1. Developmental Biology
  2. Neuroscience
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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
Research Article
Cite this article as: eLife 2017;6:e18128 doi: 10.7554/eLife.18128
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 region surrounding the striola is the extrastriola (ES). Boxed area shown at higher magnification in C. (C) Higher magnification of schematic in B with slices through the level of type II HC nuclei (left panel) and the level of the SC nuclei (right panel). Nuclei in blue, HC perinuclear cytoplasm (left panel) and basolateral processes (right panel) in magenta, and SC cytoplasm in grey (xy view). (D) Schematic of a cross-section (xz view) of the adult mouse utricle. II, type II HCs; I, type I HCs. Double lines indicate positions of the xy views shown in C, with the upper set of lines referring the left panel in C, and the lower set of lines referring to the right panel in C. The bracket indicates the level of the SC nuclei, the focal plane of xy confocal optical images in E–E’’’, H–J, K’–K’’. In panels E–K”, F-actin was labeled with phalloidin (green) and HCs were labeled with anti-myosin VIIa antibodies (Myo, magenta), except there is no myosin label in panel J. In E–E’”, blue label is DAPI. In H–K”, each blue label is a different cell-selective marker. (E–E’’’) Confocal xy optical sections of the SC nuclear layer in an adult Swiss Webster utricle. (E) Two ectopic HCs (arrowheads) are located next to F-actin-rich phagosomes (arrows). Inset, a ring-shaped phagosome not associated with a HC. See Video 1 for a 3D reconstruction of a phagosome targeting a HC, and see Video 2 for all xy images in the z-series of E. Myosin-labeled HC cytoplasm that is not surrounding a nucleus corresponds to type II HC basolateral processes (see 1C, right panel). (E’–E”’) Higher magnification of the two HCs indicated by arrowheads in E. Arrowheads in E” point to the nucleus of each ectopic HC. (F) Transverse section of an adult Swiss Webster mouse utricle showing an ectopic HC (black arrow) located in the SC nuclear layer that has condensed chromatin. The ectopic cell is surrounded by a calyx (black arrowhead), typical of a type I HC. The calyx appears as an electron-lucent ring around the cell, which has minimal cytoplasm and contains numerous electron-dense mitochondria (small gray dots). A calyx surrounding a normally localized type I HC (I) is indicated by the white arrow. Several normally positioned type I and II HCs are indicated (I, II over nucleus). Inset, higher magnification of the ectopic type I HC. (G) Several HCs and F-actin-rich phagosomes are shown in this projection image of a Swiss Webster macula. Two F-actin spikes are indicated by arrowheads. White circles indicate the area of 3 type II HCs for reference. (H–K’’) Phagosomes co-localized with markers of type I or II HCs in xy confocal slices at the level of SC nuclei in Swiss Webster utricles. (H) Two ectopic HCs (arrowheads) have POU4F3-positive nuclei (blue) and are connected to a large ring-shaped phagosome. (I) Two ectopic HCs associated with a basket-like phagosome. One HC has a SOX2-negative nucleus (arrow, lacking blue label), and the other HC has a SOX2-positive nucleus (arrowhead, blue). (J) Three ectopic HCs (arrowheads) associated with phagosomes are calretinin-positive (blue). Arrow indicates an example of a F-actin spike. (K) Tenascin (Ten, blue) immunolabeling in normally localized HCs (arrowhead). (K’,K”) Tenascin labeling (blue) is evident in two ectopic HCs (arrowheads) co-localized with phagosomes (green). Scale bar shown in E is 10 μm for E, 7 µm for E’–F, 3.5 µm for F inset, and 14 µm for G. Scale bar shown in H is 5 µm for H–K’’.

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 nuclei (DAPI, blue). (A’,A”) are yz and xz optical (cross) sections of the HC indicated in A by the arrowhead. Note the F-actin spike is beneath the apical surface, which is highlighted by the row of brightly labeled stereocilia (green) at the top of A’ and A”. See Video 3 for all xy images in the z-series of A. HC, HC nuclear layer; SC, SC nuclear layer. (B–B”) A F-actin spike (green, arrowhead) in an ectopic HC (Myo, magenta) from a C57Bl/6J mouse is shown in 3 views. (B) is a xy optical section at the level of SC nuclei (DAPI, blue). (B’,B”) are yz and xz optical sections of the HC shown in B. Arrow points to a basket-like phagosome. (C–D’’) Confocal optical sections from two normal Swiss Webster mouse utricles labeled with F-actin, antibodies to myosin VIIa, and with either anti-espin antibodies (C–C”) or anti-PMCA2 antibodies (D–D”). The 3 panels for each utricle (C–C” or D-D”) show different label combinations for the same field, as indicated. All panels are focused on the SC layer, except the boxed insets, which are focused on stereocilia. Arrowheads in C,D and C”,D” point to ectopic HCs (Myo, magenta in C and D), while arrows in all panels point to F-actin spikes (green in C,D and white in C’,D’). Scale bar in A is 3 µm and applies to A–B”. Scale bar in C is 10 µm and applies to C–D”, including insets.

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 = 3–8 mice per group (see Figure 3—source data 1). Within each strain, the number of phagosomes did not increase significantly over time. CBA/CaJ and C57Bl/6J mice had similar phagosome numbers (10–15 per utricle), but Swiss Webster mice had significantly more phagosomes per utricle (45–65) than the other strains at every age, as determined by two-way ANOVA (p=0.0189 for age and p<0.0001 for strain followed by Bonferroni’s multiple comparisons test; ****p<0.0001). (B,B’) Confocal projection image of the utricular macula from an adult Swiss Webster mouse showing that F-actin-rich phagosomes (green) are concentrated in the peristriolar region of the extrastriola (ES) but are largely absent from the calbindin-labeled striola (S, blue) and the peripheral-most portion of the ES. The projection image was constructed from just beneath the stereocilia through the SC layer to avoid obstruction of the phagosomes by the F-actin-rich stereocilia. Scale bar is 100 μm. Arrowheads in B,B’ point to a portion of the utricular macula that sustained damage during dissection; this region is rich in F-actin but is not a phagosome.

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 sensory epithelium of mice that received no tamoxifen (A) or mice that received tamoxifen (B). S, striola; SE, sensory epithelium; TE, transitional epithelium. See Figure 4—source data 1 for quantification of tdTomato-positive SCs in extrastriolar and striolar regions. (C–E’) Higher magnification optical sections of a Plp1-CreERT2:ROSA26tdTomatomouse utricular macula at one week post tamoxifen showing overlap between tdTomato-labeled SC cytoplasm (magenta) and a F-actin-rich phagosome (green, arrows). (C) Xy view with double lines indicating where cross-sectional images were created in D-E’. (D,D’) Yz view of same area in C.( E,E’) Xz view of same area in C. Very bright green labeling in D and E is F-actin in the stereocilia bundles of HCs. (F,G) Confocal optical sections of an adult Swiss Webster utricle. (F) Two F-actin-rich phagosomes (green, arrows) in the SC nuclear layer (DAPI, blue) did not co-label for antibodies to IBA1, a macrophage/monocyte lineage marker (magenta). (F’) An IBA1-positive cell (magenta, arrowhead) resided in the connective tissue under the phagosomes shown in F. (G) Xz view of the field shown in F,F’. The white dotted line indicates the border between sensory epithelium (SE) and connective tissue (CT). Arrows and arrowhead indicate the same cells shown in F,F’. (H) Confocal projection image of the utricular macula from an adult Lfng-eGFP mouse showing eGFP-positive SCs (green) in the sensory epithelium (SE). S, Striola. (I–I”’) Confocal optical sections of the same field from the extrastriolar region of a Lfng-eGFP utricle, with eGFP in green and F-actin in blue. (I) Xz view through the SE and CT of the utricle. (I’,I”) Xy views of the SC layer in the SE. (I”’) Xy view of the CT. The dotted line in I is the approximate location of the basal lamina, between the SE and the CT. Two phagosomes (arrowheads, F-actin, blue) are flanked by eGFP-positive SCs (green) and are also co-labeled with eGFP (green); co-labeled phagosomes appear cyan. One phagosome (arrow in I’,I”) is eGFP-negative and not flanked by eGFP-positive SCs. The double lines in I’–I”’ indicate the position of the xz view shown in I. Scale bar in A is 100 µm and applies to A,B,H. Scale bar in C is 5 µm and applies to C–G. Scale bar in I is 5 µm and applies to I–I”’.

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 F-actin (green) and antibodies to PCDH15-CD2 (magenta). Some PCDH15-CD2 labeling occurred in other places on the epithelial surface, but our analysis was restricted to stereocilia only. White lines indicate the approximate location of edge of the utricular macula. (C) Schematized map of a utricle from a representative adult Swiss Webster mouse. HCs with PCDH15-CD2-positive stereocilia are depicted by black dots. S, striola. (D) Number of PCDH15-CD2-expressing HC stereocilia bundles per utricle in normal adult CBA/CaJ and Swiss Webster mice, where there was no statistically significant difference (determined by an unpaired two-tailed Student’s t-test; p=0.1635, see Figure 5—source data 1). Data are expressed as mean +1 standard deviation. (E) Confocal projection image through the utricular macula of a 6-week-old Atoh1GFP/GFP mouse expressing ATOH1-GFP fusion protein (green). S, striola. (F,G) Schematics of cross-sectional views through a 6-week-old Atoh1GFP/GFP mouse utricle shown in F’–F’” and G’–G’”. (F’–F”’) Confocal optical section of a HC [arrowhead; myosin VIIa (Myo), magenta; DAPI, blue] that expressed ATOH1-GFP (green). (G’–G”’) Confocal optical section of a SC (arrow; DAPI, blue) that expressed ATOH1-GFP (green). HC, HC nuclear layer; SC, SC nuclear layer; BL, basal lamina. Scale bar in B is 12 µm and applies to A,B. Scale bar in E is 100 µm. Scale bar in G’’’ is 10 μm and applies to F’–G’’’.

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 age (corresponding to 1, 4, 10, and 15 weeks post tamoxifen). Control animals (age-matched Plp1-CreERT2:ROSA26tdTomato mice that did not receive tamoxifen) were sacrificed at the same ages. (B–C”’) Examples of a tdTomato-labeled (magenta) type I HC (B–B”’) and type II HC (C–C”’) from a Plp1-CreERT2:ROSA26tdTomato mouse utricle at 10 weeks post tamoxifen, which were classified according to criteria defined in Materials and methods, Figure 1D, and Video 4. (B,B’ and C,C’) xy slices taken at the levels indicated in the schematic to the left. Myosin VIIa (Myo) is in green and DAPI is in blue. Thin arrows in B,B’ point to two type I HCs, at the level of the neck (B) and the nucleus (B’). Only the type I HC on the right is tdTomato-positive, which is most evident in its nucleus (B’). Fat arrows in C,C’ point to two type II HCs, at the level of the neck (C) and the nucleus (C’). Only the type II HC on the left is tdTomato-positive. (B”,C”) Xz view of the same cells shown in B–B’ and C–C’, providing perspective on their morphology and lamination. Labels indicate the approximate positions of the nuclei for each cell type (HCII, type II HC; HCI, type I HC; and SC, SC). (B”’,C”’): Same images as B”,C”, but with Myo labeling only. Scale bar in C is 10 µm and applies to B–C’. Scale bars in B’’,C’’ are 10 µm and apply respectively to B’’’ and C’’’. It is important to note that, in thin optical slices such as these, myosin VIIa labeling intensity varied across cells, independent of tdTomato labeling intensity. For example, in panels C” and C”’, the type II HC on the left is brighter than the type II HC on the right. Further, in panels B’,B”, the myosin VIIa labeling for type I HC perinuclear cytoplasm was relatively weak, and labeling at the neck (B) was more robust. In cases such as this one, other morphological criteria—nuclear position, relative neck thickness, and presence/absence of a basolateral process—were essential for cell-typing. (D) Total number of tdTomato-expressing HCs per utricle, categorized by HC type [type I (black), type II (blue), and ‘unknown’ (orange)]. Patterned bars = control Plp1-CreERT2:ROSA26tdTomato mice that did not receive tamoxifen. Solid bars = Plp1-CreERT2:ROSA26tdTomato mice that received tamoxifen at 6 weeks of age. Data are expressed as mean ±1 standard deviation for n = 4–8 mice (see Figure 6—source data 1). *p<0.05; ****p<0.0001 as determined by a two-way ANOVA (p<0.0001 for treatment/age; p<0.0001 for HC type) followed by Tukey’s multiple comparisons post-test. (E) Linear regression analysis of tdTomato-expressing type II HCs per utricle demonstrated an increase in labeled cells with time (data correspond to D, blue solid bars). The calculated slope was 1.97 type II HCs per week, and the R2 value was 0.691. Data are expressed as mean ±1 standard deviation with dotted lines representing the 95% confidence interval. (F) Map of a representative Plp1-CreERT2:ROSA26tdTomato utricle injected with tamoxifen at 6 weeks of age and analyzed 15 weeks later. tdTomato-labeled HCs are depicted by magenta dots. S, striola.

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, 10, 15, and 32 weeks post tamoxifen). (B,C) Confocal projection images of whole utricles from 7-week-old Atoh1-CreERTM:ROSA26tdTomato mice showing the numbers and distribution of tdTomato-positive SCs (magenta) in the utricular sensory epithelium of mice that received no tamoxifen (B) or mice that received tamoxifen (C) S, striola. (D–F”) Examples of tdTomato-labeled type I and type II HCs. (D) A schematic of a cross-section through the utricular macula, with brackets indicating the optical sections generated for F–F”. I, type I HC; II, type II HC. (E) Xz view (similar to D) of images used to generate FF”, taken from the extrastriolar region of an Atoh1-CreERTM:ROSA26tdTomato mouse at one week post tamoxifen. Brackets indicate the location of optical sections shown in FF”. Arrow points to a tdTomato-positive type II HC (II) with a basolateral process (p). Arrowhead points to a tdTomato-positive type I HC (I) with a thin neck and more basally located nucleus than the type II HC. tdTomato is shown in magenta; DAPI is shown in blue. (F–F”) Progressively deeper optical xy sections through the utricular macula. Arrows and arrowheads point to same HCs as shown in E. Note that the type II HC has an apically located nucleus (arrow, [E,F]) and a basolateral process (arrows, E,F’,F”; p, E,F’’). Note that the type I HC (arrowhead, [E]) has a thin neck (arrowhead, [F]), a basally located nucleus (arrowhead, [F’]) and no basolateral process (arrowhead, [F”]). Scale bar in C is 100 μm and applies to B,C. Scale bar in E is 6 µm and applies to EF”. (G) Number of tdTomato-positive type I HCs at 1, 10, 15, and 32 weeks post tamoxifen normalized to the total number tdTomato-positive cells at each timepoint (see Figure 7–source data 1 for raw data). No significant differences in tdTomato-positive type I HCs were observed across time (determined by ANCOVA, p=0.103; n = 4). Data are expressed as mean ±1 standard deviation.

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 tdTomato, injected with diphtheria toxin (DT) at 10 weeks of age to kill HCs, and sacrificed (Sac) at 13 weeks of age (corresponding to 4 weeks post tamoxifen and 3 weeks post DT). (B) Utricles from control mice (Plp1-CreERT2:ROSA26tdTomato) that received DT injection but lacked the Pou4f3DTR allele exhibited normal-appearing HC densities [myosin VIIa (Myo), green]. S, striola. (C) Utricles from damaged Plp1-CreERT2:ROSA26tdTomato:Pou4f3DTR mice sacrificed 3 weeks post DT had fewer HCs (Myo, green). SCs were labeled with tdTomato (magenta) in both control and damaged adult mouse utricles (B,C). S, striola. (D,E) Confocal optical images of the extrastriolar region from similar utricles as shown in B and C, acquired at higher magnification. (D’,E’) Higher magnifications of the boxed areas in D,E. tdTomato-expressing HCs (arrows) were detected in control (D,D’) and damaged (E,E’) utricles. Scale bar in C is 100 µm and applies to B,C. Scale bar in E is 20 µm and applies to D,E. Scale bar in E’ is 10 µm and applies to D’,E’.( F) Total number of tdTomato-expressing HCs at 13 weeks of age (equivalent to 3 weeks post DT and 4 weeks post tamoxifen) in control (white bar, n = 3) and damaged (black bar, n = 4) utricles (see Figure 8—source data 1). Damaged utricles had significantly more tdTomato-labeled HCs compared to control utricles determined by an unpaired, two-tailed Student’s t-test (p=0.0346). Data are expressed as mean ±1 standard deviation.

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 arrowheads point to myosin VIIa-labeled HCs (Myo, magenta in A,B; white in A’,B’), while green arrows point to a nucleus (DAPI, blue in B; white in B’’) with condensed chromatin. (C) Projection image of a Pou4f3DTR utricle at 7 days post DT. Arrowheads point to F-actin-rich (green) phagosomes, while arrows point to TUNEL-labeled (magenta) DNA. Scale bar in A is 6 µm and applies to A–B”. Scale bar in C is 5 µm. (D) Total number of phagosomes per Pou4f3DTR utricle at different times post DT. See Figure 9–source data 1 for raw data. There were significantly more phagosomes at 4 and 7 days post DT compared to control littermates lacking the Pou4f3DTR allele (0 day post DT) determined by a one-way ANOVA (p<0.0001) followed by a Dunnett’s multiple comparisons test (*p<0.05: n = 3–7). Data are expressed as mean +1 standard deviation.

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, including the stereocilia. The actin spike aids the translocation of the HC to the SC nuclear layer, where SCs form an actin-rich phagosome and engulf the HC. Once the HC is removed from the sensory epithelium, an empty ring- or basket-like actin structure may remain until it is resorbed by SCs. (B,C) Model of type II HC addition. (B) Plp1-CreERT2-expressing SCs (grey) give rise to type II HCs (magenta) by first translocating their nuclei into the HC layer and becoming an intermediate cell type (dark magenta). Type II HCs that express Atoh1-CreERTM do not transdifferentiate into type I HCs. (C) Plp1-CreERT2-expressing SCs (grey) do not directly generate type I HCs (magenta) in adulthood.

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 structure with a spike, or process, that extends through the cytoplasm of the HC along its nucleus. Note the basket-like structure is not solid F-actin but has gaps that lack labeling suggestive of a lattice. All nuclei are labeled with DAPI (blue).

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 basal lamina. All nuclei are labeled with DAPI (blue), HCs are labeled with myosin VIIa antibodies (magenta), and the F-actin in phagosome basket- and ring-like structures and spikes (or processes) is labeled with phalloidin (green).

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 type I HC nuclear layers (DAPI, blue), and ends in the SC nuclear layer. A myosin VIIa labeled HC (magenta), which has a normal-appearing stereocilia bundle (below the arrow), has a F-actin spike (green, arrow) within its cytoplasm, extending the length of the HC and into the SC nuclear layer.

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 basolateral processes are located. An example of a type II HC basolateral process is shown by the arrow. Myosin VIIa antibodies were used to label all HCs (green).

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 nuclei, SC nuclei, and ends at the basal lamina. About 10 type II HCs and one type I HC (arrow) are labeled with tdTomato (red). All nuclei are labeled with DAPI (blue). At the level of the type II HC nuclei, the type I HC neck (arrow) can be seen. At the level of the SC nuclei, tdTomato-labeled type II basolateral processes are observed.

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 the utricular sensory epithelium (SE) in two strains of mice (C57Bl/6J and Swiss Webster) that were older than 6 weeks. BrdU was either injected intraperitoneally (IP) or administered per os (PO) in the drinking water. SD, one standard deviation.

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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