The role of bone morphogenetic protein 4 in inner ear development and function

Abstract

Bone Morphogenetic Protein 4 (BMP4) is a member of the TGF-beta superfamily and is known to be important for the normal development of many tissues and organs, including the inner ear. Bmp4 homozygous null mice die as embryos, but Bmp4 heterozygous null (Bmp4(+/-)) mice are viable and some adults exhibit a circling phenotype, suggestive of an inner ear defect. To understand the role of BMP4 in inner ear development and function, we have begun to study C57BL/6 Bmp4(+/-) mice. Quantitative testing of the vestibulo-collic reflex, which helps maintain head stability, demonstrated that Bmp4(+/-) mice that exhibit circling behavior have a poor response in the yaw axis, consistent with semicircular canal dysfunction. Although the hair cells of the ampullae were grossly normal, the stereocilia were greatly reduced in number. Auditory brainstem responses showed that Bmp4(+/-) mice have elevated hearing thresholds and immunohistochemical staining demonstrated decreased numbers of neuronal processes in the organ of Corti. Thus Bmp4(+/-) mice have structural and functional deficits in the inner ear.

Figure 1. Vestibulo-collic reflex is reduced in the yaw axis in circling Bmp4^+/− mice, but is normal in non-circling Bmp4^+/− mice

Ratio of mean gain of Bmp4^+/− mice to that of wild type mice measured in response to yaw (dashed lines) and pitch (solid lines) axis rotations of small, medium and large amplitudes. (A) Circling mice: Pitch axis, large, Bmp4^+/− n=4, wild type n=5; medium, Bmp4^+/− n=4; wild type n=4; small, Bmp4^+/− n=4; wild type n=4. Yaw axis, large, Bmp4^+/− n=2, wild type n=5; medium, Bmp4^+/− n=2; wild type n=5; small, Bmp4^+/− n=3; wild type n=4. (B) Non-circling mice: Pitch and Yaw axes, large, medium and small amplitudes, Bmp4^+/− n=4, wild type n=4.

Figure 2. Auditory Brainstem Response thresholds are reduced in Bmp4^+/− mice

ABR thresholds for Bmp4^+/− and wild type mice were measured at frequencies of 6, 12 and 24 kHz. Each symbol represents 1 mouse.

Figure 3. Stereocilia are reduced in number in ampullae of Bmp4^+/− mice

Whole mount of the organ of Corti or vestibular epithelia stained with phalloidin and viewed by epifluorescence. In the organ of Corti, utricle and saccule, the stereocilia are similar in Bmp4^+/− and wild type mice. In ampulla, the stereocilia are fewer in number in Bmp4^+/− mice compared to wild type mice. Scale bar, 10 μm.

Figure 4. Reduced neuronal processes in organ of Corti of Bmp4^+/− mice

Whole mount of the organ of Corti stained with antibodies to myosin VIIa (green) and neurofilament 200kD (red) and viewed by confocal microscopy. In the organ of Corti there is a reduction of neuronal processes in the Bmp4^+/− mice compared to wild type mice. (C) and (D) represent the boxed regions in (A) and (B). Scale bars: A, B 100 μm; C–D 5 μm. Boxed regions in (A) and (B) are located two-thirds into the first turn starting from the apex. In (E), hair cells (OHCs and IHCs) and neuronal processes between the outer and inner hair cells were quantified for 4 Bmp4^+/−mice with decreased neuronal processes and 4 wild type mice. P value was determined by two-tailed t test.

Figure 5. Neurogenin 1 expression is unaltered in Bmp4^+/− otocysts

Real time RT-PCR for Neurogenin 1 normalized to β-actin in E14.5 otocysts. Median expression levels indicated by bars. Bmp4^+/− n=8, wild type n=8.