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. 2010 Sep;11(3):395-406.
doi: 10.1007/s10162-010-0209-4. Epub 2010 Feb 23.

Maintained Expression of the Planar Cell Polarity Molecule Vangl2 and Reformation of Hair Cell Orientation in the Regenerating Inner Ear

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Free PMC article

Maintained Expression of the Planar Cell Polarity Molecule Vangl2 and Reformation of Hair Cell Orientation in the Regenerating Inner Ear

Mark E Warchol et al. J Assoc Res Otolaryngol. .
Free PMC article

Abstract

The avian inner ear possesses a remarkable ability to regenerate sensory hair cells after ototoxic injury. Regenerated hair cells possess phenotypes and innervation that are similar to those found in the undamaged ear, but little is known about the signaling pathways that guide hair cell differentiation during the regenerative process. The aim of the present study was to examine the factors that specify the orientation of hair cell stereocilia bundles during regeneration. Using organ cultures of the chick utricle, we show that hair cells are properly oriented after having regenerated entirely in vitro and that orientation is not affected by surgical removal of the striolar reversal zone. These results suggest that the orientation of regenerating stereocilia is not guided by the release of a diffusible morphogen from the striolar reversal zone but is specified locally within the regenerating sensory organ. In order to determine the nature of the reorientation cues, we examined the expression patterns of the core planar cell polarity molecule Vangl2 in the normal and regenerating utricle. We found that Vangl2 is asymmetrically expressed on cells within the sensory epithelium and that this expression pattern is maintained after ototoxic injury and throughout regeneration. Notably, treatment with a small molecule inhibitor of c-Jun-N-terminal kinase disrupted the orientation of regenerated hair cells. Both of these results are consistent with the hypothesis that noncanonical Wnt signaling guides hair cell orientation during regeneration.

Figures

FIG. 1.
FIG. 1.
Ototoxic injury and regeneration in organotypic cultures of the chick utricle. Hair cells are immunolabeled for calretinin (red) and stereocilia bundles, and cell–cell junctions are labeled with phalloidin (green). A Numerous hair cells (red) were present in utricles that were cultured for 8 days in normal medium. B Treatment for 24 h with 1 mM streptomycin caused a dramatic reduction in the numbers of labeled hair cells (red) and stereocilia bundles. C After 7 days recovery from streptomycin injury, numerous regenerated hair cells (red) were evident. Scale bar = 20 µm.
FIG. 2.
FIG. 2.
Quantification of the orientation of regenerated stereocilia bundles. Specimens were labeled with an antibody against acetylated tubulin, which labeled hair cell apical surfaces and kinocilia. Utricles were then imaged on an epifluoresence microscope and positioned so that their lateral–medial axis was aligned along the horizontal axis of the visual field, yielding the coordinate system shown in (A). Planar polarity was quantified from high magnification images of immunolabeled hair cells, following a method that is shown schematically in (B). Specifically, we used image analysis software to quantify the angular position of the kinocilium (labeled as “θ” in B and C) on the apical surfaces of individual hair cells. An example of an actual orientation measurement is shown in (C). Orientation data were obtained from 364 hair cells, and the distribution of those orientations is shown in the histogram in (D). Note that the orientations were clustered around 180°, with nodes at ∼150° and 210°. Those date indicate that correct stereocilia polarity is re-established after injury and regeneration in vitro.
FIG. 3.
FIG. 3.
Surgical removal of the reversal zone from cultured utricles. A In the normal utricle, the reversal zone can be identified by immunoreactivity for GATA3 (green, arrows). B This region can be excised from the utricle (using iridectomy scissors) prior to ototoxic injury and regeneration. Scale bar = 200 µm.
FIG. 4.
FIG. 4.
Orientation of regenerated hair cells after surgical removal of the reversal zone. Quantitative data on hair cell orientation were obtained from 368 hair cells that had regenerated after striola removal (n = 8 utricles). The coordinate system and quantification method were identical to those shown in Figure 2. The angular positions of labeled kinocilia were quantified (e.g., axes and arrows in B), and a histogram of the resulting orientations (C) closely resembled the distribution observed from hair cells in intact utricles (e.g., Fig. 2C). Specifically, distribution of hair cell orientations was bimodal and clustered around 180°. These data suggest that the recovery of hair cell orientation does not depend on signaling from the striolar region.
FIG. 5.
FIG. 5.
Patterns of Vangl2 expression in the undamaged chick utricle. A–D Utricles were immunolabeled for Vangl2 (red) and hair cells, and cell–cell junctions were labeled with phalloidin (green). All images are oriented such that the reversal zone is toward the right (i.e., in the coordinate system shown in Fig. 2A). Within the extrastriolar region (A, B), immunoreactivity for Vangl2 was observed at cell–cell junctions that were aligned with the reversal zone. Arrowheads in (B) point to strips of Vangl2 immunoreactivity that are approximately parallel to the reversal zone. In contrast, the reversal zone (C, D) frequently contained regions of Vangl2 expression that were perpendicular to the hair cell reversal line; examples are indicated by the arrowheads in (D). E, F Vangl2 expression at the reversal line. Hair cells were labeled with an antibody against α-spectrin (green), which is expressed on the entire apical surface except for the insertion point of the kinocilium. We observed immunoreactivity for Vangl2 (red) adjacent to the kinociliar pole of some hair cells (arrows), as well as near the opposite side of other hair cells (arrowheads). However, because it was not possible to localize the Vangl2 immunoreactivity to a specific cell surface (hair cell vs. supporting cell), we could not determine whether the cellular localization of Vangl2 changed across the reversal line. Scale bars = 10 µm.
FIG. 6.
FIG. 6.
Polarized expression of Vangl2 is maintained after severe ototoxic injury. Utricles were incubated for 24 h in 1 mM streptomycin and then cultured for an additional 48 h in streptomycin-free medium. Cell–cell junctions and surviving stereocilia were labeled with phalloidin (green), and immunoreactivity for Vangl2 is shown in red. All images are oriented in the coordinate system shown in Figure 2A. The extrastriolar region (A, B) contained a few surviving hair cells, and the expression of Vangl2 remained correctly polarized (B, arrow). The striolar region (C, D) contained no surviving hair cells, but Vangl2 expression was similar to that observed in unlesioned utricles and was often oriented perpendicular to the striola (D, arrow). Scale bar = 20 µm.
FIG. 7.
FIG. 7.
A, B Maintained expression of Vangl2 during hair cell regeneration. Utricles were allowed to recover for 7 days after streptomycin injury and were then labeled with phalloidin (green) and anti-Vangl2 (red). Immature hair cells (arrows) were observed at 7 days after streptomycin treatment; normal patterns of Vangl2 expression were also observed. C, D Localization of Vangl2 after treatment with the JNK inhibitor SP600125. Utricles were treated with streptomycin and then allowed to regenerate in vitro for 7 days. The JNK inhibitor SP600125 (15 µM) was added to the cultures for the final 3 days of recovery. Although this treatment resulted in disarray of hair cell orientation (e.g., Fig. 8), inhibition of JNK did not cause changes in the patterned expression of Vangl2 (red) at cell–cell junctions (green, labeled with phalloidin). Scale bar = 20 µm.
FIG. 8.
FIG. 8.
Inhibition of JNK disrupts the orientation of regenerated stereocilia bundles. Utricles were treated for 24 h in 1 mM streptomycin and then allowed to recover in vitro for 7 days. For the final 72 h, the specimens also received SP600125 (15 µM, a specific inhibitor of JNK, n = 10) or U0126 (10 µM, a specific inhibitor of Erk1/2, n = 8). After fixation and immunoprocessing, images of acetylated tubulin-labeled hair cells were obtained, and their angular orientations were quantified. A Histogram of the data obtained from hair cells (n = 223) in the SP600125-tretaed utricles shows a nearly random distribution of angular orientations. B Histogram of hair cell orientations (n = 264) obtained from the U0126-treated utricles shows a bimodal distribution that is centered at ∼180°. These data suggest that inhibition of JNK—but not Erk1/2—severely disrupts the orientation of regenerating hair cells.

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