Stereocilium diameter.

From high-magnification transmission electron micrographs, we measured the diameter of two to three stereocilia per hair cell at 1–2 µm from their insertion point in the cuticular plate and computed the average diameter for the three stereocilia (see Fig. 4). For each time point, we measured the stereocilium diameter of type II hair cells from WT-no DT mice and DTR mice at 28, 42, 70, and 140 d post-DT. For comparison, we also measured the diameter of type I hair cell stereocilia and supporting cell microvilli from WT-no DT mice.

The stereocilia of regenerated hair cells grew over time post-DT to dimensions consistent with WT type II stereocilia. A–E, Hair bundles from a type II hair cell in a WT-no DT mouse (A) and regenerated DTR hair cells at increasing days post-DT (B–E). A, E, Arrows, cuticular plate. Scale bar: A (for A–E), 200 nm. B, Degenerating hair cell at 7 d post-DT had fused stereocilia. C, Newly regenerating hair cell at 28 d had immature stereocilia (short, thin stereocilia; no clear staircase of rows) implanted in a developing cuticular plate. D, E, Hair bundles of regenerated hair cells at 70 d (D) and 170 d (E) were longer; at 170 d, stereocilia diameter and staircase organization resembled WT-no DT bundles (A). F, Confocal micrographs of type I (left) and regenerated (right) bundles from a DTR mouse at 70 d post-DT. Phalloidin (red) labels actin in stereocilia and network of junctional complexes of supporting cells, DAPI (blue) labels cell nuclei, and myosin VIIa antibody labels hair cell cytoplasm and not supporting cells. Bar, Measured height of tallest stereocilia in a regenerated hair cell. G, Heights of tallest stereocilia were measured from TEM micrographs and/or confocal micrographs (CMs) for mature type II hair cells and type I hair cells in WT-no DT mice, versus regenerated hair cells or surviving type I hair cells from DTR mice at 70 and 170 d post-DT. Regenerated stereocilia in all conditions resembled WT-no DT type II stereocilia and were much shorter than type I stereocilia, whether WT-no DT or surviving type I stereocilia. G, H, n for group is given just above the x-axis; *p < 0.05, **p < 0.001, ***p < 0.0001. Red brackets, NS comparisons. H, Stereocilia diameter was measured with TEM for regenerated hair cells at four times post-DT and compared with WT-no DT hair cells (types I and II) and supporting cell (SC) microvilli. SC microvilli were significantly thinner than all hair cell stereocilia except regenerating stereocilia at 28 d post-DT. Relative to WT type II stereocilia, regenerated stereocilia were thinner at 28 and 42 d post-DT and were not significantly different at 70 and 140 d post-DT. WT type I stereocilia were thicker than WT type II stereocilia and all regenerated stereocilia.