FIGURE 2.

The elimination of Neurod1 results in the rudimental spiral ganglion, disorganized sensory epithelium, and the shortened cochlea. (A–D) Representative images of whole-mount immunolabeling of the cochlear base and apex with anti-MyosinVIIa (MyoVIIa, a marker of hair cells) and anti-α-tubulin (nerve fibers) at E18.5. Arrowheads indicate the apical end. (A’–D’) Higher-magnification images show a dense network of radial fibers, three rows of outer hair cells (OHC), and one row of inner hair cells (IHC), forming the organ of Corti in the control cochlea. Note the formation of parallel outer spiral bundles turned toward the base in the control cochlea (arrow in A’). In Neurod1CKO, radial fibers are severely reduced and disorganized, turning randomly towards the base or apex (arrows in B’). The increased number of OHC rows in the apex indicates the disorganized sensory epithelium of the Neurod1CKO cochlea (D’). (E–H) The cochlea’s whole-mounted basal and apical half immunolabeled with anti-NeuN (a nuclear marker of differentiated neurons) and anti-tubulin (nerve fibers) shows NeuN⁺ neurons forming spiral ganglion in control at P0. In contrast, only a small cluster of NeuN⁺ neurons is found in the Neurod1CKO cochlea (arrow in F). Note the reduced size of the cochlea and massive reduction of innervation. (E’, F’) Higher-magnification images of the base show the aberrant distribution of NeuN⁺ neurons entangled with radial fibers in Neurod1CKO compared to the spiral ganglion neurons restricted to the Rosenthal’s canal in the control cochlea. (K) The length of the organ of Corti is significantly shorter in Neurod1CKO compared to control littermates. Error bars represent mean ± SD; unpaired t-test, ****p ≤ 0.0001 (n ≥ 5/genotype). Scale bars: 100 μm (A–H), 25 μm (A’–D’), 50 μm (E’,F’). HS, Hoechst nuclear staining.