Sox2 Induces Neuronal Formation in the Developing Mammalian Cochlea

NeuroD1 and Neurog1 induce neuronal phenotypes in nonsensory cells within the cochlea. A, Low-magnification image of a cochlear explant transfected with NeuroD1.EGFP (green) and labeled with anti-TuJ1 (red). The sensory epithelium (SE, dashed line) and two nonsensory regions, KO and the LER, are indicated. Arrows indicate multiple transfected cells that are positive for TuJ1. B, High-magnification view of three cells located in KO that have been transfected with Neurog1.EGFP (green). Each cell has extended a neurite (arrowheads). C, The same image as in B, except with TuJ1 labeling in red. Note that each cell body and neurite is positive for TuJ1. D, E, Example of a cluster of NeuroD1.EGFP-transfected cells (green) that are TuJ1 positive (red) and have developed neuronal phenotypes including extension of neurites (D, arrowheads) and the formation of a growth cone (arrow and inset in D). F, G, Cells transfected with either Neurog1.EGFP (green in F) or NeuroD1.EGFP (green in G; NeuroD1.EGFP abbreviated as NeuroD1) were also positive for Map2 (red). H, In contrast, cells transfected with Control.EGFP (green) alone were not positive for TuJ1 (red). I, J, In addition, control transfected cells had morphologies that were consistent with epithelial cells including lumenal and basal extensions. K, NeuroD1.EGFP transfected cells (green) were negative for the hair cell marker Myo6 (red) demonstrating that NeuroD1 does not induce a hair cell fate. L, M, In contrast, cells transfected with Atoh1.EGFP (green) were consistently positive for hair cell markers such as Myo7a (red in L) but were negative for TuJ1 (red in M). Scale bars: A, 20 μm; B (for B–G), 10 μm; H, 20 μm; I (for E, J, K–M) 10 μm.

Ectopic neurons exhibit electrophysiological characteristics consistent with neurons. Whole-cell recordings from cochlear explant cultures. A, B, Voltage-clamp recording from a Neurog1.EGFP-transfected cell (A) and a non-transfected cell (B). Voltage steps of −70 mV to +10 mV were delivered for duration of 100 or 20 ms (right panel in A). C, D, Current clamp recording from a different Neurog1.EGFP-transfected cell (C) and a non-transfected cell (D) demonstrate the presence of both inward currents and an outward current. Current injections of 50–250 pA were delivered for a duration of 20 ms.

SoxB1 gene expression correlates with spiral ganglion development. A–C, Cross-sections through the otocyst from E10.5 mouse embryos demonstrating the expression of Neurog1 (green in A), NeuroD1 (green in B), and Sox2 (green in C) in delaminating neuroblasts of the developing spiral ganglion (arrow in each). Filamentous actin is labeled with Phalloidin (red) in each panel. Note that Neurog1 and Sox2 are also expressed in cells within the otocyst while expression of NeuroD1 is restricted to delaminating neuroblasts. Orientation for sections A–C is indicated to the right of panel C. D–G, Low- and high-magnification cross-sections of the cochlear duct (D, F) or spiral ganglion (E, G). Coimmunolabeling of Sox2 (green in D, E) or Sox1 (green in F, G) with the neuronal marker TuJ1 (red) demonstrates that Sox2 and Sox1 are expressed in spiral ganglion neurons (arrows in D, F) at P0. H, I, Cross-sections as in D,E demonstrating the specificity of TuJ1 as a neuronal marker. There is no overlap between cells labeled with TuJ1 (green) and the glial marker Sox10 (red) in the ganglion. Scale bars: A (for A–C), 50 μm; D (for D, F, H), 50 μm; E (for E, G, I) 20 μm.

Sox1 is neither necessary nor sufficient for spiral ganglion neuron formation. A, B, Low-magnification cross-sections through the cochlea of Sox1^+/lacZ (control) or Sox1^lacZ/lacZ (mutant) mice at P0 double-immunolabeled for ΤuJ1 (green) and Sox10 (red). Spiral ganglion neurons, with processes extending to the sensory epithelium, are present and surrounded by glia in both control and mutant cochleae. Inset, Whole-mount immunolabeling of Sox1^+/lacZ (control) (A) or Sox1^lacZ/lacZ (mutant) (B) cochlea using anti-NF-200 demonstrates no defects in the pattern of innervation in the absence of Sox1. C, Transfection of nonsensory cells with Sox1.EGFP (green) is not sufficient to induce the expression of TuJ1 (red) or development of a neuronal phenotype. Scale bars: B (for A, B), 50 μm; C, 20 μm.

Sox2 is required for formation of the spiral ganglion. A, B, Low-magnification cross-sections through the cochleae of Sox2^+/+ (control) and Sox2^Lcc/Lcc (mutant) mice at E15.5. Spiral ganglion neurons (SG) are labeled with ΤuJ1 (green) and all cell nuclei are labeled with DAPI (blue). Note the complete absence of spiral ganglion neurons, including cell nuclei (arrow) in the Sox2^Lcc/Lcc cochlea. C–F, Double-immunolabeling for TuJ1 (green) and Sox10 (red) on cross-sections of Sox2^+/+ (control) and Sox2^Lcc/Lcc (mutant) cochlea at E15.5 (C, D) and P0 (E, F) demonstrates the absence of spiral ganglion neurons and glial cells in Sox2 mutants. Arrows in D and F indicate expected position of the spiral ganglion. Scale bars: B (for A, B), 50 μm; D (for C–F), 20 μm.