18.5 Fbxo11^+/+ and (F) Fbxo11^Jf/+. (E,F) Bulla mesenchyme (arrowheads) projects from the otic capsule. (G,H) Duplex ISH Bpifa1 signals are brown spots and Foxj1 signals are red spots. (G) Nasopharynx ciliated epithelium has a Foxj1 signal. (H) Higher magnification of mesenchyme-associated epithelium (MAE) and pseudostratified mucosal epithelium shows an intense Bpifa1 signal. Occasional Foxj1-positive ciliated cells are detected in mucosal epithelium (single unlabelled arrowhead indicates a Foxj1-positive cell). (I) Epithelia, muscle and blood vessels have a basal lamina that stains for laminin. (J) Higher magnification of mesenchyme, MAE and pseudostratified mucosal epithelium. Note laminin staining of capillary (horizontal arrowhead) as well as epithelial basal lamina. (K) The MAE stains for E-cadherin, (L) but wide-spectrum cytokeratin staining is absent. (M,N) P10 C57BL/6J vimentin IHC. (M) Bulla mesenchyme has vimentin-positive mesenchymal cells with cytoplasmic projections that form an interconnecting meshwork (unlabelled arrowheads). (N) Higher magnification of panel M shows that the MAE and mucosal epithelium are vimentin negative. (O-X) P11 C57BL/6J. (O,P) Two examples of partially regressed mesenchyme with irregular spiky projections (unlabelled arrowheads); (O) H&E, (P) Bpifa1/Foxj1 ISH. (Q-V) Partly regressed mesenchyme; (Q) Ki67 IHC showing a high proliferation index in MAE. (R) αSMA and (S) AQP1 are expressed in mesenchymal cells; (T) the laminin-positive basal lamina remains in place. (U) pSmad2 and (V) pSmad1/5 are widely expressed during regression. (W,X) P11 C57BL/6J promontory mucosa with non-ciliated epithelium, which in X has a moderate Ki67 index. bl, basal lamina; CK, wide-spectrum cytokeratin IHC; cp, cochlea promontory; ee, endoderm-derived epithelium; e-cad, E-cadherin IHC; m, malleus; mae, mesenchyme-associated epithelium; mes, mesenchyme; muc, mucosa; oc, otic capsule; np, nasopharynx; pe, pseudostratified epithelium; sa, stapedial artery; tm, tympanic membrane. Scale bars: (A,B,E-G,I,M,O) 500 µm; (P) 200 µm; (C,D,H,J-L,N,Q-X) 50 µm.

The ultrastructure of P0 C57BL/6J bulla mesenchyme shows a squamous epithelial margin. (A-C) Semi-thin Toluidine-Blue-stained sections showing bulla and otic capsule with bulla mesenchyme bordered by a single layer of epithelium. (A) Rectangles indicate location of panels B and C; arrow directions indicate the orientation: rostral (r), caudal (c), medial (m), lateral (l). (B) The mesenchyme is bordered by a single-layered epithelium that closely abuts the multi-layered epithelium of the tympanic membrane epithelium and outer canal. (C) A cleft (asterisk) separates the mesenchyme and facing multi-layered epithelium. (D-K) Transmission electron microscopy. (D,E) The mesenchyme-associated epithelium (MAE) and the facing multi-layered epithelium is separated by a variably wide cleft (asterisk). (F) MAE rests on a basal lamina and has an overlying effete cell (arrowhead) on its apical surface. (G) Higher magnification of panel F shows that the basal region of the MAE cell has a basal lamina and interdigitating cytoplasmic projections with a neighbouring epithelial cell. (H,I) Apical regions of the MAE cell show interdigitations, desmosome cell junctions and filopodia (arrowheads) projecting from the apical cell surface. (J) The MAE has cytoplasmic glycogen granules (arrowheads). (K) Mesenchymal cells and scattered bundles of collagen fibrils (arrowheads) in the ECM. bl, basal lamina; d, desmosome cell junction; int, interdigitation of adjoining epithelial cells; m, malleus; mae, mesenchyme-associated epithelium; mes, mesenchyme; oec, outer ear canal epithelium; oc, otic capsule; sa, stapedial artery; tm, tympanic membrane epithelium. Scale bars: (A) 500 µm, (B) 50 µm, (C) 100 µm, (D-K) 1 µm.

Bulla mesenchyme condensation. (A) Bulla mesenchyme regresses after P10 in Fbxo11^+/+ but persists in Fbxo11^Jf/+ mice. Numbers adjacent to points indicate the number of bullae examined; numbers above the symbol are for Fbxo11^+/+ and those beneath are for Fbxo11^Jf/+ bullae. (B) Mesenchyme area and (C) mesenchymal nuclei density. C57BL/6J partially regressed mesenchyme (PART) has a reduced area compared with pre-regression mesenchyme (PRE), and its mesenchymal nuclei density is increased. Remnant P13 and P15 Fbxo11^Jf/+ mesenchyme shrinks and has increased nuclei density. (D) MAE to cochlea promontory length ratios and (E) epithelial nuclei densities. C57BL/6J partially regressed mesenchyme epithelium is shorter than pre-regression mesenchyme epithelium. C57BL/6J epithelial density increases from pre- to partially regressed mesenchyme and to promontory epithelium (PE). Fbxo11^Jf/+ bullae have persistent MAE and epithelial density is significantly increased at P15. Data in histograms (B-E) are represented as points and the median. Statistical tests were chosen after performing D'Agostino and Pearson omnibus normality tests. C57BL/6J data were analyzed with Student's t-tests, or ANOVA with Tukey's multiple comparison tests (except that MAE ratios were analyzed with a Mann–Whitney test). Fbxo11^Jf/+ data were analyzed with Kruskal–Wallis tests and Dunn's multiple comparisons using P11 data as the reference control. Two-tailed tests: **P<0.01, ***P<0.001, ****P<0.0001; all other comparisons were not statistically significant.

Otitis media in adult Fbxo11^Jf/+ mice is characterized by bulla adhesions and soft-tissue mineralization. (A) P10 C57BL/6J pre-regression mesenchyme has widely spaced mesenchymal cells and Picrosirius-Red-stained collagen fibrils are in loose arrays (arrowheads). (B) P12 C57BL/6J partially regressed mesenchyme in which ECM collagen fibrils are more closely clustered (horizontal arrowhead); the periosteal connective tissue has a dense band of collagen (vertical arrowhead). (C) P15 Fbxo11^+/+ bulla with normal air-filled bulla cavity (asterisk). (D-F) P15 Fbxo11^Jf/+ remnant MAE projecting from the cochlea promontory (arrowhead in D); (E) collagen fibrils are clustered around mesenchymal cells and beneath the MAE (arrowhead) and (F) Ki67 staining shows a high proliferation rate and the epithelial cell density is focally hyperplastic (arrowheads). (G,H) P20 Fbxo11^Jf/+ remnant mesenchyme labelled in vivo with FITC­–dextran-70 shows red staining of mesenchyme ECM (arrowheads); (H) remnant mesenchyme lies against the bulla mucosa. (I,J) P21 Fbxo11^Jf/+ MAE shows Bpifa1 ISH signals (but not Foxj1 signals); (J) laminin-stained basal lamina of MAE and capillaries (unlabelled arrowhead). (K-T) P105-P119 Fbxo11 bullae. (K) Normal healthy Fbxo11^+/+ bulla with a curved tympanic membrane profile and air-filled bulla cavity (asterisk). (L-N) Fbxo11^Jf/+ bullae with adhesions that attach mucosa to tympanic membrane and indent its profile (unlabelled arrowheads in L and M); (L) inflammatory cell effusion in the bulla cavity is minimal, whereas (M) cross-linking of adhesions create compartments containing effusions of different cellularity and eosin staining intensity. (N) A higher magnification of panel M shows an adhesion attachment point with a vascular pedicel and a capillary blood vessel (unlabelled arrowhead) and mineralized foci. (O) Normal Fbxo11^+/+ bulla mucosa with a ciliated epithelium (arrowhead) and air-filled bulla cavity (asterisk). (P) Fbxo11^Jf/+ thickened mucosa with lymphoid infiltrates (arrowhead). (Q,R) Mucosal mineralization in (Q) non-ciliated and (R) ciliated epithelial regions (arrowheads). (S) Mesenchyme remnant attached to tympanic membrane; (T) Fbxo11^Jf/+ promontory epithelium has small populations of ciliated cells (arrowheads). ad, adhesion; bl, basal lamina; bb, bulla bone; cp, cochlea promontory; ic, inflammatory cells; m, malleus; mae, mesenchyme-associated epithelium; mes, mesenchyme; min, mineralization; muc, mucosa; tm, tympanic membrane. Scale bars: (C,K-M) 1000 µm; (D) 500 µm; (G,H) 200 µm; (N,S) 100 µm; (A,B,E,F,I,J,O-R,T) 50 µm.

Prevalence of otitis media in P105-P119 Fbxo11 mice and morphometric analysis of auditory-tube SMGs and auditory tubes. (A) The proportion of Fbxo11^Jf/+ bullae (n=12) with otitis media and mineralization and adhesions is high, but absent in Fbxo11^+/+ bullae (n=20). (B) Fbxo11^Jf/+ bulla mucosa is thickened. Cumulative areas of (C) auditory-tube SMGs and (D) auditory-tube lumen measured in 50-µm step sections are the same in Fbxo11^Jf/+ and Fbxo11^+/+ bullae. The histogram bar in A represents the mean and the error bars 95% confidence interval for each proportion. Frequency data were analyzed with Fisher's exact tests. Data in graphs B-D are represented as points; the error bars are mean±s.e.m. Data was analyzed with Student’s t-tests. Two-tailed tests: ns, not significant (P>0.05), ***P<0.001, ****P<0.0001.