ABSTRACT
Type I spiral ganglion neurons (SGNs) transmit sound information from cochlear hair cells to the CNS. Using transcriptome analysis of thousands of single neurons, we demonstrate that murine type I SGNs consist of subclasses that are defined by the expression of subsets of transcription factors, cell adhesion molecules, ion channels, and neurotransmitter receptors. Subtype specification is initiated prior to the onset of hearing during the time period when auditory circuits mature. Gene mutations linked to deafness that disrupt hair cell mechanotransduction or glutamatergic signaling perturb the firing behavior of SGNs prior to hearing onset and disrupt SGN subtype specification. We thus conclude that an intact hair cell mechanotransduction machinery is critical during the pre-hearing period to regulate the firing behavior of SGNs and their segregation into subtypes. Because deafness is frequently caused by defects in hair cells, our findings have significant ramifications for the etiology of hearing loss and its treatment.
Subject(s)
Hair Cells, Auditory/physiology , Hearing/physiology , Mechanotransduction, Cellular , Neurons/physiology , Signal Transduction , Spiral Ganglion/physiology , Animals , Cluster Analysis , Genetic Markers , Male , Mice , Mice, Inbred CBA , Mice, Knockout , Mutation , Neuroglia/physiology , Sequence Analysis, RNAABSTRACT
The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
Subject(s)
Deafness/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Alternative Splicing/genetics , Animals , Cell Line , Exons , Gene Expression Regulation/genetics , HEK293 Cells , Hair Cells, Auditory/physiology , Hearing/genetics , Hearing/physiology , Histone Deacetylase Inhibitors/metabolism , Histone Deacetylases/metabolism , Humans , Mice , Mice, Inbred C57BL , Neurons , RNA Splicing/genetics , Repressor Proteins/physiology , Transcription Factors , Vorinostat/pharmacologyABSTRACT
Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.
Subject(s)
Adult Stem Cells/metabolism , Ear, Inner/metabolism , Hair Cells, Auditory/physiology , Olfactory Mucosa/metabolism , Regeneration/physiology , Retina/metabolism , Animals , Cell Differentiation/genetics , Cell Proliferation/genetics , Cytokines/metabolism , Ear, Inner/cytology , Hair Cells, Auditory/cytology , Hair Cells, Auditory/metabolism , Humans , Inflammation/genetics , Inflammation/metabolism , Macrophages/metabolism , Regeneration/genetics , Retina/cytology , Signal Transduction/genetics , Signal Transduction/physiology , Wounds and Injuries/genetics , Wounds and Injuries/metabolismABSTRACT
Spontaneous neuronal activity during the development of the auditory sensory system is important in establishing mature connectivity. Wang et al. show that glia-like cells drive spontaneous spiking in neighboring cochlear inner hair cells via a process that involves osmotic cell shrinkage and the secretion of potassium ions.
Subject(s)
Ear, Inner/growth & development , Hair Cells, Auditory/cytology , AnimalsABSTRACT
A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.
Subject(s)
Hearing Loss, Noise-Induced/metabolism , Nerve Tissue Proteins/metabolism , Peroxisomes/metabolism , Proteins/metabolism , Animals , Auditory Pathways , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Hearing Loss, Noise-Induced/pathology , Humans , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Neurons/metabolism , Oxidative Stress , Proteins/geneticsABSTRACT
Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells.
Subject(s)
Ear, Inner/growth & development , Hair Cells, Auditory/cytology , Adenosine Triphosphate/metabolism , Animals , Anoctamin-1 , Chloride Channels/genetics , Chloride Channels/metabolism , Ear, Inner/cytology , Ear, Inner/metabolism , Hair Cells, Auditory/metabolism , Labyrinth Supporting Cells/cytology , Labyrinth Supporting Cells/metabolism , Mice , Mice, Knockout , Potassium/metabolism , Rats , Rats, Sprague-Dawley , Spiral Ganglion/cytology , Spiral Ganglion/metabolismABSTRACT
The genetic approach, based on the study of inherited forms of deafness, has proven to be particularly effective for deciphering the molecular mechanisms underlying the development of the peripheral auditory system, the cochlea and its afferent auditory neurons, and how this system extracts the physical parameters of sound. Although this genetic dissection has provided little information about the central auditory system, scattered data suggest that some genes may have a critical role in both the peripheral and central auditory systems. Here, we review the genes controlling the development and function of the peripheral and central auditory systems, focusing on those with demonstrated intrinsic roles in both systems and highlighting the current underappreciation of these genes. Their encoded products are diverse, from transcription factors to ion channels, as are their roles in the central auditory system, mostly evaluated in brainstem nuclei. We examine the ontogenetic and evolutionary mechanisms that may underlie their expression at different sites.
Subject(s)
Auditory Pathways/physiology , Gene Expression Regulation, Developmental , Genes , Neurogenesis/genetics , Animals , Auditory Pathways/growth & development , Biological Evolution , Cochlea/embryology , Cochlea/growth & development , Cochlea/physiology , Gene Ontology , Hair Cells, Auditory/cytology , Hair Cells, Auditory/physiology , Hearing Disorders/genetics , Humans , Ion Channels/genetics , Ion Channels/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Rhombencephalon/embryology , Rhombencephalon/growth & development , Rhombencephalon/physiology , Sensory Receptor Cells/physiology , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.
Subject(s)
Cochlea , Hair Cells, Auditory , Mitochondria , Mitochondrial Dynamics , Animals , Cochlea/embryology , Cochlea/cytology , Cochlea/growth & development , Hair Cells, Auditory/cytology , Hair Cells, Auditory/metabolism , Mitochondria/metabolism , Chick Embryo , Cell Shape , Chickens , Cell DifferentiationABSTRACT
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.
Subject(s)
Lateral Line System , Zebrafish , Animals , Zebrafish/genetics , Cell Shape , Animals, Genetically Modified , Hair Cells, Auditory/physiologyABSTRACT
Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.
Subject(s)
Cell Transdifferentiation , Ear, Inner , Hair Cells, Auditory , Regeneration , Stem Cells , Zebrafish , Animals , Regeneration/physiology , Ear, Inner/cytology , Stem Cells/cytology , Hair Cells, Auditory/cytology , Hair Cells, Auditory/physiology , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Animals, Genetically Modified , Larva/cytologyABSTRACT
To build a functional inner ear, hair cell morphology must be precisely controlled along the proximo-distal axis. A new paper in Development shows that differential mitochondrial dynamics in proximal versus distal cells impacts on the apical cell surface area - a key aspect of morphology. To find out more about this work, we spoke to first author James O'Sullivan and senior author Zoë Mann, both at King's College London, UK.
Subject(s)
Developmental Biology , Animals , Humans , Developmental Biology/history , Hair Cells, Auditory/cytology , History, 21st Century , History, 20th Century , Ear, Inner/embryology , Ear, Inner/cytology , Mitochondrial DynamicsABSTRACT
The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.
Subject(s)
Cell Lineage , Cell Polarity , Ear, Inner , Homeodomain Proteins , Transcription Factors , Animals , Mice , Cell Lineage/genetics , Cell Polarity/genetics , Ear, Inner/metabolism , Ear, Inner/embryology , Ear, Inner/cytology , Gene Expression Regulation, Developmental , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/cytology , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Mice, Transgenic , Saccule and Utricle/cytology , Saccule and Utricle/metabolism , Saccule and Utricle/embryology , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Hair cells are mechanosensors for the perception of sound, acceleration, and fluid motion. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity. The molecular constituents of the mechanotransduction channels of hair cells are not known. Here, we show that mechanotransduction is impaired in mice lacking the tetraspan TMHS. TMHS binds to the tip-link component PCDH15 and regulates tip-link assembly, a process that is disrupted by deafness-causing Tmhs mutations. TMHS also regulates transducer channel conductance and is required for fast channel adaptation. TMHS therefore resembles other ion channel regulatory subunits such as the transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits. We conclude that TMHS is an integral component of the hair cell's mechanotransduction machinery that functionally couples PCDH15 to the transduction channel.
Subject(s)
Hair Cells, Auditory/metabolism , Hearing , Mechanotransduction, Cellular , Membrane Proteins/metabolism , Animals , Cadherin Related Proteins , Cadherins/metabolism , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Mice , Mice, Knockout , Protein Precursors/metabolism , Stereocilia/metabolismABSTRACT
The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, we have identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that we identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. We not only discover ion channels and motors important for hearing, but also show that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. Our findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades.
Subject(s)
Drosophila/physiology , Signal Transduction , Animals , Axonemal Dyneins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila/anatomy & histology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Hair Cells, Auditory/metabolism , Hearing/physiology , Ion Channels/genetics , Ion Channels/metabolism , Nerve Tissue Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Rhodopsin/genetics , Rhodopsin/metabolism , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolismABSTRACT
Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.
Subject(s)
Deafness , Hair Cells, Auditory , Adult , Animals , Humans , Transcription Factors/genetics , Epithelium , Cochlea , MammalsABSTRACT
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.
Subject(s)
Deafness , Hearing Loss , Mice , Animals , Hearing Loss/genetics , Hearing Loss/metabolism , Hair Cells, Auditory/physiology , Deafness/genetics , Hair Cells, Auditory, Inner/metabolism , Cadherins/metabolism , Exons/geneticsABSTRACT
Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing "synaptopathy" and hearing loss. Cocultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that, when P3-5 denervated organs of Corti are cocultured with SGNs, newly formed IHC/SGN synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When using older organs of Corti at P10-11, synaptic activity probed by deconvolution showed more mature release properties, closer to the specialized mode of IHC synaptic transmission crucial for coding the sound signal. This functional assessment of newly formed IHC synapses developed here, provides a powerful tool for testing approaches to improve synapse regeneration.
Subject(s)
Spiral Ganglion , Synapses , Animals , Spiral Ganglion/cytology , Spiral Ganglion/physiology , Synapses/physiology , Mice , Hair Cells, Auditory, Inner/physiology , Hair Cells, Auditory, Inner/metabolism , Synaptic Transmission/physiology , Neurons/physiology , Neurons/metabolism , Regeneration/physiology , Hair Cells, Auditory/physiology , Coculture Techniques/methods , Optogenetics/methods , Nerve Regeneration/physiology , Excitatory Postsynaptic Potentials/physiology , Organ of Corti/physiology , Organ of Corti/cytology , Organ of Corti/metabolismABSTRACT
Collective cell rotations are widely used during animal organogenesis. Theoretical and in vitro studies have conceptualized rotating cells as identical rigid-point objects that stochastically break symmetry to move monotonously and perpetually within an inert environment. However, it is unclear whether this notion can be extrapolated to a natural context, where rotations are ephemeral and heterogeneous cellular cohorts interact with an active epithelium. In zebrafish neuromasts, nascent sibling hair cells invert positions by rotating ≤180° around their geometric center after acquiring different identities via Notch1a-mediated asymmetric repression of Emx2. Here, we show that this multicellular rotation is a three-phasic movement that progresses via coherent homotypic coupling and heterotypic junction remodeling. We found no correlation between rotations and epithelium-wide cellular flow or anisotropic resistive forces. Moreover, the Notch/Emx2 status of the cell dyad does not determine asymmetric interactions with the surrounding epithelium. Aided by computer modeling, we suggest that initial stochastic inhomogeneities generate a metastable state that poises cells to move and spontaneous intercellular coordination of the resulting instabilities enables persistently directional rotations, whereas Notch1a-determined symmetry breaking buffers rotational noise.
Subject(s)
Hair Cells, Auditory , Zebrafish , Animals , Microscopy, Video , Epithelium , MechanoreceptorsABSTRACT
The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.
Subject(s)
Hair Cells, Vestibular , Vestibule, Labyrinth , Humans , Organoids , Hair Cells, Auditory , Cell Differentiation/geneticsABSTRACT
Inner ear development requires the coordination of cell types from distinct epithelial, mesenchymal and neuronal lineages. Although we have learned much from animal models, many details about human inner ear development remain elusive. We recently developed an in vitro model of human inner ear organogenesis using pluripotent stem cells in a 3D culture, fostering the growth of a sensorineural circuit, including hair cells and neurons. Despite previously characterizing some cell types, many remain undefined. This study aimed to chart the in vitro development timeline of the inner ear organoid to understand the mechanisms at play. Using single-cell RNA sequencing at ten stages during the first 36â days of differentiation, we tracked the evolution from pluripotency to various ear cell types after exposure to specific signaling modulators. Our findings showcase gene expression that influences differentiation, identifying a plethora of ectodermal and mesenchymal cell types. We also discern aspects of the organoid model consistent with in vivo development, while highlighting potential discrepancies. Our study establishes the Inner Ear Organoid Developmental Atlas (IODA), offering deeper insights into human biology and improving inner ear tissue differentiation.