Mechanosensory Hair Cells Research Articles

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.

Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Transdifferentiation is temporally uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear.

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.

Multiple mechanisms of aminoglycoside ototoxicity are distinguished by subcellular localization of action.

Mechanosensory hair cells of the inner ears and lateral line of vertebrates display heightened vulnerability to environmental insult, with damage resulting in hearing and balance disorders. An important example is hair cell loss due to exposure to toxic agents including therapeutic drugs such as the aminoglycoside antibiotics such as neomycin and gentamicin and antineoplastic agents. We describe two distinct cellular pathways for aminoglycoside-induced hair cell death in zebrafish lateral line hair cells. Neomycin exposure results in death from acute exposure with most cells dying within 1 hour of exposure. By contrast, exposure to gentamicin results primarily in delayed hair cell death, taking up to 24 hours for maximal effect. Washout experiments demonstrate that delayed death does not require continuous exposure, demonstrating two mechanisms where downstream responses differ in their timing. Acute damage is associated with mitochondrial calcium fluxes and can be alleviated by the mitochondrially-targeted antioxidant mitoTEMPO, while delayed death is independent of these factors. Conversely delayed death is associated with lysosomal accumulation and is reduced by altering endolysosomal function, while acute death is not sensitive to lysosomal manipulations. These experiments reveal the complexity of responses of hair cells to closely related compounds, suggesting that intervention focusing on early events rather than specific death pathways may be a successful therapeutic strategy.

Multifunctional redox modulator prevents blast-induced loss of cochlear and vestibular hair cells and auditory spiral ganglion neurons

Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.

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.

Modern In Vitro Techniques for Modeling Hearing Loss.

Sensorineural hearing loss (SNHL) is a prevalent and growing global health concern, especially within operational medicine, with limited therapeutic options available. This review article explores the emerging field of in vitro otic organoids as a promising platform for modeling hearing loss and developing novel therapeutic strategies. SNHL primarily results from the irreversible loss or dysfunction of cochlear mechanosensory hair cells (HCs) and spiral ganglion neurons (SGNs), emphasizing the need for innovative solutions. Current interventions offer symptomatic relief but do not address the root causes. Otic organoids, three-dimensional multicellular constructs that mimic the inner ear's architecture, have shown immense potential in several critical areas. They enable the testing of gene therapies, drug discovery for sensory cell regeneration, and the study of inner ear development and pathology. Unlike traditional animal models, otic organoids closely replicate human inner ear pathophysiology, making them invaluable for translational research. This review discusses methodological advances in otic organoid generation, emphasizing the use of human pluripotent stem cells (hPSCs) to replicate inner ear development. Cellular and molecular characterization efforts have identified key markers and pathways essential for otic organoid development, shedding light on their potential in modeling inner ear disorders. Technological innovations, such as 3D bioprinting and microfluidics, have further enhanced the fidelity of these models. Despite challenges and limitations, including the need for standardized protocols and ethical considerations, otic organoids offer a transformative approach to understanding and treating auditory dysfunctions. As this field matures, it holds the potential to revolutionize the treatment landscape for hearing and balance disorders, moving us closer to personalized medicine for inner ear conditions.

Identification of multiple transcription factor genes potentially involved in the development of electrosensory versus mechanosensory lateral line organs.

In electroreceptive jawed vertebrates, embryonic lateral line placodes give rise to electrosensory ampullary organs as well as mechanosensory neuromasts. Previous reports of shared gene expression suggest that conserved mechanisms underlie electroreceptor and mechanosensory hair cell development and that electroreceptors evolved as a transcriptionally related "sister cell type" to hair cells. We previously identified only one transcription factor gene, Neurod4, as ampullary organ-restricted in the developing lateral line system of a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula). The other 16 transcription factor genes we previously validated in paddlefish were expressed in both ampullary organs and neuromasts. Here, we used our published lateral line organ-enriched gene-set (arising from differential bulk RNA-seq in late-larval paddlefish), together with a candidate gene approach, to identify 25 transcription factor genes expressed in the developing lateral line system of a more experimentally tractable chondrostean, the sterlet (Acipenser ruthenus, a small sturgeon), and/or that of paddlefish. Thirteen are expressed in both ampullary organs and neuromasts, consistent with conservation of molecular mechanisms. Seven are electrosensory-restricted on the head (Irx5, Irx3, Insm1, Sp5, Satb2, Mafa and Rorc), and five are the first-reported mechanosensory-restricted transcription factor genes (Foxg1, Sox8, Isl1, Hmx2 and Rorb). However, as previously reported, Sox8 is expressed in ampullary organs as well as neuromasts in a catshark (Scyliorhinus canicula), suggesting the existence of lineage-specific differences between cartilaginous and ray-finned fishes. Overall, our results support the hypothesis that ampullary organs and neuromasts develop via largely conserved transcriptional mechanisms, and identify multiple transcription factors potentially involved in the formation of electrosensory versus mechanosensory lateral line organs.

Localization of cadherins in the postnatal cochlear epithelium and their relation to space formation.

The sensory epithelium of the cochlea, the organ of Corti, has complex cytoarchitecture consisting of mechanosensory hair cells intercalated by epithelial support cells. The support cells provide important trophic and structural support to the hair cells. Thus, the support cells must be stiff yet compliant enough to withstand and modulate vibrations to the hair cells. Once the sensory cells are properly patterned, the support cells undergo significant remodeling from a simple epithelium into a structurally rigid epithelium with fluid-filled spaces in the murine cochlea. Cell adhesion molecules such as cadherins are necessary for sorting and connecting cells in an intact epithelium. To create the fluid-filled spaces, cell adhesion properties of adjoining cell membranes between cells must change to allow the formation of spaces within an epithelium. However, the dynamic localization of cadherins has not been properly analyzed as these spaces are formed. There are three cadherins that are reported to be expressed during the first postnatal week of development when the tunnel of Corti forms in the cochlea. In this study, we characterize the dynamic localization of cadherins that are associated with cytoskeletal remodeling at the contacting membranes of the inner and outer pillar cells flanking the tunnel of Corti.

Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells

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.

Artificial Hair Cell Sensor Based on Nanofiber-Reinforced Thin Metal Films.

Engineering artificial mechanosensory hair cells offers a promising avenue for developing diverse biosensors spanning applications from biomedicine to underwater sensing. Unfortunately, current artificial sensory hair cells do not have the ability to simultaneously achieve ultrahigh sensitivity with low-frequency threshold detection (e.g., 0.1 Hz). This work aimed to solve this gap by developing an artificial sensory hair cell inspired by the vestibular sensory apparatus, which has such functional capabilities. For device characterization and response testing, the sensory unit was inserted in a 3D printed lateral semicircular canal (LSCC) mimicking the environment of the labyrinth. The sensor was fabricated based on platinum (Pt) thin film which was reinforced by carbon nanofibers (CNFs). A Pi-shaped hair cell sensor was created as the sensing element which was tested under various conditions of simulated head motion. Results reveal the hair cell sensor displayed markedly higher sensitivity compared to other reported artificial hair cell sensors (e.g., 21.47 mV Hz-1 at 60°) and low frequency detection capability, 0.1 Hz < f < 1.5 Hz. Moreover, like the LSCC hair cells in biology, the fabricated sensor was most sensitive in a given plane of rotational motion, demonstrating features of directional sensitivity.

EpCAM regulates hair cell development and regeneration in the zebrafish lateral line.

The zebrafish lateral line mechanosensory system shares considerable morphological and molecular similarities with the inner ear. In particular, mechanosensory hair cells are responsible for transducing sensory stimuli in both structures. The epithelia cell adhesion molecule (EpCAM) is expressed in the cells of the inner ear of mammals and in the lateral lines system of fish. EpCAM regulates the many cellular functions including adhesion, migration, proliferation, and differentiation. In this study, we use the epcam jh79 mutant zebrafish line to determine that EpCAM function is required for proper development and regeneration of posterior lateral line hair cells.

Dynamic micro-optical coherence tomography enables structural and metabolic imaging of the mammalian cochlea.

Sensorineural hearing loss (SNHL) is caused by damage to the mechanosensory hair cells and auditory neurons of the cochlea. The development of imaging tools that can directly visualize or provide functional information about a patient's cochlear cells is critical to identify the pathobiological defect and determine the cells' receptiveness to emerging SNHL treatments. However, the cochlea's small size, embedded location within dense bone, and sensitivity to perturbation have historically precluded high-resolution clinical imaging. Previously, we developed micro-optical coherence tomography (μOCT) as a platform for otologic imaging in animal models and human cochleae. Here we report on advancing μOCT technology to obtain simultaneously acquired and co-localized images of cell viability/metabolic activity through dynamic μOCT (DμOCT) imaging of intracellular motion. DμOCT obtains cross-sectional images of ATP-dependent movement of intracellular organelles and cytoskeletal polymerization by acquiring sequential μOCT images and computing intensity fluctuation frequency metrics on a pixel-wise basis. Using a customized benchtop DμOCT system, we demonstrate the detailed resolution of anatomical and metabolic features of cells within the organ of Corti, via an apical cochleostomy, in freshly-excised adult mouse cochleae. Further, we show that DμOCT is capable of capturing rapid changes in cochlear cell metabolism following an ototoxic insult to induce cell death and actin stabilization. Notably, as few as 6 frames can be used to reconstruct cochlear DμOCT images with sufficient detail to discern individual cells and their metabolic state. Taken together, these results motivate future development of a DμOCT imaging probe for cellular and metabolic diagnosis of SNHL in humans.

Kremen1 regulates the regenerative capacity of support cells and mechanosensory hair cells in the zebrafish lateral line

Mechanosensory hair cells in the inner ear mediate the sensations of hearing and balance, and in the specialized lateral line sensory system of aquatic vertebrates, the sensation of water movement. In mammals, hair cells lack the ability to regenerate following damage, resulting in sensory deficits. In contrast, non-mammalian vertebrates, such as zebrafish, can renew hair cells throughout their lifespan. Wnt signaling is required for development of inner ear and lateral line hair cells and regulates regeneration. Kremen1 inhibits Wnt signaling and hair cell formation, though its role in regeneration is unknown. We used a zebrafish kremen1 mutant line to show overactive Wnt signaling results in supernumerary support cells and hair cell regeneration without increased proliferation, in contrast with the previously described role of Wnt signaling during hair cell regeneration. This work allows us to understand the biology of mechanosensory hair cells and how regeneration might be promoted following damage.

Intranasal delivery of NGF rescues hearing impairment in aged SAMP8 mice

Hearing loss impacts the quality of life and affects communication resulting in social isolation and reduced well-being. Despite its impact on society and economy, no therapies for age-related hearing loss are available so far. Loss of mechanosensory hair cells of the cochlea is a common event of hearing loss in humans. Studies performed in birds demonstrating that they can be replaced following the proliferation and transdifferentiation of supporting cells, strongly pointed out on HCs regeneration as the main focus of research aimed at hearing regeneration. Neurotrophins are growth factors involved in neuronal survival, development, differentiation, and plasticity. NGF has been involved in the interplay between auditory receptors and efferent innervation in the cochlea during development. During embryo development, both NGF and its receptors are highly expressed in the inner ears. It has been reported that NGF is implicated in the differentiation of auditory gangliar and hair cells. Thus, it has been proposed that NGF administration can decrease neuronal damage and prevent hearing loss. The main obstacle to the development of hearing impairment therapy is that efficient means of delivery for selected drugs to the cochlea are missing. Herein, in this study NGF was administered by the intranasal route. The first part of the study was focused on a biodistribution study, which showed the effective delivery in the cochlea; while the second part was focused on analyzing the potential therapeutic effect of NGF in senescence-accelerated prone strain 8 mice. Interestingly, intranasal administration of NGF resulted protective in counteracting hearing impairment in SAMP8 mice, ameliorating hearing performances (analyzed by auditory brainstem responses and distortion product otoacoustic emission) and hair cells morphology (analyzed by microscopy analysis). The results obtained were encouraging indicating that the neurotrophin NGF was efficiently delivered to the inner ear and that it was effective in counteracting hearing loss.

SoxC transcription factors shape the epigenetic landscape to establish competence for sensory differentiation in the mammalian organ of Corti

The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.

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

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

Lrrn1 Regulates Medial Boundary Formation in the Developing Mouse Organ of Corti.

One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti (OC), is the presence of precise boundaries between sensory and nonsensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells (IHCs) and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 (Lrrn1), which codes for a single-pass, transmembrane protein, is expressed before the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent nonsensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.SIGNIFICANCE STATEMENT Patterning of the developing mammalian cochlea into distinct sensory and nonsensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein Leucine Rich Repeat Neuronal 1 (LRRN1) is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells (IHCs) and adjacent nonsensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/nonsensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.