Figure 5. Otoconia seeds in the wt utricle.

Formation of the organic matrix precedes the deposition of CaCO₃ crystallites. Otoconia seeds in the E16.5 wt utricle have a high level of Oc90 (arrowhead in A), a low level of otolin (arrowhead in E), an absence of or extremely low levels of CaCO₃ (D). Bones on the same sagittal section are stained (stained tibia is shown as inset in D). Oc90 is known to be expressed all over the non-macular epithelium. (A) and (D) are the same confocal view. A-D have the same magnification and are immediately adjacent sagittal sections, as do E-G. (C, G) Sections adjacent to (A) and (E), respectively, were stained with H&E. Arrowheads show otoconia seeds. (B, F) Pre-immune sera were used in the negative controls (adjacent sections) to ensure that the staining in otoconia was not due to trapped reagents (null tissues only had a few giant crystal particles that were sometimes lost during sectioning). (H) P0 whole-mount utricle stained robustly with Alizarin Red Complexone. (I, J) Whole-mount Alizarin Red Complexone staining for otoconia seeds in the E16.5 wt (I) and null utricle (J). There is no staining in the wt vestibule, but moderate to strong staining in the null tissue. The aggregated null seeds (denoted by arrows in J) increase the amount of CaCO₃ for positive staining. Arrows at the bottom right corner indicate orientation of whole-mount tissues (A, anterior; L, lateral). Ca, canal; O, otoconia seeds; UM, utricular macula.