Aminoglycoside Damage and Hair Cell Regeneration in the Chicken Utricle

Abstract

In this study, we present a systematic characterization of hair cell loss and regeneration in the chicken utricle in vivo. A single unilateral surgical delivery of streptomycin caused robust decline of hair cell numbers in striolar as well as extrastriolar regions, which in the striola was detected very early, 6 h post-insult. During the initial 12 h of damage response, we observed global repression of DNA replication, in contrast to the natural, mitotic hair cell production in undamaged control utricles. Regeneration of hair cells in striolar and extrastriolar regions occurred via high rates of asymmetric supporting cell divisions, accompanied by delayed replenishment by symmetric division. While asymmetric division of supporting cells is the main regenerative response to aminoglycoside damage, the detection of symmetric divisions supports the concept of direct transdifferentiation where supporting cells need to be replenished after their phenotypic conversion into new hair cells. Supporting cell divisions appear to be well coordinated because total supporting cell numbers throughout the regenerative process were invariant, despite the initial large-scale loss of hair cells. We conclude that a single ototoxic drug application provides an experimental framework to study the precise onset and timing of utricle hair cell regeneration in vivo. Our findings indicate that initial triggers and signaling events occur already within a few hours after aminoglycoside exposure. Direct transdifferentiation and asymmetric division of supporting cells to generate new hair cells subsequently happen largely in parallel and persist for several days.

Keywords: EdU; S-phase; cell cycle; inner ear; otic; vestibular.

Fig. 1

In vivo single dose surgical ototoxic model for chickens. (a) Streptomycin was delivered unilaterally into the vestibular promontory of the chicken inner ear. Purple areas indicate the inner ear’s six main sensory epithelia. (b) The exposed mastoid bone of the left ear. (c) After opening the mastoid bone, the underlying tissues including the lateral canal were exposed. (d)A small opening was made to open the vestibule. The endosteum, a thin membrane that is attached to the inner surface of the vestibular bone, was kept intact to reduce potential trauma caused by leaking perilymph. (e) Streptomycin solution was slowly infused. (f) Quantification of supporting cells, hair cells, and EdU-labeled cells in whole utricles was performed on three selected extrastriolar (ES) and striolar (S) 10,000 μm² regions along the anterior-posterior axis (red dashed squares indicate the typical positions of these sampling areas). Bp, basilar papilla. Sac, saccule. Utr, utricle. A, anterior. M, medial. P, posterior. c., canal. Amp, Ampullary cupulae.

Fig. 2

Dying and regenerating hair cells post-streptomycin. (a) Mean total hair cell numbers in the utricle striola of streptomycin-treated inner ears (orange) compared to untreated (black) and PBS-treated (blue) specimens. Counted were all hair cells labeled with antibodies to MYO7A. The fraction of SOX2-labeled type II (a’) and SOX2-negative type I hair cells (a”) of untreated, streptomycin-treated, and PBS-treated utricles is indicated for each time point. (b) Mean total hair cell numbers in extrastriolar regions. Error bars represent the 95 % confidence interval. *q ≤ 0.05, **q ≤ 0.01, ***q ≤ 0.001 (for additional details, see Tables 1 and 2). Representative confocal images of the hair cell layer of untreated and streptomycin-treated utricles 2 (c) and 13 days (d) after streptomycin application. Whole utricles and xy projections (10,000 μm²) of a representative striolar and an extrastriolar region are shown as indicated with squares marked S and ES, respectively. MYO7A (red) and SOX2 (green) immunolabeling was used to identify hair cells (types I and II), as well as supporting cells. DAPI (blue) was used to stain nuclei.

Fig. 3

Supporting cell numbers are not affected during regeneration. (a) Average numbers of SOX2-labeled supporting cells in the striola do not significantly change after streptomycin application when assessed at the time points indicated. (b) Likewise, no significant changes in supporting cell numbers were observed in extrastriolar regions. Orange indicates streptomycin-treated utricles, black represents untreated, and blue indicates PBS-treated utricles. Counted were SOX2-immunopositive cell nuclei in the supporting cell layer. Error bars represent the 95 % confidence interval of the mean. Additional details are shown in Tables 1 and 2.

Fig. 4

Short interval EdU experiments reveal time course of cell cycle re-entry after damage. Quantification of EdU-labeled nuclei in the (a) striola and (b) extrastriolar regions post-streptomycin. To label DNA replication in cells, a single subcutaneous injection of EdU in PBS was administrated 24 h before analysis. For the 6 h and 1 day post-streptomycin time point, EdU was injected at time zero. When streptomycin-treated utricles (orange) were analyzed at 6 h and 1 day post-streptomycin, a significant decrease in the number of EdU-labeled nuclei was apparent in the striola as well as in extrastriolar regions compared with untreated (black) and PBS-treated (blue) utricles. Conversely, a significant increase of EdU-labeled cells in streptomycin-treated utricles in the striola as well as extrastriolar regions was observed between 2 and 5 days post-streptomycin when compared with untreated and PBS-treated utricles. Untreated and PBS-treated controls of 6 h and 3 days post-streptomycin showed no significant changes in the number of EdU-labeled cells. Cells labeled during this 24 h (6 h) EdU exposure were exclusively identified as SOX2-positive supporting cells. Error bars represent the 95 % confidence interval of the mean. **q ≤ 0.01, ***q ≤ 0.001 (for additional details, see Tables 1 and 2). Representative confocal images of the supporting cell layers of untreated and streptomycin-treated utricles 1 (c) and 3 days (d) post-streptomycin. Whole utricles and xy projections (10,000 μm²) of a representative striolar and an extrastriolar region are indicated with squares labeled S and ES, respectively. SOX2 (green) immunolabeling was used to identify supporting cells, DAPI (blue) stain was used to visualize cell nuclei, and EdU labeling (white) identified nuclei with genomic DNA that underwent replication. Notably, no EdU- and MYO7A-double-labeled hair cells were detected.

Fig. 5

Long interval EdU experiments allow cell fate determination during regeneration. A single subcutaneous injection of EdU in PBS was administrated 10 days before each analysis. Asymmetric pairs of EdU-labeled nuclei were defined as pairs consisting of one supporting cell and one hair cell juxtaposed in the supporting cell and hair cell layers, respectively. Symmetric pairs of EdU-labeled nuclei were defined as closely positioned supporting cells. Quantification of asymmetric and symmetric pairs in the striola (a) and extrastriolar (b) regions post-streptomycin. Counts from streptomycin-treated utricles are represented as orange bars compared to untreated utricles shown as black bars. Shown are the average counts from at least three independent specimens; error bars represent the 95 % confidence interval of the mean. *q ≤ 0.05, **q ≤ 0.01, ***q ≤ 0.001 (for additional details, see Tables 1 and 2). Representative confocal images of supporting cell (SC) and hair cell (HC) layers of untreated (c) and streptomycin-treated (d) utricles 11 days after streptomycin application. Whole utricles, xy projections (10,000 μm²), and x-z projections of the striola and extrastriolar regions are shown. MYO7A (red) was used to identify hair cells, SOX2 (green) was used to identify supporting cells and to distinguish hair cell subtypes, DAPI (blue) stain visualized all nuclei, and EdU-labeled nuclei indicative of S-phase re-entry are shown in white. Generally, we observed mitotic pairs of EdU-labeled nuclei. Examples of asymmetric (arrow) and symmetric (arrowhead) pairs are indicated in the xz projections.

Fig. 6

Orchestration of chicken hair cell regeneration. (a) Within a few hours after aminoglycoside-induced damage (red), mitotic turnover is globally suppressed. This suppressive period may serve as one of the earliest responses during the first steps of regeneration (blue). (b) Mitotic hair cell regeneration via asymmetric divisions of supporting cells and phenotypic conversion/transdifferentiation of supporting cells into new hair cells may occur largely in parallel. (c) We hypothesize that transdifferentiating supporting cells are being replenished by symmetric divisions of supporting cells. Illustrations were inspired by Burns and Corwin (2013) and Meyers et al. (2009)