Asymmetric vibrations in the organ of Corti by outer hair cells measured from excised gerbil cochlea.

Pending questions regarding cochlear amplification and tuning are hinged upon the organ of Corti (OoC) active mechanics: how outer hair cells modulate OoC vibrations. Our knowledge regarding OoC mechanics has advanced over the past decade thanks to the application of tomographic vibrometry. However, recent data from live cochlea experiments often led to diverging interpretations due to complicated interaction between passive and active responses, lack of image resolution in vibrometry, and ambiguous measurement angles. We present motion measurements and analyses of the OoC sub-components at the close-to-true cross-section, measured from acutely excised gerbil cochleae. Specifically, we focused on the vibrating patterns of the reticular lamina, the outer pillar cell, and the basilar membrane because they form a structural frame encasing active outer hair cells. For passive transmission, the OoC frame serves as a rigid truss. In contrast, motile outer hair cells exploit their frame structures to deflect the upper compartment of the OoC while minimally disturbing its bottom side (basilar membrane). Such asymmetric OoC vibrations due to outer hair cell motility explain how recent observations deviate from the classical cochlear amplification theory.

Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Store-Operated Ca2+ Channels Contribute to the Generation of Ca2+ Waves in Interdental Cells in the Cochleae.

Cochlear calcium (Ca2+) waves are vital regulators of the cochlear development and establishment of hearing function. Inner supporting cells are believed to be the main region generating Ca2+ waves that work as internal stimuli to coordinate the development of hair cells and the mapping of neurons in the cochlea. However, Ca2+ waves in interdental cells (IDCs) that connect to inner supporting cells and spiral ganglion neurons are rarely observed and poorly understood. Herein, we reported the mechanism of IDC Ca2+ wave formation and propagation by developing a single-cell Ca2+ excitation technology, which can easily be accomplished using a two-photon microscope for simultaneous microscopy and femtosecond laser Ca2+ excitation in any target individual cell in fresh cochlear tissues. We demonstrated that the store-operated Ca2+ channels in IDCs are responsible for Ca2+ wave formation in these cells. The specific architecture of the IDCs determines the propagation of Ca2+ waves. Our results provide the mechanism of Ca2+ formation in IDCs and a controllable, precise, and noninvasive technology to excite local Ca2+ waves in the cochlea, with good potential for research on cochlear Ca2+ and hearing functions.

Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development.

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.

Dync1li1 is required for the survival of mammalian cochlear hair cells by regulating the transportation of autophagosomes.

Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.

Tbx2 is a master regulator of inner versus outer hair cell differentiation.

The cochlea uses two types of mechanosensory cell to detect sounds. A single row of inner hair cells (IHCs) synapse onto neurons to transmit sensory information to the brain, and three rows of outer hair cells (OHCs) selectively amplify auditory inputs1. So far, two transcription factors have been implicated in the specific differentiation of OHCs, whereas, to our knowledge, none has been identified in the differentiation of IHCs2-4. One such transcription factor for OHCs, INSM1, acts during a crucial embryonic period to consolidate the OHC fate, preventing OHCs from transdifferentiating into IHCs2. In the absence of INSM1, embryonic OHCs misexpress a core set of IHC-specific genes, which we predict are involved in IHC differentiation. Here we find that one of these genes, Tbx2, is a master regulator of IHC versus OHC differentiation in mice. Ablation of Tbx2 in embryonic IHCs results in their development as OHCs, expressing early OHC markers such as Insm1 and eventually becoming completely mature OHCs in the position of IHCs. Furthermore, Tbx2 is epistatic to Insm1: in the absence of both genes, cochleae generate only OHCs, which suggests that TBX2 is necessary for the abnormal transdifferentiation of INSM1-deficient OHCs into IHCs, as well as for normal IHC differentiation. Ablation of Tbx2 in postnatal, largely differentiated IHCs makes them transdifferentiate directly into OHCs, replacing IHC features with those of mature and not embryonic OHCs. Finally, ectopic expression of Tbx2 in OHCs results in their transdifferentiation into IHCs. Hence, Tbx2 is both necessary and sufficient to make IHCs distinct from OHCs and maintain this difference throughout development.

Characterization of the microRNA transcriptomes and proteomics of cochlear tissue-derived small extracellular vesicles from mice of different ages after birth.

The cochlea is an important sensory organ for both balance and sound perception, and the formation of the cochlea is a complex developmental process. The development of the mouse cochlea begins on embryonic day (E)9 and continues until postnatal day (P)21 when the hearing system is considered mature. Small extracellular vesicles (sEVs), with a diameter ranging from 30 to 200 nm, have been considered a significant medium for information communication in both physiological and pathological processes. However, there are no studies exploring the role of sEVs in the development of the cochlea. Here, we isolated tissue-derived sEVs from the cochleae of FVB mice at P3, P7, P14, and P21 by ultracentrifugation. These sEVs were first characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Next, we used small RNA-seq and mass spectrometry to characterize the microRNA transcriptomes and proteomes of cochlear sEVs from mice at different ages. Many microRNAs and proteins were discovered to be related to inner ear development, anatomical structure development, and auditory nervous system development. These results all suggest that sEVs exist in the cochlea and are likely to be essential for the normal development of the auditory system. Our findings provide many sEV microRNA and protein targets for future studies of the roles of cochlear sEVs.

ROS-Induced Oxidative Damage and Mitochondrial Dysfunction Mediated by Inhibition of SIRT3 in Cultured Cochlear Cells.

Sensorineural hearing loss (SNHL) is one of the most common causes of disability worldwide. Previous evidence suggests that reactive oxygen species (ROS) may play an important role in the occurrence and development of SNHL, while its mechanism remains unclear. We cultured dissected organs of Corti in medium containing different concentrations (0, 0.25, 0.5, 0.75, 1, and 1.25 mM) of hydrogen peroxide (H2O2) and established a four-concentration model of 0, 0.5, 0.75, and 1 mM to study different degrees of damage. We examined ROS-induced mitochondrial damage and the role of sirtuin 3 (SIRT3). Our results revealed that the number of ribbon synapses and hair cells appeared significantly concentration-dependent decrease with exposure to H2O2. Outer hair cells (OHCs) and inner hair cells (IHCs) began to be lost, and activation of apoptosis of hair cells (HCs) was observed at 0.75 mM and 1 mM H2O2, respectively. In contrast with the control group, the accumulation of ROS was significantly higher, and the mitochondrial membrane potential (MMP) was lower in the H2O2-treated groups. Furthermore, the expression of SIRT3, FOXO3A, and SOD2 proteins declined, except for an initial elevation of SIRT3 between 0 and 0.75 mM H2O2. Administration of the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine resulted in increased damage to the cochlea, including loss of ribbon synapses and hair cells, apoptosis of hair cells, more production of ROS, and reduced mitochondrial membrane potential. Thoroughly, our results highlight that ROS-induced mitochondrial oxidative damage drives hair cell degeneration and apoptosis. Furthermore, SIRT3 is crucial for preserving mitochondrial function and protecting the cochlea from oxidative damage and may represent a possible therapeutic target for SNHL.

Protective effect of berberine chloride against cisplatin-induced ototoxicity.

BACKGROUND: Cisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia. OBJECTIVE: Organ of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP. METHODS: We investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay. RESULTS: BC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival. CONCLUSION: Our result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity.

Quantum magnetic imaging of iron organelles within the pigeon cochlea.

The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed "cuticulosomes" in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle-based magnetoreceptor candidates.

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration.

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Daple deficiency causes hearing loss in adult mice by inducing defects in cochlear stereocilia and apical microtubules.

The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple-/- mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple-/- mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

Sperm-associated antigen 6 (Spag6) mutation leads to vestibular dysfunction in mice.

Spag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 plays a role in hearing and planar cell polarity (PCP) in the cochlea of the inner ear. However, a role for Spag6 in the vestibule has not yet been explored. In the present study, the function of Spag6 in the vestibule of the inner ear was examined using Spag6-deficient mice. Our results demonstrate a vestibular disorder in the Spag6 mutants, associated with abnormal ultrastructures of vestibular hair cells and Scarpa's ganglion cells, including swollen stereocilia, decreased crista in mitochondria and swollen Scarpa's ganglion cells. Immunostaining data suggests existence of caspase-dependent apoptosis in vestibular sensory epithelium and Scarpa's ganglion cells. Our observations reveal new functions for Spag6 in vestibular function and apoptosis in the mouse vestibule.

High-throughput screening on cochlear organoids identifies VEGFR-MEK-TGFB1 signaling promoting hair cell reprogramming.

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells.

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.

GATA3 maintains the quiescent state of cochlear supporting cells by regulating p27kip1.

Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.

The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea.

Sensory hair cells (HCs) are highly susceptible to damage by noise, ototoxic drugs, and aging. Although HCs cannot be spontaneously regenerated in adult mammals, previous studies have shown that signaling pathways are involved in HC regeneration in the damaged mouse cochlea. Here, we used a Notch antagonist (DAPT), a Wnt agonist (QS11), and recombinant Sonic hedgehog (SHH) protein to investigate their concerted actions underlying HC regeneration in the mouse cochlea after neomycin-induced damage both in vivo and in vitro. With DAPT, the numbers of HCs increased, and supporting cell (SC) proliferation was seen in both the intact and damaged cochlear sensory epithelia, while these numbers were unchanged in the presence of QS11. When simultaneously treated with DAPT and QS11, the number of HCs increased dramatically, and much greater SC proliferation was seen in the cochlear epithelium. In transgenic mice with both Notch1 conditional knockout and ß-catenin over-expression, cochlear SC proliferation and HC regeneration were more obvious than in either Notch1 knockout or ß-catenin over-expressing mice separately. When cochleae were treated with DAPT, QS11, and SHH together, SC proliferation was even greater, and this proliferation was seen in both the HC region and the greater epithelial ridge. High-throughput RNA sequencing was used to identify the differentially expressed genes between all groups, and the results showed that the SHH and Wnt signaling pathways are involved in SC proliferation. Our study suggests that co-regulation of the Notch, Wnt, and SHH signaling pathways promotes extensive cell proliferation and regeneration in the mouse cochlea.

Murine cochlear cell sorting and cell-type-specific organoid culture.

Neonatal mouse cochlear duct cells can proliferate and grow in vitro into inner ear organoids. Distinctive cochlear duct cell types have different organoid formation capacities. Here, we provide a flow cytometric cell-sorting method that allows the subsequent culture of individual cochlear cell populations. For the efficient culture of the sorted cells, we provide protocols for growing free-floating inner ear organoids, the adherence of organoids to a substrate, and the expansion of organoid-derived inner ear colonies. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2021).

Opposing effects of Wnt/ß-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea.

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via ß-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.