NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development - PubMed (original) (raw)

NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development

W Y Kim et al. Development. 2001 Feb.

Abstract

A key factor in the genetically programmed development of the nervous system is the death of massive numbers of neurons. Therefore, genetic mechanisms governing cell survival are of fundamental importance to developmental neuroscience. We report that inner ear sensory neurons are dependent on a basic helix-loop-helix transcription factor called NeuroD for survival during differentiation. Mice lacking NeuroD protein exhibit no auditory evoked potentials, reflecting a profound deafness. DiI fiber staining, immunostaining and cell death assays reveal that the deafness is due to the failure of inner ear sensory neuron survival during development. The affected inner ear sensory neurons fail to express neurotrophin receptors, TrkB and TrkC, suggesting that the ability of NeuroD to support neuronal survival may be directly mediated through regulation of responsiveness to the neurotrophins.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Surface mapping the AEP and SEP from ND−/−Tg mouse and sibling controls. (A) Placement of the 64 channel epipial recording array (dots) covered a 3.5 × 3.5 mm area of the exposed right hemisphere. This placement included primary auditory cortex (A1; black region), as well as the vibrissal representation of primary and secondary somatosensory cortex (S1 and S2, respectively), the approximate borders of which were adapted from Wallace (Wallace, 1987). (B) Enlargements of the AEP from and electrode over auditory cortex (C and E; outlined traces) in ND−/−Tg mouse (thick trace) and controls (thin traces). The AEP was composed of a biphasic positive/negative fast wave labeled P1/N1 to indicate the polarity and sequence of occurrence. (C) Superimposed AEP from two siblings had a similar amplitude, morphology and spatial distribution, centered over auditory cortex in the caudolateral region of the array. (D) The SEP complex in the controls was more widespread, covering both somatosensory cortex in the caudolateral electrodes of the array, as well as motor cortex more medially. (E) In ND−/−Tg mouse, the AEP complex was absent. (F) The SEP complex in ND−/−Tg mouse was comparable in amplitude and spatial distribution to the controls.

Fig. 2

Fig. 2

NeuroD is expressed in developing inner ear and is required for proper development of the inner ear sensory neurons. (A) In situ hybridization of E10.5 wild-type mouse embryo section using antisense NeuroD RNA as a probe and alkaline phosphatase reaction for visualization. (B) E10.5 NeuroD+/− mouse embryo section stained with X-gal. (C,D) Inner ear dissected from newborn (P0) NeuroD+/− (C) and _NeuroD_−/− mice (D), and stained with X-gal as whole mount. Anterior vertical canal (AVC), horizontal canal (HC), posterior vertical canal (PVC), saccule (S), utricle (U), vestibular ganglion (vgl), geniculate ganglion (ggl), spiral ganglion (sgl), modiolus (M) and cochlear hair cells (CHC) are indicated. In _NeuroD_−/− inner ear, the spiral ganglia (sgl) staining is absent and that of vestibular ganglia (vgl) is reduced and displaced compared with the control. (E,F) The apical and middle turn of the P0 cochleae are dissected and mounted after whole-mount staining with X-gal. The absence of spiral ganglia staining in _NeuroD_−/− inner ear is evident. Hair cells expressing lacZ are present in the _NeuroD_−/− cochleae. (G,H) Part of vestibular organ containing the vestibular ganglia, utricle and saccule is dissected out and mounted after whole-mount staining with X-gal to reveal the reduction in the size of the vestibular ganglia in _NeuroD_−/− mice. Scale bars: 100 μm.

Fig. 3

Fig. 3

Innervation defects of the spiral ganglia in the _NeuroD_−/− mice revealed by DiI labeling. DiI labeling of the afferent nerve fibers (A–D) and efferent nerve fibers (E,F) innervating cochlear sensory epithelium at P0. The spiral ganglion tightly innervates the cochlear hair cells in sibling control animals (A). (B) In _NeuroD_−/− cochleae, only a few afferent fibers are present in the middle turn of the cochleae. (C,D) The higher magnification images showing the innervation to the inner and outer hair cells (ihc and ohc, respectively). (E) At P0, efferent fibers of sibling control animal reach the inner hair cells of the cochleae through out the entire epithelium. (F) In contrast, efferent fibers in the inner ear of _NeuroD_−/− mice are restricted to few fibers to the middle turn of the cochleae. _NeuroD_−/− efferent fibers fail to branch on their way from the modiolus to the cochlea. Scale bars: 100 μm.

Fig. 4

Fig. 4

The pattern of innervation and of _lacZ_-expressing hair cells (HC: blue in A–F) in the P0 vestibular epithelia. (A–F) Dense innervation of all sensory epithelia in the NeuroD+/− animals (A,C,E) and the reduced density and partial absence of nerve fibers to parts of the sensory epithelium in _NeuroD_−/− mice (B,D,F) are shown in black. Also evident is disorganized fiber projection towards the sensory epithelia, as indicated by acetylated tubulin staining of the fibers. (G,H) DiI labeling of the afferent fibers to the PVC in control sibling (G) and _NeuroD_−/− (H) ears at P0 shows significant reduction in innervation into the PVC in _NeuroD_−/− mice. (I,J) DiI-labeling of the extending fibers at E11.5 indicates that fiber projection fails to initiate towards the PVC in _NeuroD_−/− mice. Scale bar in G, 100 μm for G,H; in I, 100 μm for I,J.

Fig. 5

Fig. 5

Cell loss in the developing vestibulo-cochlear ganglia (vcg) of _NeuroD_−/− mice. (A) Cell counting was performed in the vcg at E10.5 and E11.5. By E11.5, a significant reduction in vcg cell number is evident in _NeuroD_−/− embryos. Error bars indicate standard deviations. (B,C) Cell death as detected by a TUNEL assay in the vcg at E11.5. Green indicates the fluorescein-labeled TUNEL-positive cells. Dark blue indicates nuclear staining by DAPI. Red indicates anti-β-tubulin staining (TUJ1), as visualized with rhodamine-conjugated secondary antibody. The TUNEL-positive profiles near the geniculate ganglion layer (ggl) are present in both the control (B) and _NeuroD_−/− (C) inner ears. The vcg of _NeuroD_−/− mice display an increased number of TUNEL-positive cells. (D,E) The spiral ganglia of E13.5 NeuroD+/− and _NeuroD_−/− embryos are stained using the TUJ1 antibody and visualized by DAB (3,3′ diaminobenzidene). The spiral ganglia (sgl), cochlear ducts (cd) and great petrosal nerve (gpn) are indicated. Scale bars:100 μm.

Fig. 6

Fig. 6

Expression analysis of the neurotrophin receptors, TrkB and TrkC, and their respective ligands, BDNF and NT3, in E10.5 inner ear. (A–D) Immunohistochemistry was performed on E10.5 vcg of control sibling (A,C) and _NeuroD_−/− (B,D) mice with anti-TrkC (A,B) and anti-TrkB (C,D) antibodies followed by visualization using a DAB. The arrowheads in D indicate a small number of TrkB-positive cells remaining in the _NeuroD_−/− vcg. The insets show unaltered TrkB/C staining in the trigeminal ganglion (tgg) from the same embryos. (E–H) In situ hybridization using antisense NT3 (E,F) and antisense BDNF (G,H) reveals that both ligands are expressed in the E10.5 inner ear of _NeuroD_−/− mice (F,H). Scale bar: 100 μm.

Fig. 7

Fig. 7

The inner ear hair cells develop and are maintained in the absence of innervation in _NeuroD_−/− mice. (A) Normally organized cochlear hair cells in _NeuroD_−/− mice are shown by X-gal staining. Many of the inner hair cells (IHC) and less of the outer hair cells (OHC) express various levels of lacZ. (B) Cochlear hair cells develop normally, even in 9-month-old animals in which the apical turn never received any innervation. (C–F) Electron micrographs of the posterior vertical canal (PVC) of a 9-month-old control (C) and _NeuroD_−/− mouse (D–F) show the absence of large calyxes, afferent innervation shown as empty space, around the Type I hair cells in _NeuroD_−/− mouse. The smaller size of the sensory epithelium and the absence of nerve fibers underneath the sensory epithelium are also evident. Despite the complete lack of innervation throughout embryonic and adult stages, hair cells are rather normal (D–F). Closer examination shows no nerve endings inside the sensory epithelium (D) but the presence of two types of hair cells (F): one type with stereocilia larger than kinocilia (asterisks), and the other with stereocilia the same diameter as kinocilia (# and circles). The Type I (I) and Type II (II) hair cells are marked in E. Note that the nuclei of the Type I cells are positioned deeper from the surface. In addition, the cuticular plate underneath the cilia coming out at the apex is thicker in the type I hair cells. The presence of two different types of hair cells suggests that development of the hair cells is independent of innervation. Scale bars: 100μm in A; 10 μm in B–E; 1 μm in F.

Similar articles

Cited by

References

    1. Barth DS, Di S. The functional anatomy of auditory evoked potentials in rat neocortex. Brain Res. 1991;565:109–115. - PubMed
    1. Barth DS, Kithas J, Di S. Anatomic organization of evoked potentials in rat parietotemporal cortex: Somatosensory and auditory responses. J Neurophysiol. 1993;69:1837–1849. - PubMed
    1. Bermingham NA, Hassan BA, Pricem SD, Vollrath MA, Ben-Arie N, Eatock RA, Bellen HJ, Lysakowski A, Zoghbi HY. Math1: an essential gene for the generation of inner ear hair cells. Science. 1999;284:1837–1841. - PubMed
    1. Bianchi LM, Conover JC, Fritzsch B, DeChiara T, Lindsay RM, Yancopoulos GD. Degeneration of vestibular neurons in late embryogenesis of both heterozygous and homozygous BDNF null mutant mice. Development. 1996;122:1965–1973. - PubMed
    1. Cowan CA, Yokoyama N, Bianchi LM, Henkemeyer M, Fritzsch B. EphB2 guides axons at the midline and is necessary for normal vestibular function. Neuron. 2000;26:417–430. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources