Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells.

Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This transmission necessitates rapid and sustained neurotransmitter release, which depends on a large pool of synaptic vesicles at the hair-cell presynapse. While previous work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, the mechanisms of this process in hair cells remain unclear. Our study demonstrates that the kinesin motor protein Kif1a, along with an intact microtubule network, is essential for enriching synaptic vesicles at the presynapse in hair cells. Through genetic and pharmacological approaches, we disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral-line system. These manipulations led to a significant reduction in synaptic-vesicle populations at the presynapse in hair cells. Using electron microscopy, in vivo calcium imaging, and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon-synapse function. Kif1aa mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Furthermore, kif1aa mutants exhibit impaired rheotaxis, a behaviour reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a-mediated microtubule transport is critical to enrich synaptic vesicles at the active zone, a process that is vital for proper ribbon-synapse function in hair cells. KEY POINTS: Kif1a mRNAs are present in zebrafish hair cells. Loss of Kif1a disrupts the enrichment of synaptic vesicles at ribbon synapses. Disruption of microtubules depletes synaptic vesicles at ribbon synapses. Kif1aa  mutants have impaired ribbon-synapse and sensory-system function.

Simultaneous recordings from vestibular Type I hair cells and their calyceal afferents in mice.

The vestibular hair cell receptors of anamniotes, designated Type II, are presynaptic to bouton endings of vestibular nerve distal neurites. An additional flask-shaped hair cell receptor, Type I, is present in amniotes, and communicates with a chalice-shaped afferent neuritic ending that surrounds the entire hair cell except its apical neck. Since the full repertoire of afferent fiber dynamics and sensitivities observed throughout the vertebrate phyla can be accomplished through Type II hair cell-bouton synapses, the functional contribution(s) of Type I hair cells and their calyces to vestibular performance remains a topic of great interest. The goal of the present study was to investigate electrical coupling between the Type I hair cell and its enveloping calyx in the mouse semicircular canal crista ampullaris. Since there are no gap junctions between these two cells, evidence for electrical communication would necessarily involve other mechanisms. Simultaneous recordings from the two cells of the synaptic pair were used initially to verify the presence of orthodromic quantal synaptic transmission from the hair cell to the calyx, and then to demonstrate bi-directional communication due to the slow accumulation of potassium ions in the synaptic cleft. As a result of this potassium ion accretion, the equilibrium potentials of hair cell conductances facing the synaptic cleft become depolarized to an extent that is adequate for calcium influx into the hair cell, and the calyx inner face becomes depolarized to a level that is near the threshold for spike initiation. Following this, paired recordings were again employed to characterize fast bi-directional electrical coupling between the two cells. In this form of signaling, cleft-facing conductances in both the hair cell and calyx increase, which strengthens their coupling. Because this mechanism relies on the cleft resistance, we refer to it as resistive coupling. We conclude that the same three forms of hair cell-calyceal transmission previously demonstrated in the turtle are present in the mammalian periphery, providing a biophysical basis for the exceptional temporal fidelity of the vestibular system.

The Role of Molecular and Cellular Aging Pathways on Age-Related Hearing Loss.

Aging, a complex process marked by molecular and cellular changes, inevitably influences tissue and organ homeostasis and leads to an increased onset or progression of many chronic diseases and conditions, one of which is age-related hearing loss (ARHL). ARHL, known as presbycusis, is characterized by the gradual and irreversible decline in auditory sensitivity, accompanied by the loss of auditory sensory cells and neurons, and the decline in auditory processing abilities associated with aging. The extended human lifespan achieved by modern medicine simultaneously exposes a rising prevalence of age-related conditions, with ARHL being one of the most significant. While our understanding of the molecular basis for aging has increased over the past three decades, a further understanding of the interrelationship between the key pathways controlling the aging process and the development of ARHL is needed to identify novel targets for the treatment of AHRL. The dysregulation of molecular pathways (AMPK, mTOR, insulin/IGF-1, and sirtuins) and cellular pathways (senescence, autophagy, and oxidative stress) have been shown to contribute to ARHL. However, the mechanistic basis for these pathways in the initiation and progression of ARHL needs to be clarified. Therefore, understanding how longevity pathways are associated with ARHL will directly influence the development of therapeutic strategies to treat or prevent ARHL. This review explores our current understanding of the molecular and cellular mechanisms of aging and hearing loss and their potential to provide new approaches for early diagnosis, prevention, and treatment of ARHL.

The upregulation of K+ and HCN channels in developing spiral ganglion neurons is mediated by cochlear inner hair cells.

Spiral ganglion neurons (SGNs) are primary sensory afferent neurons that relay acoustic information from the cochlear inner hair cells (IHCs) to the brainstem. The response properties of different SGNs diverge to represent a wide range of sound intensities in an action-potential code. This biophysical heterogeneity is established during pre-hearing stages of development, a time when IHCs fire spontaneous Ca2+ action potentials that drive glutamate release from their ribbon synapses onto the SGN terminals. The role of spontaneous IHC activity in the refinement of SGN characteristics is still largely unknown. Using pre-hearing otoferlin knockout mice (Otof-/-), in which Ca2+-dependent exocytosis in IHCs is abolished, we found that developing SGNs fail to upregulate low-voltage-activated K+-channels and hyperpolarisation-activated cyclic-nucleotide-gated channels. This delayed maturation resulted in hyperexcitable SGNs with immature firing characteristics. We have also shown that SGNs that synapse with the pillar side of the IHCs selectively express a resurgent K+ current, highlighting a novel biophysical marker for these neurons. RNA-sequencing showed that several K+ channels are downregulated in Otof-/- mice, further supporting the electrophysiological recordings. Our data demonstrate that spontaneous Ca2+-dependent activity in pre-hearing IHCs regulates some of the key biophysical and molecular features of the developing SGNs. KEY POINTS: Ca2+-dependent exocytosis in inner hair cells (IHCs) is otoferlin-dependent as early as postnatal day 1. A lack of otoferlin in IHCs affects potassium channel expression in SGNs. The absence of otoferlin is associated with SGN hyperexcitability. We propose that type I spiral ganglion neuron functional maturation depends on IHC exocytosis.

foxg1a is required for hair cell development and regeneration in the zebrafish lateral line.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammals, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. Our study demonstrates that mutation of foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. In developing and regenerating neuromasts, we find that loss of Foxg1a function results in reduced hair cell numbers, as well as decreased proliferation of neuromast cells. Foxg1a specifically regulates the development and regeneration of Islet1-labeled hair cells. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

Two new mouse alleles of Ocm and Slc26a5.

The genes Ocm (encoding oncomodulin) and Slc26a5 (encoding prestin) are expressed strongly in outer hair cells and both are involved in deafness in mice. However, it is not clear if they influence the expression of each other. In this study, we characterise the auditory phenotype resulting from two new mouse alleles, Ocmtm1e and Slc26a5tm1Cre. Each mutation leads to absence of detectable mRNA transcribed from the mutant allele, but there was no evidence that oncomodulin regulates expression of prestin or vice versa. The two mutants show distinctive patterns of auditory dysfunction. Ocmtm1e homozygotes have normal auditory brainstem response thresholds at 4 weeks old followed by progressive hearing loss starting at high frequencies, while heterozygotes show largely normal thresholds until 6 months of age, when signs of worse thresholds are detected. In contrast, Slc26a5tm1Cre homozygotes have stable but raised thresholds across all frequencies tested, 3 to 42 kHz, at least from 4 to 8 weeks old, while heterozygotes have raised thresholds at high frequencies. Distortion product otoacoustic emissions and cochlear microphonics show deficits similar to auditory brainstem responses in both mutants, suggesting that the origin of hearing impairment is in the outer hair cells. Endocochlear potentials are normal in the two mutants. Scanning electron microscopy revealed normal development of hair cells in Ocmtm1e homozygotes but scattered outer hair cell loss even at 4 weeks old when thresholds appeared normal, indicating that there is not a direct relationship between numbers of outer hair cells present and auditory thresholds.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

The ototoxicity of chlorinated paraffins via inducing apoptosis, oxidative stress and endoplasmic reticulum stress in cochlea hair cells.

Hearing loss is a common chronic sensory deficit that affects millions of people worldwide and has emerged as a significant public health concern. The association between environmental exposure to chemicals and the prevalence of hearing impairment has recently attracted increased attention. Chlorinated paraffins (CPs) are a type of chemical compound that has been widely used and commonly detected in samples of both environmental and human origin. The knowledge of the toxicological effects of CPs, particularly its ototoxicity, remains limited at present. In this study, six commercial CPs were selected and evaluated using cochlea hair HEI-OC1 cells for their cytotoxicity, apoptosis, DNA damage, reactive oxygen species (ROS) accumulation and oxidative response. The cytotoxicity was observed after CPs exposure at high concentrations except for C-40 and was positively related to the chlorine content (Cl-content) in both CCK-8 and trypan blue assays. All 6 CPs induced cells apoptosis through caspase-dependent apoptotic pathway. CPs exposure induced DNA damage and stimulated ROS overproduction. Antioxidant N-acetyl-L-cysteine (NAC) could reverse the cytotoxicity and ROS accumulation caused by CPs exposure. The overexpression of ATF4 and CHOP indicated that endoplasmic reticulum (ER) stress was involved in the CPs induced cytotoxicity. Thus, CPs induced cytotoxicity and apoptosis via ROS accumulation, ER stress and DNA damage and positively related to the Cl-content and our findings indicate that CPs may pose a risk of ototoxicity at environmental relevant exposure levels.

A free intravesicular C-terminal of otoferlin is essential for synaptic vesicle docking and fusion at auditory inner hair cell ribbon synapses.

Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof+/GFP mice exhibited a normal hearing function, homozygote OtofGFP/GFP mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof GFP/GFP IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.

BAIAP2L1 and BAIAP2L2 differently regulate hair cell stereocilia morphology.

Inner ear sensory hair cells are characterized by their apical F-actin-based cell protrusions named stereocilia. In each hair cell, several rows of stereocilia with different height are organized into a staircase-like pattern. The height of stereocilia is tightly regulated by two protein complexes, namely row-1 and row-2 tip complex, that localize at the tips of tallest-row and shorter-row stereocilia, respectively. Previously, we and others identified BAI1-associated protein 2-like 2 (BAIAP2L2) as a component of row-2 complex that play an important role in maintaining shorter-row stereocilia. In the present work we show that BAIAP2L1, an ortholog of BAIAP2L2, localizes at the tips of tallest-row stereocilia in a way dependent on known row-1 complex proteins EPS8 and MYO15A. Interestingly, unlike BAIAP2L2 whose stereocilia-tip localization requires calcium, the localization of BAIAP2L1 on the tips of tallest-row stereocilia is calcium-independent. Therefore, our data suggest that BAIAP2L1 and BAIAP2L2 localize at the tips of different stereociliary rows and might regulate the development and/or maintenance of stereocilia differently. However, loss of BAIAP2L1 does not affect the row-1 protein complex, and the auditory and balance function of Baiap2l1 knockout mice are largely normal. We hypothesize that other orthologous protein(s) such as BAIAP2 might compensate for the loss of BAIAP2L1 in the hair cells.

CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern.

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2 KO/+ ;Whrn wi/+ ) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2 KO/KO ;Whrn wi/wi ) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrn wi/wi mutants. Furthermore, over-expression of Whrn in Cib2 KO/KO mice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.

Upregulation of the Cav1.3 channel in inner hair cells by interleukin 6-dependent inflammaging contributes to age-related hearing loss.

Age-related hearing loss (AHL) is the most common sensory disorder amongst the older population. Inflammaging is a ≈chronic low-grade inflammation that worsens with age and is an early sign of AHL; however, the underlying mechanisms remain unclear. We used electrophysiological and genetic approaches to establish the importance of interleukin 6 (IL-6)-dependent inflammation in AHL. Elevated IL-6 in the cochlea enhanced Cav1.3 calcium channel function in the inner hair cell (IHC) synapse in mice with AHL. IL-6 upregulated the Cav1.3 channel via the Janus kinase-mitogen activated kinase pathway, causing neurotransmitter excitotoxicity and synapse impairment; IL-6 deficiency or the administration of a Cav1.3 channel blocker attenuated this age-related damage, and rescued hearing loss. Thus, IL-6-dependent inflammaging upregulated the Cav1.3 channel in IHCs, contributing to AHL. Our findings could help the comprehensive understanding of inflammaging's effects on AHL, aiding in early intervention to protect against hearing decline.

From sound waves to molecular and cellular mechanisms: Understanding noise­induced hearing loss and pioneering preventive approaches (Review).

Noise-induced hearing loss (NIHL) is a significant and urgent global public health concern, arising from prolonged exposure to elevated levels of noise. This auditory impairment harms delicate inner ear structures, particularly the essential hair cells transmitting auditory signals to the brain. Recognized by the World Health Organization as a major contributor to worldwide hearing loss, NIHL requires a comprehensive examination of its molecular and cellular mechanisms. Animal models emerge as indispensable tools for unraveling these intricacies, allowing researchers to simulate and study the impact of noise exposure on auditory structures, shedding light on the interplay of oxidative stress, inflammation and immune responses-crucial factors in NIHL progression. The present review focuses on elucidating the molecular mechanisms of NIHL, with a specific emphasis on findings derived from animal models, alongside the exploration of thorough preventive strategies, including protective measures and probing potential interventions. Understanding the molecular underpinnings not only provides insight into targeted treatment approaches, but also unlocks pathways for exploring and implementing preventive actions. This approach not only deepens the current comprehension of NIHL, but also has the potential to influence the shaping of public health policies, offering a nuanced perspective on this prevalent auditory disorder.

Dexamethasone reduces cisplatin-induced hair cell damage by inducing cisplatin resistance through metallothionein-2.

PURPOSE: Hair cell damage is a common side effect caused by the anticancer drug cisplatin (CDDP), which reduces patient quality of life. One CDDP resistance mechanism that occurs in recurrent cancers is heavy metal detoxification by metallothionein-2 (mt2). Here, we show that in zebrafish larvae, dexamethasone (DEX) reduces CDDP-induced hair cell damage by enhancing mt2 expression. METHODS: Transgenic zebrafish (cldn: gfp; atoh1: rfp) that express green and red fluorescent proteins in neuromasts and hair cells, respectively, were used. The zebrafish were pretreated with DEX at 52 h post-fertilization (hpf) for 8 h, followed by CDDP treatment for 12 h. The lateral line hair cells of CDDP-treated zebrafish at 72 hpf were observed by fluorescence microscopy. RESULTS: Reporting odds ratio (ROR) analysis using an adverse event database indicated an association between a decrease in CDDP-induced ototoxicity and DEX as an antiemetic treatment for cancer chemotherapy. Pretreatment with DEX protected 72 hpf zebrafish hair cells from CDDP-induced damage. The expression of mt2 mRNA was significantly increased by the combination of 10 µM DEX with CDDP. Gene editing of mt2 reversed the protective effect of DEX against CDDP-induced damage in hair cells. CONCLUSION: DEX protects hair cells from CDDP-induced damage through increased mt2 expression, which is a resistance mechanism for platinum-based anticancer drugs.

Mosaic Atoh1 deletion in the chick auditory epithelium reveals a homeostatic mechanism to restore hair cell number.

The mechanosensory hair cell of the vertebrate inner ear responds to the mechanical deflections that result from hearing or change in the acceleration due to gravity, to allow us to perceive and interpret sounds, maintain balance and spatial orientation. In mammals, ototoxic compounds, disease, and acoustic trauma can result in damage and extrusion of hair cells, without replacement, resulting in hearing loss. In contrast, non-mammalian vertebrates can regenerate sensory hair cells. Upon damage, hair cells are extruded and an associated cell type, the supporting cell is transformed into a hair cell. The mechanisms that can trigger regeneration are not known. Using mosaic deletion of the hair cell master gene, Atoh1, in the embryonic avian inner ear, we find that despite hair cells depletion at E9, by E12, hair cell number is restored in sensory epithelium. Our study suggests a homeostatic mechanism can restores hair cell number in the basilar papilla, that is activated when juxtracrine signalling is disrupted. Restoration of hair cell numbers during development may mirror regenerative processes, and our work provides insights into the mechanisms that trigger regeneration.

Metabolic Profiling of Cochlear Organoids Identifies α-Ketoglutarate and NAD+ as Limiting Factors for Hair Cell Reprogramming.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.

Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

Unveiling the ototoxic effects of paraquat on zebrafish larva.

Paraquat is a widely utilized herbicide in agricultural fields posing a significant impact on human health and the environment due to its potent oxidant properties. Rampant paraquat usage leads to serious health hazards to farmers and the ecosystem, particularly the water bodies. Paraquat exposure can damage dopaminergic neurons causing Parkinson's disease in humans and other animal models. Extensive research has been done regarding the mode of action, pathophysiology and molecular mechanisms of paraquat-induced Parkinson's disease. Meanwhile, the ototoxic effect of paraquat remains poorly understood. Potential ototoxins can cause sensorineural hearing loss, one of the most common sensory disabilities in humans. In this study, we investigated the harmful effects of paraquat on neuromast hair cells in zebrafish larvae, a powerful model organism for auditory research. We treated sub-lethal concentrations (125 µM to 1000 µM) of paraquat to 3 and 4 dpf zebrafish larvae to investigate its ototoxic effects via rheotaxis behavioral assay, neuromast staining and scanning electron microscopy. The behavioral assay findings showed a drastic decline in the rheotaxis behavior in all the concentrations of paraquat-treated larvae. Furthermore, DASPEI neuromast vital staining displayed a dose-dependent reduction in the neuromast hair cells as we increased the paraquat concentration. The scanning electron microscope data revealed the significant shortening of kinociliary length, a decrease in stereociliary density and changes in semilunar peridermal cell morphology signifying the damaging effects of paraquat at the cellular level. Collectively, the behavioral, anatomical and morphological studies highlight the potential ototoxic effects of paraquat on zebrafish neuromast hair cells, further signifying its potential role in causing hearing loss in humans.

Recent Progress in Generation of Inner Ear Organoid.

Inner ear organoids play a crucial role in hearing research. In comparison to other animal models and 2D cell culture systems, inner ear organoids offer significant advantages for studying the mechanisms of inner ear development and exploring novel approaches to disease treatment. Inner ear organoids derived from human cells are more closely resemble normal human organs in development and function. The 3D culture system of the inner ear organoid enhances cell-cell interactions and mimics the internal environment. In this review, the progress and limitations of organoid culture methods derived from tissue-specific progenitors and pluripotent stem cells (PSCs) are summarized, which may offer new insights into generating organoids that closely resemble the inner ear in terms of morphology and function.

Notch1 is required to maintain supporting cell identity and vestibular function during maturation of the mammalian balance organs.

The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear. Both hearing and vestibular sensory regions are composed of similar cell types, including hair cells and associated supporting cells. Recently, we showed that Notch1 is required for maintaining supporting cell survival postnatally during cochlear maturation. However, it is not known whether Notch1 plays a similar role in the balance organs of the inner ear. To characterize the role of Notch during vestibular maturation, we conditionally deleted Notch1 from Sox2-expressing cells of the vestibular organs in the mouse at P0/P1. Histological analyses showed a dramatic loss of supporting cells accompanied by an increase in type II hair cells without cell death, indicating the supporting cells are converting to hair cells in the maturing vestibular regions. Analysis of 6-week old animals indicate that the converted hair cells survive, despite the reduction of supporting cells. Interestingly, measurements of vestibular sensory evoked potentials (VsEPs), known to be generated in the striolar regions of the vestibular afferents in the maculae, failed to show a response, indicating that NOTCH1 expression is critical for striolar function postnatally. Consistent with this, we find that the specialized type I hair cells in the striola fail to develop the complex calyces typical of these cells. These defects are likely due to the reduction in supporting cells, which have previously been shown to express factors critical for the striolar region. Similar to other mutants that lack proper striolar development, Notch1 mutants do not exhibit typical vestibular behaviors such as circling and head shaking, but do show difficulties in some vestibular tests, including the balance beam and forced swim test. These results indicate that, unlike the hearing organ in which the supporting cells undergo cell death, supporting cells in the balance regions retain the ability to convert to hair cells during maturation, which survive into adulthood despite the reduction in supporting cells.