Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice

  1. Stephanie A Bucks
  2. Brandon C Cox
  3. Brittany A Vlosich
  4. James P Manning
  5. Tot B Nguyen
  6. Jennifer S Stone  Is a corresponding author
  1. Virginia Merrill Bloedel Hearing Research Center, University of Washington, United States
  2. Southern Illinois University School of Medicine, United States
10 figures, 5 videos and 1 table

Figures

Phagosomes target type I and type II HCs for clearance in adult mouse utricular maculae under normal conditions.

(A) Schematic of the inner ear with the utricle highlighted in magenta. (B) Schematic of a surface view of a utricle (xy view) with HCs in magenta. Blue outlined region denotes the striola (S). The …

https://doi.org/10.7554/eLife.18128.003
F-actin spikes are present in HCs, and the HC bundle is likely ejected prior to HC body translocation under normal conditions.

(A–A”) A F-actin spike (green, arrowhead) in a normally localized HC [myosin VIIa (Myo) magenta] from a C57Bl/6J mouse is shown in 3 views. (A) is a xy optical section at the level of type II HC …

https://doi.org/10.7554/eLife.18128.006
Phagosomes are present in several mouse strains across ages.

(A) Number of phagosomes per utricle in 3 mouse strains (CBA/CaJ, C57Bl/6J, and Swiss Webster) at 3 weeks, 5–10 weeks, and 43–46 weeks of age. Data are presented as mean ± 1 standard deviation for n …

https://doi.org/10.7554/eLife.18128.008
Figure 3—source data 1

Quantification of phagosomes in the normal utricle of3 mouse strains at 3 ages.

Mean (one standard deviation, SD) and 95% confidence interval (CI) of number of F-actin (phalloidin)-labeled phagosomes per utricle. n, number of mice.

https://doi.org/10.7554/eLife.18128.009
SCs, not macrophages, produce phagosomes in adult mouse utricles under normal conditions.

(A,B) Confocal projection images of the utricular macula from 7-week-old Plp1-CreERT2:ROSA26tdTomatomice showing the numbers and distribution of tdTomato-positive SCs (magenta) in the utricular …

https://doi.org/10.7554/eLife.18128.010
Figure 4—source data 1

Quantification of the percentage of SCs labeled with tdTomato in Plp1-CreERT2:ROSA26tdTomato utricles.

Mean percentage {(one standard deviation, SD); [95% confidence interval, CI]} of tdTomato-labeled SCs in Plp1-CreERT2:ROSA26tdTomato mice given tamoxifen at 6 weeks (wks) of age and sacrificed 1 or 15 weeks post tamoxifen (7 or 21 weeks of age, respectively). SCs were sampled in two striolar regions and 6 extrastriolar regions in each utricle (see Materials and methods). n = 3 mice at both timepoints.

https://doi.org/10.7554/eLife.18128.011
Immature HCs are found in adult mouse utricles under normal conditions.

(A,B) Confocal xy optical sections of utricles from neonatal (<postnatal day 8) (A) and adult (>6 weeks) (B) Swiss Webster mice. Some HC stereocilia bundles at each age (arrows) were co-labeled with …

https://doi.org/10.7554/eLife.18128.012
Figure 5—source data 1

Quantification of immature HC markers in the normal adult mouse utricle.

(A) Mean (one standard deviation, SD) and 95% confidence interval (CI) of number of PCDH15-CD2-labeled stereocilia bundles per utricle in two strains of mice. (B) Mean (1 SD) and 95% CI of number of ATOH1-GFP-positive cells per utricle. HCs were identified as myosin VIIa-positive cells with nuclei in the apical two-thirds of the epithelium. SCs were identified as myosin VIIa-negative cells whose bodies extend across the entire macular depth, whose nuclei are smaller than HC nuclei, and are positioned near the basal lamina. Unknown cells did not meet criteria for HCs or SCs. n, number of mice.

https://doi.org/10.7554/eLife.18128.013
SCs produce new type II HCs in adult mouse utricles under normal conditions.

(A) Experimental timeline. Plp1-CreERT2:ROSA26tdTomato mice were injected with tamoxifen (Tam) at 6 weeks of age to label SCs with tdTomato and were sacrificed (Sac) at 7, 10, 16, and 21 weeks of …

https://doi.org/10.7554/eLife.18128.014
Figure 6—source data 1

Quantification of tdTomato-labeled HCs in Plp1-CreERT2:ROSA26tdTomato utricles over time.

Mean (one standard deviation, SD) and 95% confidence interval (CI) of the number of tdTomato-labeled HCs per utricle categorized by type in Plp1-CreERT2:ROSA26tdTomato mice given tamoxifen at 6 weeks (wks) of age (right) or in age-matched littermate controls that did not receive tamoxifen (left). Un., unknown. n, number of mice.

https://doi.org/10.7554/eLife.18128.015
Atoh1-CreERTM-labeled type II HCs do not convert into type I HCs over 8 months in adult mouse utricles under normal conditions.

(A) Experimental timeline. Atoh1-CreERTM: ROSA26tdTomato mice were injected with tamoxifen (Tam) at 6 weeks of age and were sacrificed (Sac) at 7, 16, 21, and 38 weeks of age (corresponding to 1, …

https://doi.org/10.7554/eLife.18128.019
Figure 7—source data 1

Quantification of tdTomato-labeled HCs in Atoh1-CreERTM:ROSA26tdTomato utricles.

Mean (one standard deviation, SD) and 95% confidence interval (CI) of the number and percentage of tdTomato-labeled HCs per utricle, categorized by type [type I, type II, or unknown (Un.)]. Atoh1-CreERTM:ROSA26tdTomato mice were given tamoxifen at 6 weeks (wks) of age (right) or were age-matched littermate controls that did not receive tamoxifen (left). For the graph in Figure 7G, we present the number of tdTomato-positive type I HCs at 1, 10, 15, and 32 weeks post tamoxifen (shown here), normalized to the total number of tdTomato-positive cells at each timepoint. n, number of mice.

https://doi.org/10.7554/eLife.18128.020
DT-mediated HC damage increases SC-to-HC transition in adult mouse utricles.

(A) Experimental timeline. Plp1-CreERT2:ROSA26tdTomato:Pou4f3DTR (damaged) mice and Plp1-CreERT2:ROSA26tdTomato (control) mice were injected with tamoxifen (Tam) at 9 weeks of age to label SCs with …

https://doi.org/10.7554/eLife.18128.021
Figure 8—source data 1

Quantification of tdTomato-labeled HCs after HC damage in Plp1-CreERT2:ROSA26tdTomato:Pou4f3DTR and control utricles.

Mean (one standard deviation, SD) and 95% confidence interval (CI) of the number and percentage of tdTomato-labeled HCs per utricle. Plp1-CreERT2:ROSA26tdTomato:Pou4f3DTR mice (damaged) were given tamoxifen at 9 weeks of age, DT at 10 weeks of age and analyzed at 13 weeks of age. Controls were littermates that did not contain the Pou4f3DTR allele but received both the tamoxifen and DT injections. n, number of mice.

https://doi.org/10.7554/eLife.18128.022
DT-mediated HC damage induces apoptosis and a small increase in phagosome numbers in adult mouse utricles.

(A–A”) The same field of a control (Pou4f3DTR-negative) utricle, focused on the type II HC layer. (B–B”) The same field of a Pou4f3DTR utricle at 4 days post DT, focused on the HC layer. Green …

https://doi.org/10.7554/eLife.18128.023
Figure 9—source data 1

Quantification of phagosomes in Pou4f3DTR mice after HC damage.

Mean (one standard deviation, SD) and 95% confidence interval (CI) of number of F-actin (phalloidin)-labeled phagosomes per utricle. Littermates lacking the Pou4f3DTR allele were used as control and labeled as 0 day post DT. n, number of mice.

https://doi.org/10.7554/eLife.18128.024
Model of HC turnover in adult mouse utricles under normal conditions.

(A) Model of HC clearance. An actin spike (green) forms to connect SCs (grey) and a HC targeted for clearance (magenta). The apical portion of SCs converges and cleaves off the top of the HC, …

https://doi.org/10.7554/eLife.18128.025

Videos

Video 1
3D reconstruction of a phagosome in an adult Swiss Webster utricle under normal conditions.

This movie is a y-axis rotation of a 3D reconstruction of an ectopic HC (myosin VIIa, magenta) associated with a F-actin phagosome (phalloidin, green). The phagosome consists of a large basket-like …

https://doi.org/10.7554/eLife.18128.004
Video 2
Phagosomes in the SC nuclear layer of an adult Swiss Webster utricle under normal conditions.

This movie was constructed from optical sections collected from the area shown in Figure 1E. It begins at the level of type II HC nuclei and progresses through type I HC and SC nuclei, ending at the …

https://doi.org/10.7554/eLife.18128.005
Video 3
F-actin spike through a normally located HC in an adult mouse utricle under normal conditions.

This movie was constructed from optical sections collected from the area shown in Figure 2A–A’’. The movie begins above the stereocilia bundles (F-actin, green), continues through the type II and …

https://doi.org/10.7554/eLife.18128.007
Video 4
Clearly stratified type I and type II HCs in the adult mouse utricle.

This movie begins at the cuticular plate of the HCs, proceeds through the level of HC necks, type II HC nuclei (HCII), type I HC nuclei (HCI), and ends in the SC nuclear layer where type II HC …

https://doi.org/10.7554/eLife.18128.016
Video 5
Type I and type II HCs labeled with Atoh1-CreERTM:ROSA26tdTomato.

This movie was constructed from optical sections collected from the area shown in Figure 7E–F’’. The movie begins at the cuticular plate, passes through HC necks, type II HC nuclei, type I HC …

https://doi.org/10.7554/eLife.18128.018

Tables

Table 1

Detection of utricular SC proliferation under normal conditions. We used two delivery methods and 3 different time periods of exposure to administer BrdU to mice to investigate cell proliferation in …

https://doi.org/10.7554/eLife.18128.017

Number of BrdU+ cells per SE Mean (SD)

Delivery route

Delivery rate

Dose

Days of exposure

Number of mice/utricles (strain)

0.25 (0.50)

IP

1x/day

50 mg/kg

7

2/4

(C57Bl/6J)

0 (0)

IP

2x/day

50 mg/kg

3

3/3

(Swiss Webster)

0 (0)

PO

Continuous

2 mg/ml

7

7/14

(C57Bl/6J)

0 (0)

PO

Continuous

2 mg/ml

7

3/6

(Swiss Webster)

0 (0)

PO

Continuous

2 mg/ml

14

3/6

(Swiss Webster)

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