(A–P) Reconstruction of stereocilia bundles and stereocilia rootlets from P14 Triobp^{Ex9-10/ΔEx9-10} and Triobp^Ex9-10/+ IHCs and OHCs. (A, E, I, and M) Sagittal sections showing the morphology of rootlets reconstructed from FIB-SEM data sets. (B, F, J, and N) False-colored 3D reconstructions of hair bundles (blue) and rootlets (yellow and orange) were added to better visualize rootlet structure. (C, G, K, and O) Individual rootlets highlighted in orange show typical morphology for a particular genotype. (D, H, L, and P) Reconstruction of 1 stereocilium (blue) and rootlet selected in C, G, K, and O (orange) from hair cells in B, F, J, and N. The rootlet segments within stereocilia cores of Triobp^{Ex9-10/ΔEx9-10} mice in E–H and M–P are longer and thicker compared with phenotypically wild-type controls in A–D and I–L, while Triobp^{Ex9-10/ΔEx9-10} rootlets within the cuticular plate are abnormally thin. Scale bars: 1 μm. See also Supplemental Videos 1 and 2. (Q) Quantification of the shape of stereocilia and rootlets, rootlet length (red line), and stereocilium length (blue line) and volume of rootlet segment of stereocilia core (yellow) of the tallest row stereocilia of IHCs and OHCs. (R and S) Data in box-and-whisker plots are represented as mean ± SD. Data points represent length of rootlets within the cuticular plate (red) and within the stereocilia cores (blue) of IHCs or OHCs and volume of rootlets within stereocilia cores for Triobp-5^ΔEx9-10/+ mice (black; n = 54, 17, 102, 98, 21 and 39) or Triobp-5^{ΔEx9-10/ΔEx9-10} mice (purple, n = 47, 17, 120, 96, 27, and 34) at P14. ***P < 0.001 compared with control by Mann-Whitney U test. There is a significant difference in rootlet length and rootlet volume in stereocilia cores when comparing Triobp-5^ΔEx9-10/+ and Triobp-5^{ΔEx9-10/ΔEx9-10} mice, while there is no significant difference in stereocilia length.