Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation

Abstract

We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1^f/f ); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1^f/f ), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1^-/-). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei.

Keywords: Atoh1 mutation; auditory nuclei; development; ear; sensory epithelia; sensory neurons.

Figure 1

The distribution of Atoh1 LacZ is shown by ß-galactosidase staining in Atoh1 heterozygous (Atoh1^+/−; A,C) and Atoh1^−/− littermates (Atoh1^−/−; B,D) at embryonic day (E) 10.5 (A,B), E18.5. (C,D) and effects of Pax2-cre mediated deletion of Atoh1 (E,F). Between E10.5 and E18.5 the rhombic-lip shows migratory cells to the isthmus, pons, cerebellum, and cochlear nuclei in Atoh1^+/− (A,C) but not in Atoh1^−/−(B,D). Consistent with previous detailed analysis (Wang et al., ; Rose et al., 2009) there is absence of auditory nuclei in the Atoh1 null mice (C,D) leaving only the Atoh1-LacZ stain along the rhombic lip. Comparison of sections at cochlear nerve entry of control and Pax2-cre, Atoh1 f/f. shows profound reduction likely due to an unclear mix of afferent fiber loss and direct and indirect degeneration of cochlear nucleus neurons (E,F). CB, cerebellum; DCN, Dorsal cochlear nuclei; DCN, dorsal cochlear nucleus; PVCN, postero-ventral cochlear nucleus; VCN, Ventral cochlear nuclei; VIII, VIII nerve root. Bar indicates 100 μm.

Figure 2

Cochlear and Vestibular projections segregate centrally in the absence of differentiation of hair cells. At E14.5, dye injection into the cochlea and vestibular endorgans show segregation in the cochlear nucleus and vestibular nucleus in control (A) and in Atoh1-cre Atoh1^f/f mice (C) that lack differentiated hair cells. Cochlear projections remain in cochlear nuclei at E18.5 (D), but are smaller compared to control (B). Blue line in (B,D) indicates cochlear nerve diameter as an indicator of reduction spiral ganglion afferents. Cochlear afferents are colored yellow (or separately as red and green when apex and base are individually labeled, respectively), vestibular afferents are colored magenta. AVCN, Anteroventral Cochlear Nucleus; PVCN, Posteroventral Cochlear Nucleus; DCN, Dorsal Cochlear Nucleus; CN, Cochlear Nerve; VN, Vestibular Nerve; CB, Cerebellar fibers; Eff, Efferents; IX, Glossopharyngeal. Bars indicate 100 μm.

Figure 3

Inner ear projections segregate when hair cells at the periphery do not differentiate and when there is a possible delayed loss of cochlear nucleus neurons. Dye injected into the cochlea reveal that cochlear afferents remain confined to the cochlear nucleus at P7 (A). Dye injected into the base (green) and apex (red) of the cochlea reveals segregation of these afferents within the cochlear nucleus in E18.5 Pax2-cre Atoh1^f/f mice (B). Dye injected centrally into the hindbrain reveals afferent projections to vestibular endorgans and the cochlea (C). Red and Green bars indicate placement of dye for apical and basal injections, respectively, shown in (B). Note that no afferents extend to the organ of Corti in the middle turn with few afferents reaching the basal organ of Corti and expanding over the apex organ of Corti (C) as compared with controls that have afferents to all regions of the organ of Corti, including the base (inset). All epithelia are innervated despite lack of hair cell differentiation, with the most profound loss of afferents being in the saccule (S). Apical dye injection into the cochlea shows afferent and efferent labeling next to but not into the organ of Corti (D) at the approximate position indicated by the box in C. Central application of dye into vestibular nucleus/efferents in rhombomere 4 and to the cochlear nucleus/vestibular nucleus in rhombomere 5 show labeling in the facial nerve (FN), the vestibular and cochlear efferents (Eff) including efferent fibers in the commissure of van Oort (Cvo), the distinctly labeled cochlear nerve (CN, yellow) and a mix of vestibular neurons labeled by either dye application in superior and inferior vestibular ganglion (SVG, IVG) (E). Cochlear afferents are colored yellow (or separately as red and green when apex and base are individually labeled, respectively), vestibular afferents are colored magenta. AVCN, Anteroventral Cochlear Nucleus; DCN, Dorsal Cochlear Nucleus; SGN, Spiral Ganglion Neurons; RF, Radial Fibers; SVG, Superior Vestibular Ganglion; IVG, Inferior Vestibular Ganglion; IN, Intermediate nerve. Bars indicate 100 μm.

Figure 4

Central projection of inner ear afferents remains segregated even if neither hair cells nor cochlear nuclei develop. Dye injected into the cochlea and vestibular endorgans show segregation in the cochlear nucleus and vestibular nucleus in both E18.5 Atoh1 heterozygous (A) and Atoh1^−/− mice (B). Dye inserted into apex and base of the cochlea show fibers projecting to distinct medial and lateral divisions of the cochlear nuclei in both E18.5 Atoh1 heterozygous (C) and Atoh1^−/− mice (D). Cochlear afferents are colored yellow (or separately as red and green when apex and base are individually labeled, respectively), vestibular afferents are colored magenta. CN, Cochlear nucleus; VN, Vestibular nucleus. Bar indicates 100 μm.