Dcc Mediates Functional Assembly of Peripheral Auditory Circuits

Abstract

Proper structural organization of spiral ganglion (SG) innervation is crucial for normal hearing function. However, molecular mechanisms underlying the developmental formation of this precise organization remain not well understood. Here, we report in the developing mouse cochlea that deleted in colorectal cancer (Dcc) contributes to the proper organization of spiral ganglion neurons (SGNs) within the Rosenthal's canal and of SGN projections toward both the peripheral and central auditory targets. In Dcc mutant embryos, mispositioning of SGNs occurred along the peripheral auditory pathway with misrouted afferent fibers and reduced synaptic contacts with hair cells. The central auditory pathway simultaneously exhibited similar defective phenotypes as in the periphery with abnormal exit of SGNs from the Rosenthal's canal towards central nuclei. Furthermore, the axons of SGNs ascending into the cochlear nucleus had disrupted bifurcation patterns. Thus, Dcc is necessary for establishing the proper spatial organization of SGNs and their fibers in both peripheral and central auditory pathways, through controlling axon targeting and cell migration. Our results suggest that Dcc plays an important role in the developmental formation of peripheral and central auditory circuits, and its mutation may contribute to sensorineural hearing loss.

Figure 1. Disorganized SG fibers in the absence Dcc in SGNs.

(A) Microarray analysis showing fold of difference between SGN and HC populations purified from P5-P7 mouse cochleae for selected axon guidance molecules based on the RNA-sequencing data. N = 3. Bar = SD. (B) In situ hybridization of Dcc in the whole-mount cochlea at E17.5. “oc”, organ of corti; “ger”, greater epithelial ridge; “sg”, spiral ganglion. (C) In situ hybridization of Dcc in the transverse section of E17.5 cochlea. “rm”, reissner’s membrane; “sl”, spiral ligament; “sva”, stria vascularis. (D–F) Representative images of cross-sections of wild-type cochleae at E14.5, E16.5, and E18.5, immunostained with anti-Tuj1 (green) antibody and merged with differential interference contrast (DIC) images. “ce”, cochlear epithelium; “sl”, spiral ligament; “sv”, scala vestibule; “sm”, scala media; “st”, scala timpani; “mo”, modiolus; “rc”, Rosenthal’s canal. (G–I) Cross-sectional images of Dcc^−/− cochleae, treated similarly as in (D–F). White arrows mark misrouted fibers in “sl”. Blue arrows mark misrouted fibers in mesothelial cells of “sv” or “st”. Yellow arrows indicate fibers in “mo”. Green arrow marks fibers in “sm”. (J–L) High-magnification cross-sectional views of E18.5 mouse cochlea from three Dcc^−/− animals, immunostained with anti-PV (red) and anti-Tuj1 (green) antibodies. Misrouted fibers are marked in similar manners as in (H,I).All scale bars represent 50 μm.

Figure 2. Defective peripheral innervation patterns in Dcc^−/− mice.

(A–C) Representative images of whole-mount wild-type cochleae at E14.5, E16.5, and E18.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. (D–F) Images of whole-mount Dcc^−/− cochleae, stained in similar manners as in (A–C). (G–I) Higher magnification images of cochleae shown in (D–F). Arrows point to misrouted fiber bundles originating from SG. “HC”, hair cells; “sg”, spiral ganglion. (J–O) Representative z-stack projections images of the base region of wild-type and Dcc^−/− cochleae at E18.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. Images in (K,N) are the projection of a subset of z-stack images from (J,M), showing only the HC layers, while images in (L,O) exclude stacks for HC layers. All scale bars represent 50 μm.

Figure 3. Diminished afferent synaptic connections in the Dcc mutant cochlea.

(A–F) Representative images of whole-mount wild-type and Dcc^−/− cochleae at E16.5, immunostained with anti-Tuj1 (green) antibody and labelled with DiI for efferent fiber projection patterns and their superimposed images. (G–J) Representative images of cross-sections of the mid region of E18.5 wild-type and Dcc^−/− cochleae, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies, showing SG fibers innervating HCs. Images of SG fibers (green) and HCs (red) are superimposed in (H,J). “bm”, basilar membrane; “hp”, habenula perforate. (K,L) Whole-mount images from the mid-base region of wild-type and Dcc^−/− cochleae at E18.5, immunostained with anti-CtBP2 antibody. Each white spot labels one ribbon synapse. Dotted ovals indicate individual inner hair cell (IHC) nuclei. (M) Average number of ribbon synapses per IHC at E18.5. Data are shown as mean ±  SEM. *p < 0.05, t test. N = 4 cochleae for both genotypes. Scale bar represents 50 μm in (A), 20 μm in (H), and 5 μm in (K).

Figure 4. Mispositioned SGNs in the Dcc mutant cochlea.

(A–F) Representative images of whole-mount wild-type and Dcc^−/− cochleae at E14.5, E16.5, and E18.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. White arrows point to mispositioned SGN cell bodies. (G,H) Whole-mount E16.5 wild-type and Dcc^−/− cochleae, immunostained with anti-Myo6 (red) and anti-PV (white) antibodies. White dotted curve labels the lateral boundary of SG. (I) Average number of mispositioned SGN cell bodies per cochlea between in wild-type and Dcc mutant animals at different developmental time points. Data are shown as mean ±  SEM. ***p < 0.001, t test. N = 8 embryos per group for both genotypes. (J–O) Representative images of whole-mount wild-type and Dcc^−/− cochleae at E16.5, immunostained with anti-CC3 (red) and anti-Tuj1 (green) antibodies. Superimposed images of SG fibers and CC3 positive signals are shown in (L,O). Arrows point to selected mispositioned SGN cell bodies showing CC3 labeling. Arrowhead points to selective misrouted SG fibers showing CC3 labeling. White dotted curve labels the lateral boundary of SG. All scale bars represent 50 μm.

Figure 5. Developmental changes of SGN positions in the Dcc mutant cochlea.

(A,B) Representative images of whole-mount Dcc^−/− cochlea at E16.5 and E18.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. Arrows mark SGN cell bodies located in the lesser epithelial ridge (LER). (C) Average number of mispositioned SGN cell bodies in LER and GER regions per cochlea in Dcc mutants at different time points. Data are shown as mean ±  SEM. **p < 0.01, t test. N = 8 embryos per group. (D) Representative z-stack projection image of whole-mount wild-type cochleae at E16.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. (E) 3D reconstructed 30° angled transverse z-stacked image of (D). “HC”, hair cells; “sg”, spiral ganglion; “irb”, inner radial bundle; “slc”, spiral laminal canal; V, ventral; D, dorsal; A, anterior; P, posterior; M, medial; L, lateral. (F,G) Transverse views from the 3D image in (E) at horizontal planes of the indicated mispositioned SGNs by arrows in (D,E). Arrows of the same color indicate same mispositioned SGN cell bodies shown in (D–G). All scale bars represent 50 μm.

Figure 6. Atypical exit of SGNs from the Rosenthal’s canal along the central auditory pathway in the Dcc mutant.

(A–F) Cross-sectional images of E18.5 wild-type and Dcc^−/− cochleae, immunostained with anti-PV (red) antibody. Images in middle and right panels are high-magnification views of the regions marked with white dotted squares. Arrows point to some of mispositioned SGN cell bodies. “oc”, organ of corti; “rc”, Rosenthal’s canal; “can”, central auditory nerve. (G,H) Cross-sectional views of the Rosenthal’s canal region of a wild-type and Dcc^−/− cochlea at E18.5, immunostained with anti-PV (red) and anti-Tuj1 (green) antibodies. (I) Average number of mispositioned SGN cell bodies within 200 μm distance from the Rosenthal’s canal along the central auditory nerve. Data are shown as mean ±  SEM. ***p < 0.001, t test. N = 6 embryos per group for both genotypes. All scale bars represent 30 μm.

Figure 7. Disorganization of central auditory nerves towards the cochlear nucleus in the Dcc mutant embryo.

(A–D) DiI-labeled auditory nerve fibers (red) extending from the cochlea to cochlear nucleus in sagittal sections of hindbrains of wild-type and Dcc^−/− animals at E16.5, merged with the corresponding DIC image. Images in (B,D) are high-magnification views of bifurcated nerve fibers shown in (A,C). Arrows point to scattered SGN cell bodies. Arrowhead points to aberrant axon bundles drifting away from the body of central nerve. “DCN”, dorsal cochlear nucleus; “PVCN”, posteroventral cochlear nucleus; “AVCN”, anteroventral cochlear nucleus. (E,F) Sagittally sectioned E16.5 cochlear nucleus from another wild-type and Dcc^−/− animal, with auditory nerve fibers labeled with DiI. Arrows point to scattered SGN cell bodies. (G) Average number of scattered SGN cell bodies in the proximity of auditory axon terminals within the cochlear nucleus from E14.5 to E18.5 for wild-type and Dcc^−/− animals. **p < 0.01, t test. N = 5 embryos per group for both genotypes. All scale bars represent 100 μm.

Figure 8. Normal peripheral SG organization in the Ntn1 mutant cochlea.

(A) In situ hybridization of Ntn1 in the whole-mount cochlea at E17.5. “oc”, organ of corti; “ger”, greater epithelial ridge; “sg”, spiral ganglion; “rm”, reissner’s membrane. (B) Representative images of whole-mount Ntn1^−/− cochlea at E16.5, immunostained with anti-Myo6 (red) and anti-Tuj1 (green) antibodies. (C) Higher magnification images of cochleae shown in (B). All scale bars represent 50 μm.