The mammalian auditory sensory epithelium, the organ of Corti, comprises the hair cells and supporting cells that are pivotal for hearing function. The origin and development of their precursors are poorly understood. Here we show that loss-of-function mutations in mouse fibroblast growth factor receptor 1 (Fgfr1) cause a dose-dependent disruption of the organ of Corti. Full inactivation of Fgfr1 in the inner ear epithelium by Foxg1-Cre-mediated deletion leads to an 85% reduction in the number of auditory hair cells. The primary cause appears to be reduced precursor cell proliferation in the early cochlear duct. Thus, during development, FGFR1 is required for the generation of the precursor pool, which gives rise to the auditory sensory epithelium. Our data also suggest that FGFR1 might have a distinct later role in intercellular signaling within the differentiating auditory sensory epithelium.

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Interactions between FGF10 and the IIIb isoform of FGFR-2 appear to be crucial for the induction and growth of several organs, particularly those that involve budding morphogenesis. We determined their expression patterns in the inner ear and analyzed the inner ear phenotype of mice specifically deleted for the IIIb isoform of FGFR-2. FGF10 and FGFR-2(IIIb) mRNAs showed distinct, largely nonoverlapping expression patterns in the undifferentiated otic epithelium. Subsequently, FGF10 mRNA became confined to the presumptive cochlear and vestibular sensory epithelia and to the neuronal precursors and neurons. FGFR-2(IIIb) mRNA was expressed in the nonsensory epithelium of the otocyst that gives rise to structures such as the endolymphatic and semicircular ducts. These data suggest that in contrast to mesenchymal-epithelial-based FGF10 signaling demonstrated for other organs, the inner ear seems to depend on paracrine signals that operate within the epithelium. Expression of FGF10 mRNA partly overlapped with FGF3 mRNA in the sensory regions, suggesting that they may form parallel signaling pathways within the otic epithelium. In addition, hindbrain-derived FGF3 might regulate otocyst morphogenesis through FGFR-2(IIIb). Targeted deletion of FGFR-2(IIIb) resulted in severe dysgenesis of the cochleovestibular membraneous labyrinth, caused by a failure in morphogenesis at the otocyst stage. In addition to the nonsensory epithelium, sensory patches and the cochleovestibular ganglion remained at a rudimentary stage. Our findings provide genetic evidence that signaling by FGFR-2(IIIb) is critical for the morphological development of the inner ear.

In situ hybridization was used to study the site and timing of the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 5 (NT-5) mRNAs in the developing inner ear of the rat. In the sensory epithelia, the levels of NGF and NT-5 mRNAs were below the detection limit. NT-3 and BDNF mRNAs were expressed in the otic vesicle in overlapping but also in distinct regions. Later in development, transcripts were localized to the differentiating sensory and supporting cells of the auditory organ and vestibular maculae.In these sensory epithelia, the intensity of NT-3 mRNA expression decreased in parallel with maturation. The expression of BDNF mRNA was restricted to the sensory cells of both the auditory and vestibular organs, including ampullary cristae. In bioassays, BDNF and NT-3, but not NGF, at physiological concentrations induced neurite outgrowth from the statoacoustic ganglion explants. These results demonstrate that NT-3 and BDNF, rather than NGF and NT-5, are the primary neurotrophins present in the target fields of the cochlear and vestibular neurons. Expression of NT-3 and BDNF mRNAs in the otic vesicle before and during the ingrowth of neurites from the statoacoustic ganglion suggests that NT-3 or BDNF or both may serve as chemoattractants for the early nerve fibers. The results also suggest that these neurotrophins have a role in later development of the cochlear and vestibular neurons.Development of the vertebrate inner ear from the otic placode into cochlear and vestibular sensory organs, including their respective ganglia, is a complex process which requires the action of several factors regulating proliferation and differentiation (1). By synthesizing controlled quantities and types of neurotrophic factors, peripheral and central targets of sensory systems can selectively support appropriate numbers and kinds of neurons that innervate them (2).In avian and rat embryos, low-affinity nerve growth factor (NGF) receptor (gp75LNGFR) and its mRNA have been localized to the otic placode and otic vesicle, to nonsensory cells of the developing organ of Corti, and to developing inner ear ganglia (3-5). Binding studies have demonstrated the presence of NGF receptors in the statoacoustic ganglion (SAG) and its target, the otic vesicle (6-8). In addition, an otic conditioned medium-and NGF-induced neurite outgrowth from the SAG has been shown in vitro (9, 10). These data suggest that NGF exerts some action on the developing inner ear. However, to date the NGF family of factors, the neurotrophins, includes five members: NGF (11), brain-derived neurotrophic factor (BDNF) (12, 13), neurotrophin 3 (NT-3) (14-17), neurotrophin 4 (NT-4) (18), and neurotrophin 5 (NT-5) (19), sharing almost 60%o amino acid identity. All neurotrophins (not shown for NT-5) bind to gp75LNGFR with low-affinity kinetics (18,20).Furthermore, the members of the Trk family of proteintyrosine kinase receptors likewise bind neurotrophins with low and high affinity (21-23). Th...

We have studied the mechanisms of auditory hair cell death after insults in vitro and in vivo. We show DNA fragmentation of hair cell nuclei after ototoxic drug and intense noise trauma. By using phospho-specific c-Jun-N-terminal kinase (JNK) and c-Jun antibodies in immunohistochemistry, we show that the JNK pathway, associated with stress, injury, and apoptosis, is activated in hair cells after trauma. CEP-1347, a derivative of the indolocarbazole K252a, is a small molecule that has been shown to attenuate neurodegeneration by blocking the activation of JNK (). Subcutaneously delivered CEP-1347 attenuated noise-induced hearing loss. The protective effect was demonstrated by functional tests, which showed less hearing threshold shift in CEP-1347-treated than in nontreated guinea pigs, and by morphometric methods showing less hair cell death in CEP-1347-treated cochleas. In organotypic cochlear cultures, CEP-1347 prevented neomycin-induced hair cell death. In addition to hair cells, CEP-1347 promoted survival of dissociated cochlear neurons. These results suggest that therapeutic intervention in the JNK signaling cascade, possibly by using CEP-1347, may offer opportunities to treat inner ear injuries.

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Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell typespecific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.

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