Sox10 conditional mutants have no Schwann cells and show different distribution of neurons.

<p>These sections are cut parallel to the facial nerve (FN) to obtain cross sections through the vestibular (Scarpa’s) ganglion and cochlear nerve between the ear and the brain. Vestibular ganglion (VG) neurons are anterior and adjacent to the cochlear nerve (CN) in control mice (A, A’). In Sox10 mutants, vestibular and spiral ganglion neurons co-mingle next to the facial nerve (B). Schwann cells with small, elongated and heterochromatin rich nuclei are readily visible between nerve fibers (arrows in C) and the developing spiral ganglion (E) in control mice. No such cells exist in the Sox10 mutants (D, F). Abbreviations: CN, cochlear nerve; FN, facial nerve; SPG; spiral ganglion; VG, vestibular ganglion. Bar indicates 100 um in A–F.</p

Conditional Sox10 mutants reveal differential migration of spiral ganglion neurons.

<p>p75^NTR<i>in situ</i> hybridization labels all sensory neurons of the ear and the inner pillar cells of the organ of Corti <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094580#pone.0094580-vonBartheld1" target="_blank">[40]</a>. Labeling both cell types with a single marker allows for measuring the distance between the spiral ganglion neurons (SPG) and the inner pillar cells (IPC) (lilac bar in A’, B’, A”, B”). Note the altered ganglion cell distribution (A, B) and the 4 times increase in distance between the SPG and the cochlear duct (green bar), but constant distance to the inner pillar cells (red bar) in these littermates. Abbreviations: BT, basal tip; VG, vestibular ganglion; U, utricle; SPG, spiral ganglion neurons; IPC, inner pillar cells. Bar indicates 100 um.</p

Development of spiral ganglion neurons as revealed with Mmp-24 <i>in situ</i> hybridization.

<p>The metalloproteinase Mmp-24 is an early marker for sensory neurons of the ear (A–D) and also labels the organ of Corti at later stages (C, D). This marker reveals that developing spiral ganglion neurons are differently distributed inside the developing cochlea from E14.5 (A, B) to E18.5 (C, D): Instead of dispersing along the cochlea (A, C) neurons lump together in the center of the cochlea (B, D). Whereas spiral ganglion neurons are in control mice at a nearly constant distance from the cochlea (C’) all spiral ganglion neurons aggregate in the center of the basal turn in the Wnt1-cre;Sox10^f/f littermates (D’). Abbreviations: OC, organ of Corti; PC, posterior crista; AC, anterior crista; HC, horizontal crista; U, utricle; S, saccule; SPG, spiral ganglion; VG, vestibular ganglion. Bar indicates 100 μm.</p

Sox10 CKO mice show unusual distribution of anti-tubulin labeled fibers.

<p>In the control mice, neurofilament and tubulin double labeled fibers (A,A’, A”) extend in radial fiber bundles to the hair cells of the organ of Corti (indicated by yellow dotted line). In contrast, in Sox10 conditional mutants radial fibers are disorganized as revealed by either anti-tubulin (B, C) or anti-neurofilament (B’, C’). Superimposing the double labeling shows nearly complete overlap of fiber staining with either antibody in control (A”) but segregation of many tubulin labeled fibers in Sox10 mutants, in particular those to the lateral wall (B”, C”). Abbreviations: IGSB, intraganglionic spiral bundle; RF, radial fibers; OC, organ of Corti. Bars indicate 100 μm.</p

Sox10 mutants have unusual innervation patterns.

<p>Immunocytochemistry of Sox10 conditional knockout mice show normal organization of organ of Corti hair cells (A, A,’B, B’). Myo7a (red) labels one row of inner hair cells (IHC) and three rows of outer hair cells (OHC; A’, B’). Short radial fibers (RF) project from spiral ganglion neurons (SPG) to the organ of Corti (lilac line in C), indicated by the Myo7a labeled hair cells (HC in C, E). In contrast, Sox10 conditional mutants have elongated radial fibers (lilac line in D) and have no intraganglionic spiral bundle (IGSB) formed by efferent axons in the displaced spiral ganglion (C, D). The regular spacing of radial bundles is disrupted in the Sox10 mutant mice and fibers overshoot the organ of Corti hair cells (HC) to end in the lateral wall (LW in E, F). Abbreviations: HC, hair cells; RF, radial fibers; IGSB, intraganglionic spiral bundle; SPG, spiral ganglion; S, saccule; LW, lateral wall. Bar indicates100 μm.</p

Immunochemistry of a coronally sectioned ear shows differences in spiral ganglion cell distribution and projection.

<p>Spiral ganglion neurons (SGN) are inside the otic capsule in the control ear (A) but have migrated to the center of the spiral and partially outside the ear in the Sox10 mutant (B, dotted line). The dotted circle in (A) indicates spiral ganglion location and is shown in (B) to indicate the position of Rosenthal’s canal. Most spiral ganglion neurons are tubulin positive and few are neurofilament positive in control (C, C’, C”) and Sox10 mutant mice (D, D’, D”). However, while all labeled fibers run together from spiral ganglion neurons to innervate hair cells in control mice (C, C’ C”), many tubulin labeled afferent fibers of Sox10 mutant mice bypass the organ of Corti (D, D’, D”). Abbreviations: OC, organ of Corti; HC, hair cells; LW, lateral wall. Bar indicates 100 μm.</p

Afferents to vestibular epithelia are reduced and disorganized.

<p>Neural crest-derived Sox10 positive cells play a guidance function in the vestibular innervation of the ear. Compared with control mice of E18.5 (A), Sox10 conditional mutants show a reduced innervation to the canal cristae(B, D) and disorganized fibers routing from one canal crista to another and occasionally to nowhere (B, D). Note the smaller size of epithelia and fiber projections in the younger Sox10 mutant (B). Abbreviations: PC, posterior crista; AC, anterior crista; HC, horizontal crista; U, utricle. Bar indicates 100 μm.</p

Sox10 conditional mutants have no Schwann cells and show different distribution of neurons.

<p>These sections are cut parallel to the facial nerve (FN) to obtain cross sections through the vestibular (Scarpa’s) ganglion and cochlear nerve between the ear and the brain. Vestibular ganglion (VG) neurons are anterior and adjacent to the cochlear nerve (CN) in control mice (A, A’). In Sox10 mutants, vestibular and spiral ganglion neurons co-mingle next to the facial nerve (B). Schwann cells with small, elongated and heterochromatin rich nuclei are readily visible between nerve fibers (arrows in C) and the developing spiral ganglion (E) in control mice. No such cells exist in the Sox10 mutants (D, F). Abbreviations: CN, cochlear nerve; FN, facial nerve; SPG; spiral ganglion; VG, vestibular ganglion. Bar indicates 100 um in A–F.</p

ErbB2 mutants with Sox10 conditional mutants have similar unusual spiral ganglion position but differ in fiber projections and apoptotic neuron ratio.

<p>Injection of lipophilic dyes into cochlear nuclei label spiral ganglion cells in the center of cochlea (A, B) and elongated radial fibers (RF). Only Sox10 mutants show a massive projection of spiral ganglion afferents to the lateral wall (insert in B). ErbB2 mutants have a severe reduction in radial fibers with very few extending into the organ of Corti (shown by PLP-eGFP) and the lateral wall (LW). In contrast, Sox10 mutants have a denser organ of Corti innervation (shown with Myo7a immunoreactivity in D) and many fibers to the lateral wall. One in 5 spiral ganglion neurons of ErbB2 mutants is apoptotic (E) and rare apoptotic profiles are also found in Sox10 mutants (F). Markers for apoptosis such as anti-activated caspase3 immunocytochemistry (G’) and PSVue (G”) confirm the occasional dying cell (G, G’”) in Sox10 conditional mutant mice. Abbreviations: LW, lateral wall; OC, organ of Corti; PLP-GFP, phospholipoprotein-green fluorescent protein; RF, radial fiber. Bar indicates 100 μm in A, B, D, 50 μm in C, E, F and 10 μm in G.</p

Conditional deletion of Sox10 does not affect the central projections of the cochlear nerve.

<p>Lipophilic-dye-soaked filter strips (NeuroVue maroon) were applied into the cochlear to label cochlear afferents to the cochlear nuclei (A) including the antero-ventral cochlear nucleus (AVCN) and dorsal cochlear nucleus (DCN). Cochlear afferents project normally in the Sox10 mutants (B). Olivocochlear efferent fibers (Eff) exit normally with the vestibular nerve root in the control and mutant mice(C, D). Abbreviations: CN, cochlear nerve. Bar indicates 100 μm.</p