Hair Bundle Research Articles

Emx2 regulates hair cell rearrangement but not positional identity within neuromasts.

Each hair cell (HC) precursor of zebrafish neuromasts divides to form two daughter HCs of opposite hair bundle orientations. Previously, we showed that transcription factor Emx2, expressed in only one of the daughter HCs, generates this bidirectional HC pattern (Jiang et al., 2017). Here, we asked whether Emx2 mediates this effect by changing location of hair bundle establishment or positions of HCs since daughter HCs are known to switch positions with each other. We showed this HC rearrangement, redefined as two processes named Rock and Roll, is required for positional acquisition of HCs. Apical protrusion formation of nascent HCs and planar polarity signaling are both important for the Rock and Roll. Emx2 facilitates Rock and Roll by delaying apical protrusion of its nascent HCs but it does not determine HCs' ultimate positions, indicating that Emx2 mediates bidirectional HC pattern by changing the location where hair bundle is established in HCs.

Recent advances in cochlear hair cell nanophysiology: subcellular compartmentalization of electrical signaling in compact sensory cells.

In recent years, genetics, physiology, and structural biology have advanced into the molecular details of the sensory physiology of auditory hair cells. Inner hair cells (IHCs) and outer hair cells (OHCs) mediate two key functions: active amplification and non-linear compression of cochlear vibrations by OHCs and sound encoding by IHCs at their afferent synapses with the spiral ganglion neurons. OHCs and IHCs share some molecular physiology, e.g. mechanotransduction at the apical hair bundles, ribbon-type presynaptic active zones, and ionic conductances in the basolateral membrane. Unique features enabling their specific function include prestin-based electromotility of OHCs and indefatigable transmitter release at the highest known rates by ribbon-type IHC active zones. Despite their compact morphology, the molecular machineries that either generate electrical signals or are driven by these signals are essentially all segregated into local subcellular structures. This review provides a brief account on recent insights into the molecular physiology of cochlear hair cells with a specific focus on organization into membrane domains.

Rbm24a Is Necessary for Hair Cell Development Through Regulating mRNA Stability in Zebrafish.

Hair cells in the inner ear and lateral lines are mechanosensitive receptor cells whose development and function are tightly regulated. Several transcription factors as well as splicing factors have been identified to play important roles in hair cell development, whereas the role of RNA stability in this process is poorly understood. In the present work, we report that RNA-binding motif protein 24a (Rbm24a) is indispensable for hair cell development in zebrafish. Rbm24a expression is detected in the inner ear as well as lateral line neuromasts. Albeit rbm24a deficient zebrafish do not survive beyond 9 days post fertilization (dpf) due to effects outside of the inner ear, rbm24a deficiency does not affect the early development of inner ear except for delayed otolith formation and semicircular canal fusion. However, hair cell development is severely affected and hair bundle is disorganized in rbm24a mutants. As a result, the auditory and vestibular function of rbm24a mutants are compromised. RNAseq analyses identified several Rbm24a-target mRNAs that are directly bound by Rbm24a and are dysregulated in rbm24a mutants. Among the identified Rbm24a-target genes, lrrc23, dfna5b, and smpx are particularly interesting as their dysregulation might contribute to the inner ear phenotypes in rbm24a mutants. In conclusion, our data suggest that Rbm24a affects hair cell development in zebrafish through regulating mRNA stability.

Mechanical Frequency Tuning by Sensory Hair Cells, the Receptors and Amplifiers of the Inner Ear

We recognize sounds by analyzing their frequency content. Different frequency components evoke distinct mechanical waves that each travel within the hearing organ, or cochlea, to a frequency-specific place. These signals are detected by hair cells, the ear's sensory receptors, in response to vibrations of mechanically sensitive antennas termed hair bundles. An active process enhances the sensitivity, sharpens the frequency tuning, and broadens the dynamic range of hair cells through several mechanisms, including active hair-bundle motility. A dynamic interplay between negative stiffness mediated by ion channels’ gating forces and delayed force feedback owing to myosin motors and channel reclosure by calcium ions brings the hair bundle to the vicinity of an oscillatory instability—a Hopf bifurcation. Operation near a Hopf bifurcation provides nonlinear generic features that are characteristic of hearing. Multiple gradients at molecular, cellular, and supercellular scales tune hair cells to characteristic frequencies that cover our auditory range.

Hydrodynamic Elastocapillary Morphing of Hair Bundles.

We report polymorphic self-assembly of hair arranged in hollow bundles driven by capillarity, hydrodynamics, and elasticity. Bundles emerging from a liquid bath shrink but remain hollow at slow drainage due to the negative pressure of the menisci trapped between the hairs. The timescale allows the collective stiffening of the fibers to resist closure. At fast drainage, the bundles fully close before the liquid can drain through the hair. A liquid column trapped in the hole closes the bundle while the lubricated hairs still behave softly. Scaling laws predict this reversible hair polymorphism.

Dynamics of Mechanically Coupled Hair-Cell Bundles of the Inner Ear

The high sensitivity and effective frequency discrimination of sound detection performed by the auditory system rely on the dynamics of a system of hair cells. In the inner ear, these acoustic receptors are primarily attached to an overlying structure that provides mechanical coupling between the hair bundles. Although the dynamics of individual hair bundles has been extensively investigated, the influence of mechanical coupling on the motility of the system of bundles remains underdetermined. We developed a technique of mechanically coupling two active hair bundles, enabling us to probe the dynamics of the coupled system experimentally. We demonstrated that the coupling could enhance the coherence of hair bundles’ spontaneous oscillation, as well as their phase-locked response to sinusoidal stimuli, at the calcium concentration in the surrounding fluid near the physiological level. The empirical data were consistent with numerical results from a model of two coupled nonisochronous oscillators, each displaying a supercritical Hopf bifurcation. The model revealed that a weak coupling can poise the system of unstable oscillators closer to the bifurcation by a shift in the critical point. In addition, the dynamics of strongly coupled oscillators far from criticality suggested that individual hair bundles may be regarded as nonisochronous oscillators. An optimal degree of nonisochronicity was required for the observed tuning behavior in the coherence of autonomous motion of the coupled system.

Loss of Baiap2l2 destabilizes the transducing stereocilia of cochlear hair cells and leads to deafness.

Key points Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded ‘staircase’ structure.The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin‐binding proteins, many of which are still unidentified.We identify a new stereociliary protein, the I‐BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs).We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age.We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8.We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin‐based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase‐like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I‐BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post‐hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The "handshake" that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca2+ chelators. Using hair bundles from the rat's cochlea and the bullfrog's sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca2+ chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca2+ chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca2+ concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.

Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding

Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.

The Anatomical and Physiological Basis of Clinical Tests of Otolith Function. A Tribute to Yoshio Uchino.

Otolithic receptors are stimulated by gravitoinertial force (GIF) acting on the otoconia resulting in deflections of the hair bundles of otolithic receptor hair cells. The GIF is the sum of gravitational force and the inertial force due to linear acceleration. The usual clinical and experimental tests of otolith function have used GIFs (roll tilts re gravity or linear accelerations) as test stimuli. However, the opposite polarization of receptors across each otolithic macula is puzzling since a GIF directed across the otolith macula will excite receptors on one side of the line of polarity reversal (LPR at the striola) and simultaneously act to silence receptors on the opposite side of the LPR. It would seem the two neural signals from the one otolith macula should cancel. In fact, Uchino showed that instead of canceling, the simultaneous stimulation of the oppositely polarized hair cells enhances the otolithic response to GIF—both in the saccular macula and the utricular macula. For the utricular system there is also commissural inhibitory interaction between the utricular maculae in each ear. The results are that the one GIF stimulus will cause direct excitation of utricular receptors in the activated sector in one ear as well as indirect excitation resulting from the disfacilitation of utricular receptors in the corresponding sector on the opposite labyrinth. There are effectively two complementary parallel otolithic afferent systems—the sustained system concerned with signaling low frequency GIF stimuli such as roll head tilts and the transient system which is activated by sound and vibration. Clinical tests of the sustained otolith system—such as ocular counterrolling to roll-tilt or tests using linear translation—do not show unilateral otolithic loss reliably, whereas tests of transient otolith function [vestibular evoked myogenic potentials (VEMPs) to brief sound and vibration stimuli] do show unilateral otolithic loss. The opposing sectors of the maculae also explain the results of galvanic vestibular stimulation (GVS) where bilateral mastoid galvanic stimulation causes ocular torsion position similar to the otolithic response to GIF. However, GVS stimulates canal afferents as well as otolithic afferents so the eye movement response is complex.

Interaction of protocadherin-15 with the scaffold protein whirlin supports its anchoring of hair-bundle lateral links in cochlear hair cells

The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.

Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2.

Directional sensitivity of hair cells (HCs) is conferred by the aymmetric apical hair bundle, comprised of a kinocilium and stereocilia staircase. The mother centriole (MC) forms the base of the kinocilium and the stereocilia develop adjacent to it. Previously, we showed that transcription factor Emx2 reverses hair bundle orientation and its expression in the mouse vestibular utricle is restricted, resulting in two regions of opposite bundle orientation (Jiang et al., 2017). Here, we investigated establishment of opposite bundle orientation in embryonic utricles by live-imaging GFP-labeled centrioles in HCs. The daughter centriole invariably migrated ahead of the MC from the center to their respective peripheral locations in HCs. Comparing HCs between utricular regions, centriole trajectories were similar but they migrated toward opposite directions, suggesting that Emx2 pre-patterned HCs prior to centriole migration. Ectopic Emx2, however, reversed centriole trajectory within hours during a critical time-window when centriole trajectory was responsive to Emx2.

Exposure to colistin impairs skin keratinocytes and lateral-line hair cells in zebrafish embryos

Environmental contamination by antibiotics has become a global issue. Colistin, a cationic antimicrobial polypeptide, has been widely used in human/veterinary medicine, and growth promotion in aquaculture. However, no study has been conducted to test the toxic effects of colistin on aquatic animals. In this study, we examined the effects of colistin on zebrafish embryos. Zebrafish embryos were incubated in different concentrations (0, 0.01, 0.1, 1, 2, 3, and 10 μM) of colistin for 96 h. Colistin increased the mortality rate in a dose-dependent manner (LC50 was 3.0 μM or 3.5 mg L−1), but it did not change the hatching rate, heart rate, body length, eye size, or yolk size of embryos. However, colistin impaired keratinocytes and lateral-line hair cells in the skin of embryos. Colistin (at concentrations ≥0.1 μM) decreased the number of FM1-43-labeled hair cells and reduced the mechanotransduction-mediated Ca2+ influx at hair bundles, suggesting that sublethal concentrations of colistin can impair lateral line function. To investigate the lethal injury, morphological changes were sequentially observed in post-hatched embryos subjected to lethal concentrations of colistin. We found that skin keratinocytes were severely damaged and detached after exposure, leading to hypotonic swelling of the yolk sac, loss of ion contents, cell lysis, and eventual death. This study revealed that acute colistin exposure can impair skin cells and pose a threat to fish survival.

Assessing the Reliability of Mobility Interpretation From a Multi-Isotope Hair Profile on a Traveling Individual

Forensic practitioners, archeologists, and ecologists increasingly use hair isotope profiles and isotope databases and maps to reconstruct the life history of unidentified individuals. Relationships between hair isotope profiles with travel history have been primarily investigated through controlled laboratory experiments. However, those controlled studies do not reflect the complex life history of modern individuals who often travel between multiple locations over different periods. Here, we collect one bundle of hairs from a volunteer whose primary residence is in Ottawa (Canada) but who traveled to multiple destinations over a period of 18 months. Those travels include multi-week trips to distant locations and multi-day trips to more local areas. We use multi-isotope profiles on the individual’s hairs coupled with isotope databases across the world to reconstruct travel history at sub-monthly temporal resolution. We compare the isotopic interpretation of mobility with the recorded travel history. A prominent shift in δ2H values is interpreted as a westward movement towards central Canada, which corresponds to a month-long road-trip to the Prairie. We observed a marked negative excursion in δ13C values along the hairs profile, which was related to a multi-week-long trip to several countries in Europe. Except for an exceptionally variable interval likely driven by health issues, δ15N values show very little fluctuation across the entire profile, indicating consumption of consistent amounts of animal or marine-based protein at different locations. The isotopic shifts in the proximal part of the hair reveal a clearer picture of traveling destinations than the shifts in the distal part, which have larger uncertainty in terms of timing and amplitude. Except for the last couple of months before collection, 87Sr/86Sr ratios show little variation throughout the profile likely due to recent exchange with Ottawa tap water during bathing or showering. The different 87Sr/86Sr ratios in the month preceding collection appear to partially preserve the 87Sr/86Sr ratio of the volunteer’s diet. This study demonstrates the interest of using multi-isotope systems and large isotopic databases to reconstruct individual mobility. This study also underlines the challenges in linking isotope data to mobility, particularly in rapidly growing keratinous tissues.

Functional, Morphological, and Evolutionary Characterization of Hearing in Subterranean, Eusocial African Mole-Rats

Naked mole-rats are highly vocal, eusocial, subterranean rodents with, counterintuitively, poor hearing. The causes underlying their altered hearing are unknown. Moreover, whether altered hearing is degenerate or adaptive to their unique lifestyles is controversial. We used various methods to identify the factors contributing to altered hearing in naked and the related Damaraland mole-rats and to examine whether these alterations result from relaxed or adaptive selection. Remarkably, we found that cochlear amplification was absent from both species despite normal prestin function in outer hair cells isolated from naked mole-rats. Instead, loss of cochlear amplification appears to result from abnormal hair bundle morphologies observed in both species. By exploiting a well-curated deafness phenotype-genotype database, we identified amino acid substitutions consistent with abnormal hair bundle morphology and reduced hearing sensitivity. Amino acid substitutions were found in unique groups of six hair bundle link proteins. Molecular evolutionary analyses revealed shifts in selection pressure at both the gene and the codon level for five of these six hair bundle link proteins. Substitutions in three of these proteins are associated exclusively with altered hearing. Altogether, our findings identify the likely mechanism of altered hearing in African mole-rats, making them the only identified mammals naturally lacking cochlear amplification. Moreover, our findings suggest that altered hearing in African mole-rats is adaptive, perhaps tailoring hearing to eusocial and subterranean lifestyles. Finally, our work reveals multiple, unique evolutionary trajectories in African mole-rat hearing and establishes species members as naturally occurring disease models to investigate human hearing loss.

Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling.

In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gβγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.

Molecular Crux of Hair Cell Mechanotransduction Machinery

Although 62 years have elapsed since the first report of hereditary deafness in a mouse strain, the molecular mechanism of hair cell mechanotransduction remains elusive. Three recent studies present crucial insights into the molecular crux of hair cell mechanotransduction machinery.

Ammonia exposure impairs lateral-line hair cells and mechanotransduction in zebrafish embryos

Ammonia (including NH3 and NH4+) is a major pollutant of freshwater environments. However, the toxic effects of ammonia on the early stages of fish are not fully understood, and little is known about the effects on the sensory system. In this study, we hypothesized that ammonia exposure can cause adverse effects on embryonic development and impair the lateral line system of fish. Zebrafish embryos were exposed to high-ammonia water (10, 15, 20, 25, and 30 mM NH4Cl; pH 7.0) for 96 h (0–96 h post-fertilization). The body length, heart rate, and otic vesicle size had significantly decreased with ≥15 mM NH4Cl, while the number and function of lateral-line hair cells had decreased with ≥10 mM NH4Cl. The mechanoelectrical transduction (MET) channel-mediated Ca2+ influx was measured with a scanning ion-selective microelectrode technique to reveal the function of hair cells. We found that NH4+ (≥5 mM NH4Cl) entered hair cells and suppressed the Ca2+ influx of hair cells. Neomycin and La3+ (MET channel blockers) suppressed NH4+ influx, suggesting that NH4+ enters hair cells via MET channels in hair bundles. In conclusion, this study showed that ammonia exposure (≥10 mM NH4Cl) can cause adverse effects in zebrafish embryos, and lateral-line hair cells are sensitive to ammonia exposure.

Disruption of tmc1/2a/2b Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

Distinct roles of stereociliary links in the nonlinear sound processing and noise resistance of cochlear outer hair cells

Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.