Purinergic Signaling Controls Spontaneous Activity in the Auditory System throughout Early Development.

Spontaneous bursts of electrical activity in the developing auditory system arise within the cochlea before hearing onset and propagate through future sound-processing circuits of the brain to promote maturation of auditory neurons. Studies in isolated cochleae revealed that this intrinsically generated activity is initiated by ATP release from inner supporting cells (ISCs), resulting in activation of purinergic autoreceptors, K+ efflux, and subsequent depolarization of inner hair cells. However, it is unknown when this activity emerges or whether different mechanisms induce activity during distinct stages of development. Here we show that spontaneous electrical activity in mouse cochlea from both sexes emerges within ISCs during the late embryonic period, preceding the onset of spontaneous correlated activity in inner hair cells and spiral ganglion neurons, which begins at birth and follows a base to apex developmental gradient. At all developmental ages, pharmacological inhibition of P2Y1 purinergic receptors dramatically reduced spontaneous activity in these three cell types. Moreover, in vivo imaging within the inferior colliculus revealed that auditory neurons within future isofrequency zones exhibit coordinated neural activity at birth. The frequency of these discrete bursts increased progressively during the postnatal prehearing period yet remained dependent on P2RY1. Analysis of mice with disrupted cholinergic signaling in the cochlea indicate that this efferent input modulates, rather than initiates, spontaneous activity before hearing onset. Thus, the auditory system uses a consistent mechanism involving ATP release from ISCs and activation of P2RY1 autoreceptors to elicit coordinated excitation of neurons that will process similar frequencies of sound.SIGNIFICANCE STATEMENT In developing sensory systems, groups of neurons that will process information from similar sensory space exhibit highly correlated electrical activity that is critical for proper maturation and circuit refinement. Defining the period when this activity is present, the mechanisms responsible and the features of this activity are crucial for understanding how spontaneous activity influences circuit development. We show that, from birth to hearing onset, the auditory system relies on a consistent mechanism to elicit correlate firing of neurons that will process similar frequencies of sound. Targeted disruption of this activity will increase our understanding of how these early circuits mature and may provide insight into processes responsible for developmental disorders of the auditory system.

The Notch Ligand Jagged1 Is Required for the Formation, Maintenance, and Survival of Hensen's Cells in the Mouse Cochlea.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2CreERT2/+::Jag1loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1 Finally, using Fgfr3-iCreERT2 ::Jag1loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.SIGNIFICANCE STATEMENT Hensen's cells play an essential role in the development and homeostasis of the cochlea. Defects in the biophysical or functional properties of Hensen's cells have been linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly little is known about the molecular mechanisms that guide their development. Morphologic and fate-mapping analyses in our study revealed that, in the absence of the Notch ligand JAGGED1, Hensen's cells died or converted into Claudius cells, which are specialized epithelium-like cells outside the sensory epithelium. Confirming a link between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, auditory phenotyping revealed that JAGGED1's function in supporting cells is necessary for low-frequency hearing.

Generation of inner ear hair cells by direct lineage conversion of primary somatic cells.

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

Worldwide, hearing loss is the most common loss of sensation. Most cases of hearing loss are due to the death of specialized hair cells found deep inside the ear. These hair cells convert sounds into nerve impulses which can be understood by the brain. Hair cells naturally degrade as part of aging and can be damaged by other factors including loud noises, and otherwise therapeutic drugs, such as those used in chemotherapy for cancer. In humans and other mammals, once hair cells are lost they cannot be replaced. Hair cells have often been studied using mice, but the small number of hair cells in their ears, and their location deep inside the skull, makes it particularly difficult to study them in this way. Scientists are seeking ways to grow hair cells in the laboratory to make it easier to understand how they work and the factors that contribute to their damage and loss. Different cell types in the body are formed in response to specific combinations of biological signals. Currently, scientists do not have an efficient way to grow hair cells in the laboratory, because the correct signals needed to create them are not known. Menendez et al. have now identified four proteins which, when activated, convert fibroblasts, a common type of cell, into hair cells similar to those in the ear. These proteins are called Six1, Atoh1, Pou4f3 and Gfi1. Menendez et al. termed the resulting cells induced hair cells, or iHCs for short, and analyzed these cells to identify those characteristics that are similar to normal hair cells, as well as their differences. Importantly, the iHCs were found to be damaged by the same chemicals that specifically harm normal hair cells, suggesting they are useful test subjects. The ability to create hair cells in the laboratory using more easily available cells has many uses. These cells can help to understand the normal function of hair cells and how they become damaged. They can also be used to test new drugs to assess their success in preventing or reversing hearing loss. These findings may also lead to genetic solutions to curing hearing loss.

Purinergic signaling in cochlear supporting cells reduces hair cell excitability by increasing the extracellular space.

Neurons in developing sensory pathways exhibit spontaneous bursts of electrical activity that are critical for survival, maturation and circuit refinement. In the auditory system, intrinsically generated activity arises within the cochlea, but the molecular mechanisms that initiate this activity remain poorly understood. We show that burst firing of mouse inner hair cells prior to hearing onset requires P2RY1 autoreceptors expressed by inner supporting cells. P2RY1 activation triggers K+ efflux and depolarization of hair cells, as well as osmotic shrinkage of supporting cells that dramatically increased the extracellular space and speed of K+ redistribution. Pharmacological inhibition or genetic disruption of P2RY1 suppressed neuronal burst firing by reducing K+ release, but unexpectedly enhanced their tonic firing, as water resorption by supporting cells reduced the extracellular space, leading to K+ accumulation. These studies indicate that purinergic signaling in supporting cells regulates hair cell excitability by controlling the volume of the extracellular space.

As the brain develops, billions of cells respond to genetic and environmental cues to form the trillions of connections that make up its neural networks. However, before these brain circuits can respond to real life stimuli, their connections are refined by bursts of electrical activity. For example, sensory cells in the ear produce bursts of spontaneous electrical activity that mimic those made by sounds. This activity allows the neural network in the hearing system to 'practice' responding to sounds. However, the origin of these electrical bursts is unusual as they do not start in the sensory cells themselves, but are initiated by the non-sensory cells around them. Past research has shown that as the ear develops these non-sensory cells, or supporting cells, release regular doses of a molecule called ATP. The supporting cells then detect their own ATP release using specialized receptor proteins on their surface. This self-stimulation causes the supporting cells to release potassium ions that interact with the sensory cells and trigger bursts of electrical activity. However, the identity of this ATP-detecting receptor was not known, and without this information it was unclear how the electrical activity starts and why it happens in rhythmic bursts. To fill this knowledge gap, Babola et al. measured electrical activity in ear cells isolated from mice, and examined nerve cell activity in live mice during this critical stage of development. This revealed that the bursts of activity in the ear depend on a receptor called P2RY1 which can be found on the supporting cells located next to sensory cells. When P2RY1 is activated it triggers the release of calcium ions inside the supporting cells. This opens channels in the cell membrane, allowing the potassium ions to flow out and electrically activate the sensory cells. But, when the potassium ions leave the supporting cells, water is drawn out with them, causing the cells to shrink and the space around the cells to get bigger. As a result, the released potassium ions disperse more quickly, moving away from the sensory cells and stopping the burst in electrical activity. Conversely, when P2RY1 is inhibited, this causes the supporting cells to swell, trapping potassium ions near the sensory cells and making them fire continuously. This indicates that bursts in electrical activity are controlled by the rhythmic swelling and shrinking of supporting cells. Although supporting cells cannot detect sound themselves, they seem to play a crucial role in developing the hearing system. A better understanding of these cells could therefore aid research into hearing problems without a known cause such as hypersensitivity to sound, tinnitus, and complex auditory processing disorders in children.

Multiple supporting cell subtypes are capable of spontaneous hair cell regeneration in the neonatal mouse cochlea.

Supporting cells (SCs) are known to spontaneously regenerate hair cells (HCs) in the neonatal mouse cochlea, yet little is known about the relative contribution of distinct SC subtypes which differ in morphology and function. We have previously shown that HC regeneration is linked to Notch signaling, and some SC subtypes, but not others, lose expression of the Notch effector Hes5 Other work has demonstrated that Lgr5-positive SCs have an increased capacity to regenerate HCs; however, several SC subtypes express Lgr5. To further investigate the source for spontaneous HC regeneration, we used three CreER lines to fate-map distinct groups of SCs during regeneration. Fate-mapping either alone or combined with a mitotic tracer showed that pillar and Deiters' cells contributed more regenerated HCs overall. However, when normalized to the total fate-mapped population, pillar, Deiters', inner phalangeal and border cells had equal capacity to regenerate HCs, and all SC subtypes could divide after HC damage. Investigating the mechanisms that allow individual SC subtypes to regenerate HCs and the postnatal changes that occur in each group during maturation could lead to therapies for hearing loss.

Ontogenetic development of the inner ear saccule and utricle in the Lusitanian toadfish: Potential implications for auditory sensitivity.

Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.

Micro-optical coherence tomography of the mammalian cochlea.

The mammalian cochlea has historically resisted attempts at high-resolution, non-invasive imaging due to its small size, complex three-dimensional structure, and embedded location within the temporal bone. As a result, little is known about the relationship between an individual's cochlear pathology and hearing function, and otologists must rely on physiological testing and imaging methods that offer limited resolution to obtain information about the inner ear prior to performing surgery. Micro-optical coherence tomography (µOCT) is a non-invasive, low-coherence interferometric imaging technique capable of resolving cellular-level anatomic structures. To determine whether µOCT is capable of resolving mammalian intracochlear anatomy, fixed guinea pig inner ears were imaged as whole temporal bones with cochlea in situ. Anatomical structures such as the tunnel of Corti, space of Nuel, modiolus, scalae, and cell groupings were visualized, in addition to individual cell types such as neuronal fibers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed image stacks and endoscopic perspective videos, represents an improvement over previous efforts using conventional OCT. These are the first µOCT images of mammalian cochlear anatomy, and they demonstrate µOCT's potential utility as an imaging tool in otology research.

Responses to cell loss become restricted as the supporting cells in mammalian vestibular organs grow thick junctional actin bands that develop high stability.

Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti.

Atonal homolog1 (Atoh1) encodes a basic helix-loop-helix protein that is the first transcription factor to be expressed in differentiating hair cells. Previous work suggests that expression of Atoh1 in prosensory precursors is necessary for the differentiation and survival of hair cells, but it is not clear whether Atoh1 is required exclusively for these processes, or whether it regulates other functions later during hair cell maturation. We used EGFP-tagged Atoh1 knock-in mice to demonstrate for the first time that Atoh1 protein is expressed in hair cell precursors several days before the appearance of differentiated markers, but not in the broad pattern expected of a proneural gene. We conditionally deleted Atoh1 at different points in hair cell development and observe a rapid onset of hair cell defects, suggesting that the Atoh1 protein is unstable in differentiating hair cells and is necessary through an extended phase of their differentiation. Conditional deletion of Atoh1 reveals multiple functions in hair cell survival, maturation of stereociliary bundles, and auditory function. We show the presence of distinct critical periods for Atoh1 in each of these functions, suggesting that Atoh1 may be directly regulating many aspects of hair cell function. Finally, we show that the supporting cell death that accompanies loss of Atoh1 in hair cells is likely caused by the abortive trans-differentiation of supporting cells into hair cells. Together our data suggest that Atoh1 regulates multiple aspects of hair cell development and function.

Coupling the cell cycle to development and regeneration of the inner ear.

Cell cycle exit and acquirement of a postmitotic state is essential for the proper development of organs. In the present review, we examine the role of the cell cycle control in the sensory epithelia of the mammalian inner ear. We describe the roles of the core cell cycle regulators in the proliferation of prosensory cells and in the initiation and maintenance of terminal mitosis of the sensory epithelia. We also discuss how other intracellular signalling may influence the cell cycle. Finally, we address the question of whether manipulations of the cell cycle may have the potential to create replacement cells for the damaged inner sensory epithelia.

Notch signaling during cell fate determination in the inner ear.

In the inner ear, Notch signaling has been proposed to specify the sensory regions, as well as regulate the differentiation of hair cells and supporting cell within those regions. In addition, Notch plays an important role in otic neurogenesis, by determining which cells differentiate as neurons, sensory cells and non-sensory cells. Here, I review the evidence for the complex and myriad roles Notch participates in during inner ear development. A particular challenge for those studying ear development and Notch is to decipher how activation of a single pathway can lead to different outcomes within the ear, which may include changes in the intrinsic properties of the cell, Notch modulation, and potential non-canonical pathways.

Inner ear supporting cells: rethinking the silent majority.

Sensory epithelia of the inner ear contain two major cell types: hair cells and supporting cells. It has been clear for a long time that hair cells play critical roles in mechanoreception and synaptic transmission. In contrast, until recently the more abundant supporting cells were viewed as serving primarily structural and homeostatic functions. In this review, we discuss the growing information about the roles that supporting cells play in the development, function and maintenance of the inner ear, their activities in pathological states, their potential for hair cell regeneration, and the mechanisms underlying these processes.

Selective ablation of pillar and deiters' cells severely affects cochlear postnatal development and hearing in mice.

Mammalian auditory hair cells (HCs) are inserted into a well structured environment of supporting cells (SCs) and acellular matrices. It has been proposed that when HCs are irreversibly damaged by noise or ototoxic drugs, surrounding SCs seal the epithelial surface and likely extend the survival of auditory neurons. Because SCs are more resistant to damage than HCs, the effects of primary SC loss on HC survival and hearing have received little attention. We used the Cre/loxP system in mice to specifically ablate pillar cells (PCs) and Deiters' cells (DCs). In Prox1CreER(T2)+/-;Rosa26(DTA/+) (Prox1DTA) mice, Cre-estrogen receptor (CreER) expression is driven by the endogenous Prox1 promoter and, in presence of tamoxifen, removes a stop codon in the Rosa26(DTA/+) allele and induces diphtheria toxin fragment A (DTA) expression. DTA produces cell-autonomous apoptosis. Prox1DTA mice injected with tamoxifen at postnatal days 0 (P0) and P1 show significant DC and outer PC loss at P2-P4, that reaches ∼70% by 1 month. Outer HC loss follows at P14 and is almost complete at 1 month, while inner HCs remain intact. Neural innervation to the outer HCs is disrupted in Prox1DTA mice and auditory brainstem response thresholds in adults are 40-50 dB higher than in controls. The hearing deficit correlates with loss of cochlear amplification. Remarkably, in Prox1DTA mice, the auditory epithelium preserves the ability to seal the reticular lamina and spiral ganglion neuron counts are normal, a key requirement for cochlear implant success. In addition, our results show that cochlear SC pools should be appropriately replenished during HC regeneration strategies.

Structure and mechanics of supporting cells in the guinea pig organ of Corti.

The mechanical properties of the mammalian organ of Corti determine its sensitivity to sound frequency and intensity, and the structure of supporting cells changes progressively with frequency along the cochlea. From the apex (low frequency) to the base (high frequency) of the guinea pig cochlea inner pillar cells decrease in length incrementally from 75-55 µm whilst the number of axial microtubules increases from 1,300-2,100. The respective values for outer pillar cells are 120-65 µm and 1,500-3,000. This correlates with a progressive decrease in the length of the outer hair cells from >100 µm to 20 µm. Deiters'cell bodies vary from 60-50 µm long with relatively little change in microtubule number. Their phalangeal processes reflect the lengths of outer hair cells but their microtubule numbers do not change systematically. Correlations between cell length, microtubule number and cochlear location are poor below 1 kHz. Cell stiffness was estimated from direct mechanical measurements made previously from isolated inner and outer pillar cells. We estimate that between 200 Hz and 20 kHz axial stiffness, bending stiffness and buckling limits increase, respectively,~3, 6 and 4 fold for outer pillar cells, ~2, 3 and 2.5 fold for inner pillar cells and ~7, 20 and 24 fold for the phalangeal processes of Deiters'cells. There was little change in the Deiters'cell bodies for any parameter. Compensating for effective cell length the pillar cells are likely to be considerably stiffer than Deiters'cells with buckling limits 10-40 times greater. These data show a clear relationship between cell mechanics and frequency. However, measurements from single cells alone are insufficient and they must be combined with more accurate details of how the multicellular architecture influences the mechanical properties of the whole organ.

Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium.

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27(Kip1), a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27(Kip1), p27(Kip1)-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27(Kip1) gradually declined with age. In addition, deletion of Sox2 or p27(Kip1) did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27(Kip1) promoter support that Sox2 directly activates p27(Kip1) transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27(Kip1) to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27(Kip1) expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

Mature mice lacking Rbl2/p130 gene have supernumerary inner ear hair cells and supporting cells.

Adult mammalian auditory hair cells (HCs) and their associated supporting cells (SCs) do not proliferate, and HC death leads to irreversible neurosensory hearing loss and balance impairment. In nonmammalian vertebrates, loss of HCs induces mitotic proliferation of adjacent nonsensory SCs and/or direct SC transdifferentiation to generate replacement cells. This results in the structural and functional recovery of the nonmammalian sensory systems. Potential replacement of mammalian auditory HCs, either by transplanting cells or by transforming existing cells through molecular therapy, has long been proposed. However, HC replacement strategies with clear therapeutic potential remain elusive. The retinoblastoma (pRB) family of cell cycle regulators, Rb1, Rbl1 (p107), and Rbl2 (p130), regulate the G(1)- to S-phase transition in proliferating cells. In the inner ear, the biochemical and molecular pathways involving pRBs, particularly p107 and p130, are relatively unexplored and their therapeutic suitability is yet to be determined. In this study, we analyzed the cochleae of adult p130 knock-out (p130(-/-)) mice and showed that lack of the p130 gene results in extra rows of HCs and SCs in the more apical regions of the cochlea. No evidence of transdifferentiation of these supernumerary SCs into HCs was observed in the p130(-/-) mouse. Nevertheless, unscheduled proliferation of SCs in the adult p130(-/-) cochlea coupled to downregulation of bona fide cell cycle inhibitors provides a mechanistic basis for the role of p130 as a regulator of SC and HC mitotic quiescence in the more apical regions of the cochlea. Interestingly, p130(-/-) mice exhibited nearly normal peripheral auditory sensitivity.

Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.

Epithelial homeostasis is essential for sensory transduction in the auditory and vestibular organs of the inner ear, but how it is maintained during trauma is poorly understood. To examine potential repair mechanisms, we expressed ß-actin-enhanced green fluorescent protein (EGFP) in the chick inner ear and used live-cell imaging to study how sensory epithelia responded during aminoglycoside-induced hair cell trauma. We found that glial-like supporting cells used two independent mechanisms to rapidly eliminate dying hair cells. Supporting cells assembled an actin cable at the luminal surface that extended around the pericuticular junction and constricted to excise the stereocilia bundle and cuticular plate from the hair cell soma. Hair bundle excision could occur within 3 min of actin-cable formation. After bundle excision, typically with a delay of up to 2-3 h, supporting cells engulfed and phagocytosed the remaining bundle-less hair cell. Dual-channel recordings with ß-actin-EGFP and vital dyes revealed phagocytosis was concurrent with loss of hair cell integrity. We conclude that supporting cells repaired the epithelial barrier before hair cell plasmalemmal integrity was lost and that supporting cell activity was closely linked to hair cell death. Treatment with the Rho-kinase inhibitor Y-27632 did not prevent bundle excision but prolonged phagocytic engulfment and resulted in hair cell corpses accumulating within the epithelium. Our data show that supporting cells not only maintain epithelial integrity during trauma but suggest they may also be an integral part of the hair cell death process itself.

The challenge of hair cell regeneration.

Sensory hair cells of the inner ear are responsible for translating auditory or vestibular stimuli into electrical energy that can be perceived by the nervous system. Although hair cells are exquisitely mechanically sensitive, they can be easily damaged by excessive stimulation by ototoxic drugs and by the effects of aging. In mammals, auditory hair cells are never replaced, such that cumulative damage to the ear causes progressive and permanent deafness. In contrast, non-mammalian vertebrates are capable of replacing lost hair cells, which has led to efforts to understand the molecular and cellular basis of regenerative responses in different vertebrate species. In this review, we describe recent progress in understanding the limits to hair cell regeneration in mammals and discuss the obstacles that currently exist for therapeutic approaches to hair cell replacement.

Notch regulation of progenitor cell behavior in quiescent and regenerating auditory epithelium of mature birds.

Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signaling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signaling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage.

Transdifferentiation and its applicability for inner ear therapy.

During normal development, cells divide, then differentiate to adopt their individual form and function in an organism. Under most circumstances, mature cells cannot transdifferentiate, changing their fate to adopt a different form and function. Because differentiated cells cannot usually divide, the repair of injuries as well as regeneration largely depends on the activation of stem cell reserves. The mature cochlea is an exception among epithelial cell layers in that it lacks stem cells. Consequently, the sensory hair cells that receive sound information cannot be replaced, and their loss results in permanent hearing impairment. The lack of a spontaneous cell replacement mechanism in the organ of Corti, the mammalian auditory sensory epithelium, has led researchers to investigate circumstances in which transdifferentiation does occur. The hope is that this information can be used to design therapies to replace lost hair cells and restore impaired hearing in humans.