Figure 5.
Extrastriolar Type II hair cells gained a full or partial calyx afferent following Sox2 deletion. A1, A3, In Sox2 WT mice at four months post-tamoxifen, Tomato-positive hair cells (magenta, arrows) were Type II-like and lacked a calyx (labeled with antibodies to Tubb3, green). By contrast, Type I hair cells (arrowheads), which were Tomato-negative, had a calyx. A2, A4, Same fields as A1, A3 but just the Tubb3 channel. B1, B3, In Sox2 CKO mice at four months post-tamoxifen, some Tomato-positive hair cells (magenta, arrows) were Type I-like, with most of their cell body enwrapped by a calyx. B2, B4, Same fields as B1, B3 but just the Tubb3 channel. C, Depiction of the appearance of cells in Sox2 WT mice, with dimorphic afferent nerve (AffD, green) surrounding a Type I hair cell as a calyx and contacting a Tomato-positive Type II hair cell as bouton terminals. D, Graph shows the number of extrastriolar Tomato-labeled hair cells per utricle (mean ± SD) with a calyx in Sox2 WT and CKO mice at different times post-tamoxifen. Each point is the average number for each mouse in the group. N = 3 mice for all groups; **p < 0.01, ***p < 0.001. E, Image from a Sox2 CKO mouse at four months post-tamoxifen showing a Tomato-positive cell with a partial calyx (arrow). xy, top-down view, while xz and yz are slices of the same field in each plane. F, Drawing of Tomato-positive hair cell partially enveloped by calyx nerve afferent (green), similar to what is shown for a Sox2 CKO mouse in panel E. G–O, TEM images of cells from the extrastriolar region. All afferent nerve terminals are pseudo-colored in blue. G–I, Exemplary Type I and Type II hair cells from Sox2 WT mice. Calyx afferents (c) fully enwrap the Type I hair cell body, up to the neck (arrows in I). Bouton afferents (b and arrowheads in G) only contact Type II hair cells. J, K, N, O, Examples of presumed converting Type II hair cells (II-c) with a partial calyx in Sox2 CKO mice at one or nine months (m) post-tamoxifen. Arrowheads point to places where the calyx is lacking; in one case (N), the hair cell cytoplasm bulges between the two pieces of calyx material. L, M, Examples of presumed Type I hair cell precursors (I-prec) with a partial calyx in developing mouse utricles at P18 (L) and P14 (M). Arrowheads point to places where the calyx is lacking. I, Type I hair cell; II, Type II hair cell; sc, supporting cell; bm, basal membrane; bp, basolateral process. Scale bar in A1: 6 µm (A1–B4); in E: 6 µm; in G: 5 µm (G, I, J, L, M, O) and 1 µm (N); and in H: 3 µm (H, K).