Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea.

The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate (PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them potential targets for regenerative therapies.

Sox2 is required for sensory organ development in the mammalian inner ear.

Sensory hair cells and their associated non-sensory supporting cells in the inner ear are fundamental for hearing and balance. They arise from a common progenitor, but little is known about the molecular events specifying this cell lineage. We recently identified two allelic mouse mutants, light coat and circling (Lcc) and yellow submarine (Ysb), that show hearing and balance impairment. Lcc/Lcc mice are completely deaf, whereas Ysb/Ysb mice are severely hearing impaired. We report here that inner ears of Lcc/Lcc mice fail to establish a prosensory domain and neither hair cells nor supporting cells differentiate, resulting in a severe inner ear malformation, whereas the sensory epithelium of Ysb/Ysb mice shows abnormal development with disorganized and fewer hair cells. These phenotypes are due to the absence (in Lcc mutants) or reduced expression (in Ysb mutants) of the transcription factor SOX2, specifically within the developing inner ear. SOX2 continues to be expressed in the inner ears of mice lacking Math1 (also known as Atoh1 and HATH1), a gene essential for hair cell differentiation, whereas Math1 expression is absent in Lcc mutants, suggesting that Sox2 acts upstream of Math1.

Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture.

UNLABELLED: The Bronx waltzer (bv) deaf mouse is characterized by massive degeneration of the primary auditory receptors, the inner hair cells, which occurs during the time of expected afferent synaptogenesis. The process is associated with degeneration and protracted division of the normally postmitotic afferent spiral ganglion neurons. To investigate the potential role of neurotrophins in the afferent synaptogenesis of inner hair cells, we exposed bv newborn cochleas in organotypic culture to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF), and also to gamma aminobutyric acid (GABA), for up to 8 days. The study was done using light and electron microscopy. Only about 20% of the inner hair cells survived in culture, regardless of the treatment, similar to the number in the intact mutant in our colony. Depending on the exogenous treatment, this population consisted of either innervated ultrastructurally normal cells or denervated dedifferentiated cells wrapped-in lieu of nerve endings-by the supporting inner phalangeal and border cells. In the control and GABA cultures, inner hair cells were mostly denervated. BDNF and NT-3 alone or combined increased synaptogenesis and hair cell survival only during the first 3 days (by about 10%); however, the cells became denervated by 8 postnatal (PN). Only NGF induced stable innervation and differentiation of neurosensory relationships, including supernumerary innervation characteristic of the intact bv. Denervation among the remaining 20% of inner hair cells induced a reactive wrapping by inner phalangeal and border cells which evidently extended inner hair cell survival. Immunocytochemical studies of these reactive supporting cells were done in the intact (8 PN) mutant cochlea. The supporting cells that provide sustenance to the denervated inner hair cells displayed strong BDNF (and possibly NT-3) immunoreactivity. Subsequently, we revealed the presence of all three neurotrophins in the inner hair cell region of the developing (1-8 PN) cochlea of the normal ICR mouse. The inner hair cells expressed all three neurotrophins; BDNF prevailed in the inner phalangeal cells, NT-3 in the pillar cells and inner phalangeal cells, and NGF in the pillar cells. IN CONCLUSION: initially, the 80% loss of inner hair cells is apparently caused by their failed afferent synaptogenesis. Exogenous neurotrophins influence synaptogenesis in the bv in culture, but NGF alone is successful in promoting stable neurosensory relationships. The presence of neurotrophins in supporting cells in the normal and degenerating cochlea indicates their role in the sustenance of inner hair cells.

Localization of efferent neurotransmitters in the inner ear of the homozygous Bronx waltzer mutant mouse.

Naturally occurring mutant mice provide an excellent model for the study of genetic malformations of the inner ear. Mice homozygous for the Bronx waltzer (bv/bv) mutation are severely hearing impaired or deaf and exhibit a 'waltzing' gait. Functional aspects of cochlear and vestibular efferents in the bv/bv mutant mouse are not well known. The present study was designed to evaluate several candidates of efferent neurotransmitters or neuromodulators including choline acetyltransferase (ChAT), gamma-aminobutyric acid (GABA), and calcitonin gene-related peptide (CGRP) in the inner ear of the bv/bv mutant mouse. Ultrastructural investigations at both light and electron microscopic level were performed. Ultrastructural morphologic evaluations of the cochlea and the vestibular end-organs were also undertaken. It is demonstrated that ChAT, GABA and CGRP immunoreactivities are present in the cochlea and in vestibular end-organs of bv/bv mutant mice. In the organ of Corti, immunoreactivity of ChAT, GABA and CGRP is confined to the inner spiral fibers, tunnel-crossing fibers, and the vesiculated nerve endings synapsing with outer hair cells. Interestingly, immunoreactivity was detectable even where inner hair cells appeared missing. Results also revealed malformations of the outer hair cells with synaptic contacts to efferent nerve endings consistently intact. In the neurosensory epithelia of the vestibular end-organs, the presence of ChAT, GABA, and CGRP immunoreactivity was localized at the vestibular efferents, with the exception of the macula of saccule. In one 8-month-old macula of utricle where the depletion of hair cells appeared highest, ChAT immunostaining was still discernible. Ultrastructural investigation demonstrated that vesiculated efferent nerve endings make synaptic contact with the outer hair cells in the organ of Corti and with type II hair cells in the vestibular end-organs. The present study provides further support that the efferent system in the bv/bv mutant inner ear is morphologically as well as functionally mature. These findings also demonstrate that if and when the onset of efferent degeneration in the bv/bv mutant inner ear occurs, it transpires subsequent to pathological conditions in the hair cells. The present findings give further indication that the efferent systems of the bv/bv mutant inner ear are independent of the afferent systems in many aspects including development, maturation as well as degeneration.

Elongation of hair cell stereocilia is defective in the mouse mutant whirler.

The recessive mouse mutant whirler (wi) shows no response to sound and exhibits circling and head-tossing behaviour, indicative of both auditory and vestibular dysfunction. The wi mutation maps genetically to mouse chromosome 4. We examined the organ of Corti of whirler mutants to explore the possibility that the wi mutation affects sensory hair cells. Scanning electron microscopy (SEM) reveals that the specialised microvilli (stereocilia) that are projected by the sensory hair cells and are vital for sound transduction are abnormal in wi homozygotes. Specifically, wi homozygous inner hair cell (IHC) stereocilia are approximately half the length of equivalent stereocilia in heterozygous littermates. They are arranged normally into ranks, but the gradation in height and width of stereocilia in adjacent ranks is less prominent in wi homozygotes. Analysis of IHC stereocilia during the course of their development shows that, by embryonic day 18.5, mutant stereocilia are already significantly shorter than those in controls. Mutant stereocilia elongate at a normal rate, at least until postnatal day 1, but prematurely stop elongating between postnatal days 1 and 4. Stereocilia length then decreases. At postnatal day 15, outer hair cell (OHC) stereocilia in wi homozygotes appear short and are arranged in a rounded, "U" shape rather than the normal "W" or "V" shape. Eventually, both IHCs and OHCs degenerate. We show that the whirler locus encodes a protein(s) required for the elongation and maintenance of IHC and OHC stereocilia.