Fig. 2: Failure of cerebellar afferent neurons to reach Emx2 KO maculae.

a–b”, c–d” Neuronal tracing with NeuroVue® Red (magenta)- and NeuroVue® Maroon (green)-soaked filter papers inserted into the respective brainstem and cerebellum of hemi-sectioned heads of control (a–b”) and Emx2 KO (c–d”) at E16.5. b, d are higher magnifications of the maximum-intensity projection images of a, c. b’, b”, d’, d” are z-stack images of areas indicated with yellow dotted lines in b, d, respectively. In control, dye traced from the cerebellum (green) is segregated to the lateral region of utricle (b’, b”, white dotted bracket), whereas tracing from the brainstem is largely restricted to the medial utricle (a, n = 5). Dyes from both cerebellum and brainstem reached the sensory epithelium with some appeared vacuolated, which are likely due to surrounding bodies of HCs (b’, b”, yellow arrowheads, see insets). In Emx2 KO utricles (c, n = 7), dye traced from the cerebellum (green) is much reduced and failed to reach the lateral utricle, whereas this region is filled with dye traced from the brainstem (magenta), some of which show a vacuolated appearance (d’, yellow arrowhead, inset), similar to controls (b’, b”). White arrowheads in a, c point to dye from the cerebellum reaching the anterior crista in both control and Emx2 KO ears. e, f Schematic summary of hair bundle orientation and afferent neuronal innervation pattern in control and Emx2 KO utricles. Anterior crista (ac), lateral crista (lc), and utricle (ut). Scale bar = 100 μm in a–d.