Article Figures & Data
Figures
- Figure 1.
Excision lesions in embryonic utricles close rapidly. A, Scanning electron micrographs of utricles from E18 mice that received excision lesions and were fixed after the indicated time in culture. At 1 h after lesioning, there was a large open lesion exposing the underlying stromal matrix. The lesion contracted by 10 h after lesioning and was fully closed within 24 h, with the location of the lesion covered over by supporting cells with greatly expanded apical surfaces. B, Individual micrographs from a time-lapse recording of an E18 mouse utricle after lesioning. The lesion rapidly contracted and completely closed by 18 h after the lesioning. C, Closure of lesions measured from simultaneous time-lapse recordings of seven E18 lesioned mouse utricles. The lesion area is plotted every 2 h for each of the utricles, and the average of all seven utricles is plotted (thick black line). Closure of lesions took an average of 17.5 ± 1.5 h.
- Figure 2.
Closure of lesions in embryonic utricles occurs via movement and cell shape change of cells at the edge of the lesion. A, Frames from a time-lapse recording of a closing of a lesion in the utricle from an E18 transgenic mouse that expresses GFP under control of the β-actin promoter. The GFP is expressed in a mosaic pattern, allowing tracking of individual cell movements (arrows). The trajectory of the cells in the 9 and 18 h images is marked with a dashed line. Cells well outside the lesion moved little during closure of lesions, whereas cells at the leading edge of the lesion moved into the center of the lesion. B, A vector plot showing the distance and direction of movement of 59 cells whose positions were tracked over 15 h of closure of a lesion in a P0 GFP mouse utricle. The cells began at the back of the arrows and moved to the tip of the arrowheads over the 15 h, with each arrow representing the movement of a single cell in the epithelium. The dashed line indicates the initial location of the lesion. Cells at the edge of the lesion moved substantially inward, whereas cells distant from the lesion moved less. C, Histogram of distance moved from 75 cells from each of six E18 GFP mosaic utricles (tracked as in B) plotted based on the original distance from the centroid of the lesion (mean ± SEM). Cells at the edge of the lesion, between 75 and 125 μm of the centroid of the lesion, move significantly farther than cells >125 μm from the lesion (*p < 0.005). D, Fluorescent micrograph showing occludin labeling of the tight junctions between the epithelial cells in an E18 utricle fixed 24 h after lesioning. The occludin staining demarcates the lateral edge of the apical surface of the cells, revealing the expansion of supporting cells at the center of the lesion. E, Comparison of planar surface area versus distance to the center of the lesion for 627 cells in an E18 utricle, showing that cells that moved into center of the lesion site took on greatly expanded apical surface area, whereas more distant cells retained a compact shape. F, Tracings of five cells at the leading edge of the lesion of an E18 GFP mosaic utricle taken every 3 h showing their expansion as the lesion closes.
- Figure 3.
Cells at the lesion site proliferate after wound closure in embryonic mouse utricles. A, Immunolabeling of an unlesioned E19 mouse utricle cultured for 72 h. Anti-occludin shows the apical surface area of the cells, and anti-calretinin labels the hair cells. Anti-BrdU labels the nuclei of cells that have passed through S-phase during the 72 h of culturing. Few BrdU-positive cells are found within the sensory epithelium. B, Immunolabeling of an E19 mouse utricle cultured for 72 h after excision lesioning. The lesion region in the center of the epithelium shows supporting cells with expanded surface area and extensive BrdU incorporation. C, Quantification of BrdU-positive nuclei in two 30,000 μm2 regions for unlesioned and lesioned utricles: a central circular region over the lesion or center of the unlesioned utricle, and a concentric peripheral region (mean ± SEM). There are few BrdU-positive nuclei in the unlesioned utricles in either the central or peripheral regions, but there is a significant increase in the number of BrdU-positive nuclei in the central region of lesioned utricles (*p < 0.05).
- Figure 4.
Lesion closure in embryonic utricles occurs via actin purse-string contraction. A, An E18 utricle fixed 6 h after lesioning and triple labeled for phalloidin to label actin, anti-occludin to label tight junctions, and anti-calretinin to label hair cells. An actin purse string runs along the leading edge of the lesion (arrows). B, Individual frames from a time-lapse series showing closure of lesions in a utricle cultured in control media containing 0.1% DMSO. The lesion closed within 24 h. C, Frames from a time-lapse series showing lesion closure in a utricle cultured in media containing 1 μm cytochalasin B (CytoB). After 24 h, the lesion had neither rounded up nor closed. C', After washout of cytochalasin B containing media and replacement with control media, the lesion shown in C contracted and closed 13 h after washout. D, Quantification of lesion size from simultaneous time-lapse recordings of utricles cultured in DMSO control media (dashed line) or cytochalasin B (solid line). The mean ± SEM are plotted every 4 h, showing that lesion closure is inhibited by cytochalasin B but recovers after washout of cytochalasin B.
- Figure 5.
Y-27632 inhibits contraction but not formation of the actin ring. A, A control E18 utricle fixed 10 h after lesioning and labeled with phalloidin, demonstrating formation of a leading edge actin ring. B, An E18 utricle treated with 1 μm cytochalasin D and fixed 10 h after lesioning. Phalloidin labeling shows that cytochalasin blocked formation of the actin ring, leaving an irregular-shaped lesion. C, An E18 utricle treated with 50 μm Y-27632 and fixed 10 h after lesioning. Phalloidin shows that an actin ring formed and that the edge of the lesion rounded up and became smooth.
- Figure 6.
Closure of lesions in embryonic utricles requires activity of ROCK but not activity of MLCK. A, Individual frames from a time-lapse recording of closure of lesions in an E18 utricle cultured in control media. The lesion closes completely within 24 h. B, Individual frames from a time-lapse recording of closure of lesions in an E18 utricle cultured in the presence of 50 μm of the ROCK inhibitor Y-27632 for 24 h. Lesion healing is inhibited in the presence of Y-27632, although the lesion edge becomes smooth and round. Cyto D, Cytochalasin D. B', Frames from the time-lapse shown in B after washout of Y-27632 and replacement with control media. The effects of Y-27632 are completely reversible, with closure occurring within 10 h of washout. C, Quantification of closure of lesions from simultaneous time-lapse recordings from E18 utricles cultured in control media or media containing 50 μm Y-27632 for 24 h; mean ± SEM is plotted every 2 h. Although the control utricles healed normally and fully closed within 24 h, utricles cultured in Y-27632 showed little closure of lesions but healed rapidly after washout of the drug. D, Frames from a time-lapse recording of an E18 utricle cultured in 20 μm ML-7. The lesion rounds up and contracts normally, fully closing within 24 h. E, Quantification of closure of lesions from simultaneous time-lapse recordings of E18 utricles cultured in 20 μm ML-7 for 24 h; mean ± SEM are plotted every 4 h showing normal closure of the lesions.
- Figure 7.
Cells from the sensory macula of 2-week-old mouse utricles lose the capacity to close lesions, corresponding with a change in actin concentration. A, Phalloidin labeling of a P15 utricle cultured for 48 h after lesioning, demonstrating that the lesion had not closed. The sensory epithelium (SE) and the nonsensory epithelium (NSE) are labeled. B, Higher-magnification view of phalloidin labeling of actin in a P19 utricle cultured 24 h after lesioning showing that, although there was little contraction, there was a buildup of actin along the leading edge of the lesion (arrows). C, Phalloidin labeling of 2-week-old utricles cultured for 36 h after a lesion that extended out of the sensory epithelium into the nonsensory epithelium. Although the sensory epithelium had not closed in on the lesion, cells from the nonsensory epithelium migrated into the lesion (arrow). D–H, Cells in the sensory epithelium have enhanced cortical actin belts compared with cells in the nonsensory epithelium in mature utricles. D, A P0 utricle labeled with phalloidin, showing that the immature utricular epithelium had a comparable level of actin and thickness of the cortical belts between the sensory epithelium and the nonsensory epithelium. E, A P17 utricle labeled with anti-occludin shows that the concentration of occludin at the tight junction and thickness of the tight junctions are also comparable for cells in the sensory epithelium and nonsensory epithelium. F, A P17 utricle labeled with phalloidin reveals the sharp increase in labeling intensity and thickness of the cortical actin belts for the cells within the sensory epithelium compared with the cells in the nonsensory epithelium. G, Quantification of the ratio of labeling intensity for sensory epithelium compared with nonsensory epithelium for P0 phalloidin labeling, P17 occludin labeling, and P17 phalloidin labeling. H, Quantification of the junctional width for cells in the sensory epithelium and nonsensory epithelium for 10 cell junctions in the sensory epithelium and 10 cell junctions in the nonsensory epithelium per utricle for P0 phalloidin labeling (n = 5 utricles), P17 occludin labeling (n = 7 utricles), and P17 phalloidin labeling (n = 6 utricles). The width of the actin belts in the P17 sensory epithelium is significantly greater than the other junctional complexes (*p < 0.005).
- Figure 8.
Closure of lesions and reentry into the cell cycle in mature utricles is stimulated by LPA. A, Phalloidin labeling of actin in a utricle from a P15 mouse cultured for 48 h after lesioning in control media. The lesion has not closed. B, Phalloidin labeling of actin in a utricle from a P15 mouse cultured in media containing 10 μm LPA for 48 h after lesioning. LPA stimulated closure of the lesion and resulted in supporting cells with expanded apical surfaces covering over the lesion site. C, Two utricles from P16 mice, lesioned and cultured for 4 d control media with BrdU, labeled with anti-occludin (white), anti-BrdU (green), and anti-calretinin (red). Large open lesions are still visible (asterisks), although a few of the supporting cells at the lesion edge that spread have taken up BrdU. D, Two utricles from P16 mice, lesioned and cultured for 4 d in BrdU and 10 μm LPA, labeled as in C. The lesions have fully closed, and many of the spread cells have reentered the cell cycle, as indicated by BrdU labeling.
- Figure 9.
In mature utricles that have been treated with LPA, proliferation is strongly correlated with whether the cells have an expanded planar area. A, A utricle from a P16 mouse, lesioned and cultured for 7 d in 10 μm LPA, labeled with anti-occludin (white), anti-BrdU (green), and anti-calretinin (red). Many of the cells at the center of the lesion had reentered the cell cycle. B, Representative tracing of cell surface areas based on occludin labeling for a single line of cells from a P16 mouse utricle cultured as in A. Filled cells had a BrdU-positive nucleus, whereas outlined cells did not have a BrdU-positive nucleus. C, Composite plot of cell surface area versus distance along the x-axis for 303 cells from three utricles traced as in B, revealing that BrdU-positive cells were predominantly the large cells covering over the lesion (filled circles represent BrdU-positive cells, and × signs represent BrdU-negative cells). D, A histogram showing the percentage of cells that are BrdU-positive for three bins of cell area. A total of 90% cells with a surface area <100 μm2 were BrdU negative, whereas 85% cells with a surface area >300 μm2 were BrdU positive.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Video 1: Wound closure in E18 utricles. Time-lapse images were recorded simultaneously for four utricles every 30 minutes for 24 hours. The supporting cells around the lesion close over the wound and reconstitute epithelial integrity. Time is compressed 18,000x.
- supplemental material - Supplemental Video 2: Wound closure is blocked by cytochalasin B. Time-lapse images were recorded simultaneously for the three utricles cultured in 1 �M cytochalasin B and the three utricles cultured in 0.1% DMSO, one frame every 30 minutes for 24 hours. While all of the utricles cultured in DMSO have fully closed lesions within the 24 hour record, all of the cytochalasin-treated utricles have open lesions with irregular edges. Following the initial 24 hour recording, the cytochalasin-containing media was washed out and replaced with control media. Time-lapse recording was restarted on the utricles, and revealed normal wound closure in the previously blocked utricles. Time is compressed 18,000x.
- supplemental material - Supplemental Video 3: Wound closure is inhibited by Y-27632. Time-lapse images were recorded simultaneously for the three utricles cultured in 50 �M Y-27632. The lesions in the Y-27632-treated utricles become regular, smooth, and circular, but do not contract during the initial 24 hour incubation. At the end of the 24 hour recording, Y-27632 was washed out and time-lapse recording restarted. The lesions rapidly close following washout of the inhibitor. Time is compressed 18,000x.
