(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.