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. 2019 Jan;57(1):e23252.
doi: 10.1002/dvg.23252. Epub 2018 Oct 4.

A requirement for Fgfr2 in middle ear development

Diana Rigueur  1   2 , Ryan R Roberts  1   2 , Lauren Bobzin  1   2 , Amy E Merrill  1   2
Affiliations

A requirement for Fgfr2 in middle ear development

Diana Rigueur et al. Genesis. 2019 Jan.

Abstract

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Keywords: Fgfr2; auditory ossicles; craniofacial development; joint development; skeletal development.

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Figures

Figure 1.
Figure 1.
Wnt1-Cre; Fgfr2flx/flx mice have decreased auditory function. Auditory Brainstem Response (ABR) testing of control and Wnt1-Cre; Fgfr2flx/flx littermates at P30 (n=6). Mean values plus or minus standard error are plotted. **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 2.
Figure 2.
The auditory bulla is dysmorphic in Wnt1-Cre; Fgfr2flx/flx mice. (A, C) μCT of P30 skulls of control and Wnt1-Cre; Fgfr2flx/flx littermates (n=3). (B, D) Higher magnification rendering of tympanic bulla and middle ear ossicles (boxed regions from A and C). (E) Measurements of the auditory canal area from control and Wnt1-Cre; Fgfr2flx/flx littermates (n=3). Lines in the inset image shows the area measured. (E) Measurements of the auditory bulla volume from control and Wnt1-Cre; Fgfr2flx/flx littermates (n=3), as shown in the inset image. ***p < 0.001 and ****p < 0.0001.
Figure 3.
Figure 3.
The tympanic ring and retrotympanic process are hypoplastic in Wnt1-Cre; Fgfr2flx/flx mice. (A, B) Sagittal surface μCT rendering of the auditory bulla in control and Wnt1-Cre; Fgfr2flx/flx littermates at P30 (n=3). The tympanic ring is pseudo-colored red and retrotympanic process is pseudo-colored blue. (C, D) Alcian blue cartilage stain of the developing auditory bulla in control and Wnt1-Cre; Fgfr2flx/flx littermates at P5 (n=3). The membrane bones of the retrotympanic process and tympanic ring are demarcated with dotted lines. The white arrow indicates the most dorsal tip of the tympanic ring. i, incus; m, malleus; rt, retrotympanic process; s, stapes; and t, tympanic ring.
Figure 4.
Figure 4.
The airspace cavities in Wnt1-Cre; Fgfr2flx/flx mice are reduced in size and dysmorphic. (A, B) Coronal orthogonal μCT slices through the middle ear at P30 in control and Wnt1-Cre; Fgfr2flx/flx littermates (n=3). The cavities of the external (e) and middle ear (m) are indicated in the control. Asterisk denotes abnormal middle ear airspace in mutant. (C, D) Histological sections stained with Hematoxylin-Eosin at P13 in control and Wnt1-Cre; Fgfr2flx/flx littermates (n=3). In panel D, the asterisks indicate abnormalities in the ear air space, arrowheads show the close proximity of the malleus to the cochlea, and the arrow marks mesenchymal adhesion between the tympanic membrane and malleus. c, cochlea; e, external ear cavity; m, middle ear cavity; and tm, tympanic membrane.
Figure 5.
Figure 5.
The ossicles of Wnt1-Cre; Fgfr2flx/flx mice exhibit ectopic bone at the sites of tendon/ligament attachment and within the incudomalleal joint. (A, B) μCT renderings of the internal and base surfaces of the ear ossicles at P30 in control and Wnt1-Cre; Fgfr2flx/flx littermates (n = 3). Alizarin red and Alcian blue staining of ossicles disarticulated from control and Wnt1-Cre; Fgfr2flx/flx littermates at P30 (C, D) (n = 3) and P10 (E, F) (n = 3). Open arrowheads indicate ectopic bone at tendon/ligament attachment sites, arrows mark the ectopic bone nodule within the incudomalleal joint, and arrowheads denote ectopic bone on the manubrium. i, incus; m, malleus; and s, stapes.
Figure 6.
Figure 6.
Ectopic cartilage forms within the synovial joint between the malleus and incus in Wnt1-Cre; Fgfr2flx/flx mice. Histological sections of the middle ear stained with HBQ at P9 (A, C) (n=4) and P7 (B, D) (n=4) in control and Wnt1-Cre; Fgfr2flx/flx littermates. A’-D’ are higher magnification images of the incudomalleal joint region demarcated by the boxes in their corresponding lower magnification images. Arrows indicate normal joint space and asterisks mark the ectopic cartilage nodule. i, incus; and m, malleus.
Figure 7.
Figure 7.
Specification of incudomalleal joint is abnormal in Wnt1-Cre; Fgfr2flx/flx embryos (A, C) Whole mount Alcian blue staining of the cartilage anlagen for the malleus and incus at E16.5 in control and Wnt1-Cre; Fgfr2flx/flx embryos (n=3). (B, D) Section in situ hybridization of Gdf5 in the incudomalleal joint at E16.5 in control and Wnt1-Cre; Fgfr2flx/flx embryos (n=3). Arrow indicates the developing incudomalleal joint. In panel D, the asterisk indicates reduced Gdf5 expression in the joint interzone. i, incus; and m, malleus.
Figure 8.
Figure 8.
Ectopic cartilage within incudomalleal joint of Wnt1-Cre; Fgfr2flx/flx develops from Scx+ cells. (A, C) Safranin O staining of the incudomalleal joint at E18.5 in control and Wnt1-Cre; Fgfr2flx/flx embryos (n=4). (B, D) Immunofluorescent staining for Scx-GFP (green)(B, B”) and anti-Fgfr2 (red)(B’, B”) in the incudomalleal joint at E18.5 in control and Wnt1-Cre; Fgfr2flx/flx embryos (n=4). In panels C, D, D’ and D”, the asterisks indicate the developing ectopic cartilage. i, incus; jc, joint capsule; and m, malleus.
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