DEV001925 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. TMRD labeling of the neurons in the apical and basal turn regions of E18 and P6 mouse cochlea. This maximal intensity projection of TMRD labeling demonstrates the efficient and specific labeling of the nerve fibers with TMRD throughout the cochlea. (A-D) TMRD labeling in the E18 cochlea. (A,C) An overview of TMRD labeling in the apical (A) and basal (C) turns of the E18 cochlea in a whole-mount preparation. (B,D) TMRD-labeled neurites in the E18 organ of Corti (o/C) exhibited a low density of type I nerve fibers innervating the outer hair cells (ohc) in the apical turn (B), but a higher density of type I fibers in the basal turn (D) with an unorganized innervation pattern. (E-H) TMRD labeling in the P6 cochlea. (E,G) An overview of TMRD labeling in the apical (E) and basal (G) turns of the P6 cochlea in a whole-mount preparation. (F) Details of TMRD-labeled neurites in the P6 organ of Corti demonstrated that in the apical turn, type I nerve fibers still formed three rows of outer spiral bundles innervating outer hair cells in addition to the innervation under inner hair cells (ihc), reflecting the later maturation of the apical cochlea. (H) In the basal turn of P6 organ of Corti, when neurite extension, refinement and retraction is complete, the TMRD-labeled nerve fibers were at maximum density under the inner hair cells (ihc), with efferent tunnel crossing fibers projecting to the outer hair cell region. SGN, spiral ganglion neurons. Scale bar: 50 μm for A,C,E,G; 10 μm for B,D,F,H.

• Supplemental Figure S2 -

  Fig. S2. Peripherin immunolabeling in the P0 and P12 rat cochlea. (A-D) As described by Hafidi et al. (Hafidi et al., 1993), strong peripherin immunoreactivity was evident on the spiral ganglion neurites beneath both the inner (ihc) and outer (ohc) hair cells and in the majority of the neurons in the P0 rat cochlea. (E-H) Transient peripherin expression in the inner spiral plexus (isp) under the inner hair cells was absent at P12, and peripherin expression was restricted to a subgroup of spiral ganglion neurons − type II spiral ganglion neurons (sgn). osb, outer spiral bundles. Scale bar: 10 μm for A,C,E,G; 25 μm for B,D,F,G. This replication of the Hafidi et al. data for peripherin expression by spiral ganglion neurons in the developing rat cochlea further validates the specificity of the peripherin immunolabeling of the mouse cochlea reported in the present study.

• Movie 1 -

  Movie 1. 3D reconstruction of afferent innervation of the mouse cochlea at post-natal day 3 (P3). This movie provides a 360° rotational view of the afferent innervation of the organ of Corti shown in Fig. 5C. This movie reveals the pathways for type I spiral ganglion neurites (TMRD-labeled, red) and type II spiral ganglion neurites (peripherin-immunolabeled, green) under the hair cells and in the osseous spiral lamina. The movie shows that single type II neurites travel diagonally, independently beneath the type I neurites in the osseous spiral lamina and remain spatially distinct within the outer spiral bundles under the outer hair cells.

• Movie 2 -

  Movie 2. 3D reconstruction of afferent innervation of the mouse cochlea at P6. This movie provides a 360° perspective of Fig. 5F. The type I spiral ganglion neurites (TMRD-labeled, red) and type II spiral ganglion neurites (peripherin-immunolabeled, green) are spatially distinguishable beneath the inner hair cells and in the osseous spiral lamina.