Gata3 is required for the functional maturation of inner hair cells and their innervation in the mouse cochlea.

KEY POINTS: The physiological maturation of auditory hair cells and their innervation requires precise temporal and spatial control of cell differentiation. The transcription factor gata3 is essential for the earliest stages of auditory system development and for survival and synaptogenesis in auditory sensory afferent neurons. We show that during postnatal development in the mouse inner ear gata3 is required for the biophysical maturation, growth and innervation of inner hair cells; in contrast, it is required only for the survival of outer hair cells. Loss of gata3 in inner hair cells causes progressive hearing loss and accounts for at least some of the deafness associated with the human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. The results show that gata3 is critical for later stages of mammalian auditory system development where it plays distinct, complementary roles in the coordinated maturation of sensory hair cells and their innervation. ABSTRACT: The zinc finger transcription factor gata3 regulates inner ear development from the formation of the embryonic otic placode. Throughout development, gata3 is expressed dynamically in all the major cochlear cell types. Its role in afferent formation is well established but its possible involvement in hair cell maturation remains unknown. Here, we find that in heterozygous gata3 null mice (gata3+/- ) outer hair cells (OHCs) differentiate normally but their numbers are significantly lower. In contrast, inner hair cells (IHCs) survive normally but they fail to acquire adult basolateral membrane currents, retain pre-hearing current and efferent innervation profiles and have fewer ribbon synapses. Targeted deletion of gata3 driven by otoferlin-cre recombinase (gata3fl/fl otof-cre+/- ) in IHCs does not affect OHCs or the number of IHC afferent synapses but it leads to a failure in IHC maturation comparable to that observed in gata3+/- mice. Auditory brainstem responses in gata3fl/fl otof-cre+/- mice reveal progressive hearing loss that becomes profound by 6-7 months, whilst distortion product otoacoustic emissions are no different to control animals up to this age. Our results, alongside existing data, indicate that gata3 has specific, complementary functions in different cell types during inner ear development and that its continued expression in the sensory epithelium orchestrates critical aspects of physiological development and neural connectivity. Furthermore, our work indicates that hearing loss in human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome arises from functional deficits in IHCs as well as loss of function from OHCs and both afferent and efferent neurons.

Phantom tones and suppressive masking by active nonlinear oscillation of the hair-cell bundle.

Processing of two-tone stimuli by the auditory system introduces prominent masking of one frequency component by the other as well as additional "phantom" tones that are absent in the sound input. Mechanical correlates of these psychophysical phenomena have been observed in sound-evoked mechanical vibrations of the mammalian cochlea and are thought to originate in sensory hair cells from the intrinsic nonlinearity associated with mechano-electrical transduction by ion channels. However, nonlinearity of the transducer is not sufficient to explain the rich phenomenology of two-tone interferences in hearing. Here we show that active oscillatory movements of single hair-cell bundles elicit two-tone suppression and distortions that are shaped by nonlinear amplification of periodic stimuli near the characteristic frequency of spontaneous oscillations. When both stimulus frequencies enter the bandwidth of the hair-bundle amplifier, two-tone interferences display level functions that are characteristic both of human psychoacoustics and of in vivo mechanical measurements in auditory organs. Our work distinguishes the frequency-dependent nonlinearity that emerges from the active process that drives the hair bundle into spontaneous oscillations from the passive nonlinear compliance associated with the direct gating of transduction channels by mechanical force. Numerical simulations based on a generic description of an active dynamical system poised near an oscillatory instability--a Hopf bifurcation--account quantitatively for our experimental observations. In return, we conclude that the properties of two-tone interferences in hearing betray the workings of self-sustained "critical" oscillators, which function as nonlinear amplifying elements in the inner ear.

Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses.

Otoferlin, a C2-domain-containing Ca(2+) binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otof(-/-)) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps in Otof(-/-) mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca(2+) entry, flowing through voltage-activated Ca(2+) channels, and exocytosis. This linear Ca(2+) dependence was observed when changing the Ca(2+) channel open probability or the Ca(2+) flux per channel during various test potentials. In Otof(-/-) hair cells, exocytosis displays slower kinetics, reduced Ca(2+) sensitivity, and nonlinear Ca(2+) dependence, despite morphologically normal synapses and normal Ca(2+) currents. We conclude that otoferlin is essential for a high-affinity Ca(2+) sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse.

Vestibular evoked myogenic potentials are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.

This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 +/- 1.2/4000 microm(2) in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 +/- 1.8/4000 microm(2)). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.

Sphingosine 1-phosphate (S1P) signaling is required for maintenance of hair cells mainly via activation of S1P2.

Hearing requires the transduction of vibrational forces by specialized epithelial cells in the cochlea known as hair cells. The human ear contains a finite number of terminally differentiated hair cells that, once lost by noise-induced damage or toxic insult, can never be regenerated. We report here that sphingosine 1-phosphate (S1P) signaling, mainly via activation of its cognate receptor S1P2, is required for the maintenance of vestibular and cochlear hair cells in vivo. Two S1P receptors, S1P2 and S1P3, were found to be expressed in the cochlea by reverse transcription-PCR and in situ hybridization. Mice that are null for both these receptors uniformly display progressive cochlear and vestibular defects with hair cell loss, resulting in complete deafness by 4 weeks of age and, with complete penetrance, balance defects of increasing severity. This study reveals the previously unknown role of S1P signaling in the maintenance of cochlear and vestibular integrity and suggests a means for therapeutic intervention in degenerative hearing loss.

Neurovestibular modulation of circadian and homeostatic regulation: vestibulohypothalamic connection?

Chronic exposure to increased force environments (+G) has pronounced effects on the circadian and homeostatic regulation of body temperature (T(b)), ambulatory activity (Act), heart rate, feeding, and adiposity. By using the Brn 3.1 knockout mouse, which lacks vestibular hair cells, we recently described a major role of the vestibular system in mediating some of these adaptive responses. The present study used the C57BL6JEi-het mouse strain (het), which lacks macular otoconia, to elucidate the contribution of specific vestibular receptors. In this study, eight het and eight WT mice were exposed to 2G for 8 weeks by means of chronic centrifugation. In addition, eight het and eight WT mice were maintained as 1G controls in similar conditions. Upon 2G exposure, the WT exhibited a decrease in T(b) and an attenuated T(b) circadian rhythm. Act means and rhythms also were attenuated. Body mass and food intake were significantly lower than the 1G controls. After 8 weeks, percent body fat was significantly lower in the WT mice (P < 0.0001). In contrast, the het mice did not exhibit a decrease in mean T(b) and only a slight decrease in T(b) circadian amplitude. het Act levels were attenuated similarly to the WT mice. Body mass and food intake were only slightly attenuated in the het mice, and percent body fat, after 8 weeks, was not different in the 2G het group. These results link the vestibular macular receptors with specific alterations in homeostatic and circadian regulation.

Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants.

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Time-dependent response of vestibular hair cells of guinea pigs following high-dose applications of streptomycin.

Apoptosis has been reported to occur in vestibular hair cells following aminoglycoside treatment and is suggested to play a predominant role in deletion of affected hair cells. However, the type of cell death occurring during an acute phase of vestibular damage following high-dose application of streptomycin has not yet been determined. Hence, in this study we examined the cell death mode of vestibular hair cells during the acute phase. The numbers of hair cell nuclei stained by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method and residual hair cells were quantitatively analysed. Hoechst nuclear staining was used for analysis of the nuclear morphology of affected hair cells. TUNEL staining of hair cell nuclei and lost hair cells began to appear 6 h after streptomycin treatment and increased with more exposure time. Apoptotic nuclear features could also be found from 6 h after streptomycin treatment. These findings support the thesis that apoptosis is a predominant cell death mode in degeneration of vestibular hair cells due to streptomycin ototoxicity.

Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs.

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.