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. 2011 Feb;92(1):66-71.
doi: 10.1111/j.1365-2613.2010.00751.x. Epub 2010 Dec 13.

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice

Denise Yan  1 , Kazusaku Kamiya, Xiao Mei Ouyang, Xue Zhong Liu
Affiliations

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice

Denise Yan et al. Int J Exp Pathol. 2011 Feb.

Abstract

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. An important finding from mouse models and molecular studies is that the USH proteins are integrated into a protein network that regulates inner ear morphogenesis. To understand further the function of harmonin in the pathogenesis of USH1, we have generated a targeted null mutation Ush1c mouse model. Here, we examine the effects of null mutation of the Ush1c gene on subcellular localization of Myo7a, Pcdh15 and Sans in the inner ear. Morphology and proteins distributions were analysed in cochlear sections and whole mount preparations from Ush1c(-/-) and Ush1c(-/+) controls mice. We observed the same distribution of Myo7a throughout the cytoplasm in knockout and control mice. However, we detected Pcdh15 at the base of stereocilia and in the cuticular plate in cochlear hair cells from Ush1c(+/-) controls, whereas in the knockout Ush1c(-/-) mice, Pcdh15 staining was concentrated in the apical region of the outer hair cells and no defined staining was detected at the base of stereocilia nor in the cuticular plate. We showed localization of Sans in the stereocilia of controls mouse cochlear hair cells. However, in cochleae from Ush1c(-/-) mice, strong Sans signals were detected towards the base of stereocilia close to their insertion point into the cuticular plate. Our data indicate that the disassembly of the USH1 network caused by absence of harmonin may have led to the mis-localization of the Protocadherin 15 and Sans proteins in the cochlear hair cells of Ush1c(-/-) knockout mice.

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Figures

Figure 1
Figure 1
Abnormalities at the apical surface of outer hair cells (OHC) and structural defects of the hair cells in Ush1c−/− mice at PD21. Cochlear whole mounts were stained with phalloidin (red) to reveal F-actin in stereocilia and an antibody against myosin 7a (green) to show the basal structure of the hair cells in Ush1c+/− (a, c and e) and Ush1c−/− mice (b, d and f). Stereocilia defects were observed in the middle part of the stereocilia bundles of the OHCs (d, arrows) that were not detected in stereocilia of inner hair cells (IHC; b). c and d show magnified images corresponding to the boxed areas in a and b respectively. The confocal analysis at the basal level of hair cells with nuclear staining (e and f) (DAPI, Blue). Bars = 20 μm.
Figure 2
Figure 2
Localization of Protocadherin 15 (green) in the cochlear hair cells of Ush1c-knockout mice at PD21. Cross sections of the organ of Corti were stained with an antibody to Protocadherin 15 (green). In Ush1c+/− mice (a and c), expression of Protocadherin 15 was localized at the base of stereocilia (left panel, arrow) and in the cuticular plate. In contrast, in Ush1c−/− mice (b and d), Protocadherin 15 immunoreactivity appears (arrowhead) diffuse above nuclei (DAPI-Blue). Bars = 20 μm.
Figure 3
Figure 3
Localization of Sans (green) in the cochlear hair cells of Ush1c−/− mice at PD21. Cross sections of the organ of Corti of Ush1c+/− (a and c) and Ush1c−/− (b and d) mice were stained with an antibody to Sans (green). c and d are higher magnification images of a and b respectively. Arrowheads and arrows indicate inner hair cells and outer hair cells (OHC) respectively. Nuclei were stained by DAPI (Blue). Sans was localized in the stereocilia bundles in Ush1c+/− cochlear hair cells at PD21 (c). However, in Ush1c−/− mice, strong signals were observed towards the base of stereocilia close to their insertion into the cuticular plate with a slight cytoplasmic staining of OHC (d). Bars indicate 20 μm.
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