BAIAP2L2 Inactivation Does Not Affect Stereocilia Development or Maintenance in Vestibular Hair Cells

Abstract

Hair cells are mechanosensitive cells in the inner ear, characterized by dozens to hundreds of actin-based stereocilia and one tubulin-based kinocilium on the apical surface of each cell. Two types of hair cells, namely cochlear hair cells and vestibular hair cells (VHCs), are responsible for the sensation of sound and balancing information, respectively. In each hair cell, the stereocilia are organized into rows of increasing heights with the mechano-electrical transduction (MET) channels localized at the tips of shorter-row stereocilia. A so-called "row 2 protein complex" also localizes at the tips of shorter-row mechanotransducing stereocilia, which plays important roles in the maintenance of mechanotransducing stereocilia. Recently, we and others identified BAIAP2L2 as a new component of row 2 complex. Baiap2l2 inactivation causes degeneration of the mechanotransducing stereocilia in cochlear hair cells, and leads to profound hearing loss in mice. In the present work, we examined the role of BAIAP2L2 in the VHC stereocilia. Confocal microscopy reveals that BAIAP2L2 immunoreactivity is localized at the tips of shorter-row stereocilia in VHCs. However, stereocilia development and maintenance are unaffected in Baiap2l2^-/- VHCs. Meanwhile, MET function of VHCs as well as vestibular functions are also unaffected in Baiap2l2^-/- mice. Further investigations show that the stereociliary tip localization of CAPZB2, another known row 2 complex component, is not affected in Baiap2l2^-/- VHCs, consistent with the unaltered stereocilia morphology. Taken together, our present data show that BAIAP2L2 inactivation does not affect vestibular hair cell stereocilia.

Keywords: BAIAP2L2; CAPZB2; inner ear; stereocilia; vestibular hair cells.

FIGURE 1

BAIAP2L2 is localized at the tips of shorter-row stereocilia in VHCs. Whole-mount immunostaining using a specific anti-BAIAP2L2 antibody (green) was performed to examine the localization of BAIAP2L2 in the stereocilia of utricular (A) and saccular (B) hair cells. Steoreociliary F-actin core was visualized using TRITC-conjugated phalloidin (red). The genotypes and ages of mice are indicated. Scale bar, 2 μm.

FIGURE 2

Stereocilia morphology is unaffected in VHCs of Baiap2l2 knockout mice. SEM was performed to examine the stereocilia morphology of P8 utricle (A), P8 saccule (B), P30 utricle (C), and P30 saccule (D) in mice of different genotypes as indicated. In each panel, low-magnification images and high magnification images of type I and II VHCs are shown at the top and bottom, respectively. Scale bar, 5 μm (in low-magnification images) and 1 μm (in high-magnification images).

FIGURE 3

Vestibular function is unaffected in Baiap2l2 knockout mice. (A,B) FM1-43FX uptake by P30 utricular (A) and saccular (B) hair cells from Baiap2l2^+/– or Baiap2l2^–/– mice was examined using confocal microscope. Scale bars, 10 μm. (C,D) FM1-43FX uptake was quantified according to the results from (A,B), respectively. (E–G) The vestibular function of Baiap2l2^+/– or Baiap2l2^–/– mice of different ages was evaluated by examining circling stereotyped movement (E), swimming test (F), and rotarod test (G). Lhfpl5^–/– mice were included as positive control. Numbers of animals in each group are indicated in brackets. ns, not significant; ***p < 0.001.

FIGURE 4

Stereociliary tip localization of CAPZB2 and EPS8 is unaffected in VHCs of Baiap2l2 knockout mice. Whole-mount immunostaining and confocal microscopy were performed to examine the localization of CAPZB2 in utricular (A) and saccular (B) hair cells of P30 Baiap2l2^+/+ or Baiap2l2^–/– mice. Similar experiments were performed to examine the localization of EPS8 in utricular (C) and saccular (D) hair cells of P30 Baiap2l2^+/+ or Baiap2l2^–/– mice. TRITC-conjugated phalloidin was used to visualize stereociliary F-actin core. Scale bars, 2 μm.