Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability.

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

Hair cell regeneration, reinnervation, and restoration of hearing thresholds in the avian hearing organ.

Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells' bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4-5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.

Genetic correction of induced pluripotent stem cells from a DFNA36 patient results in morphologic and functional recovery of derived hair cell-like cells.

BACKGROUND: TMC1 is one of the most common deafness genes causing DFNA36. Patient-derived human induced pluripotent stem cells (iPSCs) provide an opportunity to modelling diseases. TMC1 p.M418K mutation in human is orthologous to Beethoven mice. Here, we investigated the differentiation, morphology and electrophysiological properties of hair cell-like cells (HC-like cells) derived from DFNA36 patient. METHODS: Inner ear HC-like cells were induced from iPSCs derived from DFNA36 (TMC1 p.M418K) patient (M+/-), normal control (M+/+) and genetic corrected iPSCs (M+/C). Immunofluorescence, scanning electron microscopy and whole-cell patch-clamp were used to study the mechanism and influence of TMC1 p.M418K mutation. RESULTS: In this study we successfully generated HC-like cells from iPSCs with three different genotypes. HC-like cells from M+/- showed defected morphology of microvilli and physiological properties compared to M+/+. HC-like cells from M+/C showed recovery in morphology of microvilli and physiological properties. CONCLUSIONS: Our results indicate that TMC1 p.M418K mutation didn't influence inner ear hair cell differentiation but the morphology of microvilli and electrophysiological properties and gene correction induced recovery. CRISPR/Cas9 gene therapy is feasible in human patient with TMC1 p.M418K mutation.

Transmembrane Channel-Like (Tmc) Subunits Contribute to Frequency Sensitivity in the Zebrafish Utricle.

Information about dynamic head motion is conveyed by a central "striolar" zone of vestibular hair cells and afferent neurons in the inner ear. How vestibular hair cells are tuned to transduce dynamic stimuli at the molecular level is not well understood. Here we take advantage of the differential expression pattern of tmc1, tmc2a, and tmc2b, which encode channel subunits of the mechanotransduction complex in zebrafish vestibular hair cells. To test the role of various combinations of Tmc subunits in transducing dynamic head movements, we measured reflexive eye movements induced by high-frequency stimuli in single versus double tmc mutants. We found that Tmc2a function correlates with the broadest range of frequency sensitivity, whereas Tmc2b mainly contributes to lower-frequency responses. Tmc1, which is largely excluded from the striolar zone, plays a minor role in sensing lower-frequency stimuli. Our study suggests that the Tmc subunits impart functional differences to the mechanotransduction of dynamic stimuli.Significance Statement Information about dynamic head movements is transmitted by sensory receptors, known as hair cells, in the labyrinth of the inner ear. The sensitivity of hair cells to fast or slow movements of the head differs according to cell type. Whether the mechanotransduction complex that converts mechanical stimuli into electrical signals in hair cells participates in conveying frequency information is not clear. Here we find that the transmembrane channel-like 1/2 genes, which encode a central component of the complex, are differentially expressed in the utricle and contribute to frequency sensitivity in zebrafish.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear.

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.

The sensitivity of mechanoelectrical transduction response phase to acoustic overstimulation is calcium-dependent.

The Mechanoelectrical transduction (MET) channels of the mammalian hair cells are essential for converting sound stimuli into electrical signals that enable hearing. However, the impact of acoustic overstimulation, a leading cause of hearing loss, on the MET channel function remains poorly understood. In this study, I investigated the effect of loud sound-induced temporary threshold shift (TTS) on the transduction response phase across a wide range of sound frequencies and amplitudes. The results demonstrated an increase in the transduction response phase following TTS, indicating altered transduction apparatus function. Further investigations involving the reduction of extracellular calcium, a known consequence of TTS, replicated the observed phase changes. Additionally, reduction of potassium entry confirmed the specific role of calcium in regulating the transduction response phase. These findings provide novel insights into the impact of loud sound exposure on hearing impairment at the transduction apparatus level and highlight the critical role of calcium in modulating sound transduction. Considering that over 1 billion teenagers and young adults globally are at risk of hearing loss due to unsafe music listening habits, these results could significantly enhance awareness about the damaging effects of loud sound exposure.

Repeated boat noise exposure damages inner ear sensory hair cells and decreases hearing sensitivity in Atlantic croaker (Micropogonias undulatus).

Anthropogenic noise is becoming a major underwater pollutant because of rapidly increasing boat traffic worldwide. But its impact on aquatic organisms remains largely unknown. Previous studies have focused mainly on high-frequency and impulsive noises (i.e. sonar); however, boat noise is more pervasive, continuous, and its highest intensity and component frequencies overlap the auditory bandwidth of most fishes. We assessed the impacts of boat noise on saccular sensory hair cell density and hearing thresholds of a soniferous species, Atlantic croaker (Micropogonias undulatus). In two laboratory experiments, individuals were subjected to simulated boat noise: a single 15-min exposure and 3 days of intermittent noise (simulating passing vessels). Immediately after both experiments, fish were either (1) tested for hearing sensitivity with auditory evoked potential (AEP) tests or (2) euthanized for fluorescent phalloidin and TUNEL labeling for hair cell density counts. Relative to controls, no differences were observed in auditory thresholds nor hair cell density between individuals subjected to a single 15-min noise exposure. However, fish from the 3-day experiment showed decreased sensory hair cell density, increased apoptotic cells, and higher hearing thresholds than control fish at 300, 800 and 1000 Hz. Our results demonstrate that impacts from boat noise depend upon the duration and frequency of exposure. For a species reliant on vocalization for communication, these impacts may hinder spawning success, increase predation risks and significantly alter the ecosystem.

Anion efflux mediates transduction in the hair cells of the zebrafish lateral line.

Hair cells are the principal sensory receptors of the vertebrate auditory system, where they transduce sounds through mechanically gated ion channels that permit cations to flow from the surrounding endolymph into the cells. The lateral line of zebrafish has served as a key model system for understanding hair cell physiology and development, often with the belief that these hair cells employ a similar transduction mechanism. In this study, we demonstrate that these hair cells are exposed to an unregulated external environment with cation concentrations that are too low to support transduction. Our results indicate that hair cell excitation is instead mediated by a substantially different mechanism involving the outward flow of anions. Further investigation of hair cell transduction in a diversity of sensory systems and species will likely yield deep insights into the physiology of these unique cells.

Pioneer statoacoustic neurons guide neuroblast behaviour during otic ganglion assembly.

Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development.

Differential regulation of hair cell actin cytoskeleton mediated by SRF and MRTFB.

The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various cell types. However, the specific contribution of MRTF-SRF in hair cells remains unknown. In this study, we showed that hair cell-specific deletion of Srf or Mrtfb, but not Mrtfa, leads to similar defects in the development of stereocilia dimensions and the maintenance of cuticular plate integrity. We used fluorescence-activated cell sorting-based hair cell RNA-Seq analysis to investigate the mechanistic underpinnings of the changes observed in Srf and Mrtfb mutants, respectively. Interestingly, the transcriptome analysis revealed distinct profiles of genes regulated by Srf and Mrtfb, suggesting different transcriptional regulation mechanisms of actin cytoskeleton activities mediated by Srf and Mrtfb. Exogenous delivery of calponin 2 using Adeno-associated virus transduction in Srf mutants partially rescued the impairments of stereocilia dimensions and the F-actin intensity of cuticular plate, suggesting the involvement of Cnn2, as an Srf downstream target, in regulating the hair bundle morphology and cuticular plate actin cytoskeleton organization. Our study uncovers, for the first time, the unexpected differential transcriptional regulation of actin cytoskeleton mediated by Srf and Mrtfb in hair cells, and also demonstrates the critical role of SRF-CNN2 in modulating actin dynamics of the stereocilia and cuticular plate, providing new insights into the molecular mechanism underlying hair cell development and maintenance.

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 °C, and gently suspended in culture medium using a 200 µL pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached >90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Gradients of glucose metabolism regulate morphogen signalling required for specifying tonotopic organisation in the chicken cochlea.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

ZBTB20 is essential for cochlear maturation and hearing in mice.

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral nonsensory region. Here, we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear nonsensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated, but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane (TM), a flattened spiral prominence (SP), and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the nonsensory epithelium covering the outer border Claudius cells, outer sulcus root cells, and SP epithelial cells. Transcriptome analysis shows that ZBTB20 regulates genes encoding for TM proteins in the greater epithelial ridge, and those preferentially expressed in root cells and SP epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral nonsensory domain.

Preservation of developmental spontaneous activity enables early auditory system maturation in deaf mice.

Intrinsically generated neural activity propagates through the developing auditory system to promote maturation and refinement of sound processing circuits prior to hearing onset. This early patterned activity is induced by non-sensory supporting cells in the organ of Corti, which are highly interconnected through gap junctions containing connexin 26 (Gjb2). Although loss of function mutations in Gjb2 impair cochlear development and are the most common cause of congenital deafness, it is not known if these variants disrupt spontaneous activity and the developmental trajectory of sound processing circuits in the brain. Here, we show in a new mouse model of Gjb2-mediated congenital deafness that cochlear supporting cells adjacent to inner hair cells (IHCs) unexpectedly retain intercellular coupling and the capacity to generate spontaneous activity, exhibiting only modest deficits prior to hearing onset. Supporting cells lacking Gjb2 elicited coordinated activation of IHCs, leading to coincident bursts of activity in central auditory neurons that will later process similar frequencies of sound. Despite alterations in the structure of the sensory epithelium, hair cells within the cochlea of Gjb2-deficient mice were intact and central auditory neurons could be activated within appropriate tonotopic domains by loud sounds at hearing onset, indicating that early maturation and refinement of auditory circuits was preserved. Only after cessation of spontaneous activity following hearing onset did progressive hair cell degeneration and enhanced auditory neuron excitability manifest. This preservation of cochlear spontaneous neural activity in the absence of connexin 26 may increase the effectiveness of early therapeutic interventions to restore hearing.

Reprogramming by drug-like molecules leads to regeneration of cochlear hair cell-like cells in adult mice.

Strategies to overcome irreversible cochlear hair cell (HC) damage and loss in mammals are of vital importance to hearing recovery in patients with permanent hearing loss. In mature mammalian cochlea, co-activation of Myc and Notch1 reprograms supporting cells (SC) and promotes HC regeneration. Understanding of the underlying mechanisms may aid the development of a clinically relevant approach to achieve HC regeneration in the nontransgenic mature cochlea. By single-cell RNAseq, we show that MYC/NICD "rejuvenates" the adult mouse cochlea by activating multiple pathways including Wnt and cyclase activator of cyclic AMP (cAMP), whose blockade suppresses HC-like cell regeneration despite Myc/Notch activation. We screened and identified a combination (the cocktail) of drug-like molecules composing of small molecules and small interfering RNAs to activate the pathways of Myc, Notch1, Wnt and cAMP. We show that the cocktail effectively replaces Myc and Notch1 transgenes and reprograms fully mature wild-type (WT) SCs for HC-like cells regeneration in vitro. Finally, we demonstrate the cocktail is capable of reprogramming adult cochlea for HC-like cells regeneration in WT mice with HC loss in vivo. Our study identifies a strategy by a clinically relevant approach to reprogram mature inner ear for HC-like cells regeneration, laying the foundation for hearing restoration by HC regeneration.

[Progress of research on the role of Atoh1 gene in the regeneration of mammalian auditory hair cells].

Atoh1 gene encodes a helix-loop-helix transcription factor which is involved in the generation and differentiation of mammalian auditory hair cells and supporting cells, and regulation of the proliferation of cochlear cells, therefore plays an important role in the pathogenesis and recovery of sensorineural deafness. This study reviews the progress of the Atoh1 gene in hair cell regeneration, with the aim of providing a reference for the study of hair cell regeneration gene therapy for sensorineural deafness.

RBM24 is required for mouse hair cell development through regulating pre-mRNA alternative splicing and mRNA stability.

As the sensory receptor cells in vertebrate inner ear and lateral lines, hair cells are characterized by the hair bundle that consists of one tubulin-based kinocilium and dozens of actin-based stereocilia on the apical surface of each hair cell. Hair cell development is tightly regulated, and deficits in this process usually lead to hearing loss and/or balance dysfunctions. RNA-binding motif protein 24 (RBM24) is an RNA-binding protein that is specifically expressed in the hair cells in the inner ear. Previously, we showed that RBM24 affects hair cell development in zebrafish by regulating messenger RNA (mRNA) stability. In the present work, we further investigate the role of RBM24 in hearing and balance using conditional knockout mice. Our results show that Rbm24 knockout results in severe hearing and balance deficits. Hair cell development is significantly affected in Rbm24 knockout cochlea, as the hair bundles are poorly developed and eventually degenerated. Hair bundle disorganization is also observed in Rbm24 knockout vestibular hair cells, although to a lesser extent. Consistently, significant hair cell loss is observed in the cochlea but not vestibule. RNAseq analysis identified several genes whose mRNA stability or pre-mRNA alternative splicing is affected by Rbm24 knockout. Among them are Cdh23, Pcdh15, and Myo7a, which have been shown to play important roles in stereocilia development as well as mechano-electrical transduction. Taken together, our present work suggests that RBM24 is required for mouse hair cell development through regulating pre-mRNA alternative splicing as well as mRNA stability.