Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

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.

ZBTB20 is essential for cochlear maturation and hearing in mice.

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral nonsensory region. Here, we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear nonsensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated, but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane (TM), a flattened spiral prominence (SP), and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the nonsensory epithelium covering the outer border Claudius cells, outer sulcus root cells, and SP epithelial cells. Transcriptome analysis shows that ZBTB20 regulates genes encoding for TM proteins in the greater epithelial ridge, and those preferentially expressed in root cells and SP epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral nonsensory domain.

Specification of neuronal subtypes in the spiral ganglion begins prior to birth in the mouse.

The afferent innervation of the cochlea is comprised of spiral ganglion neurons (SGNs), which are characterized into four subtypes (Type 1A, B, and C and Type 2). However, little is known about the factors and/or processes that determine each subtype. Here, we present a transcriptional analysis of approximately 5,500 single murine SGNs collected across four developmental time points. All four subtypes are transcriptionally identifiable prior to the onset of coordinated spontaneous activity, indicating that the initial specification process is under genetic control. Trajectory analysis indicates that SGNs initially split into two precursor types (Type 1A/2 and Type 1B/C), followed by subsequent splits to give rise to four transcriptionally distinct subtypes. Differential gene expression, pseudotime, and regulon analyses were used to identify candidate transcription factors which may regulate the subtypes specification process. These results provide insights into SGN development and comprise a transcriptional atlas of SGN maturation across the prenatal period.

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.

Helios is a key transcriptional regulator of outer hair cell maturation.

The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.

Development of the cochlea.

The cochlea, a coiled structure located in the ventral region of the inner ear, acts as the primary structure for the perception of sound. Along the length of the cochlear spiral is the organ of Corti, a highly derived and rigorously patterned sensory epithelium that acts to convert auditory stimuli into neural impulses. The development of the organ of Corti requires a series of inductive events that specify unique cellular characteristics and axial identities along its three major axes. Here, we review recent studies of the cellular and molecular processes regulating several aspects of cochlear development, such as axial patterning, cochlear outgrowth and cellular differentiation. We highlight how the precise coordination of multiple signaling pathways is required for the successful formation of a complete organ of Corti.

YAP Mediates Hair Cell Regeneration in Balance Organs of Chickens, But LATS Kinases Suppress Its Activity in Mice.

Loss of sensory hair cells causes permanent hearing and balance deficits in humans and other mammals, but for nonmammals such deficits are temporary. Nonmammals recover hearing and balance sensitivity after supporting cells proliferate and differentiate into replacement hair cells. Evidence of mechanical differences between those sensory epithelia and their supporting cells prompted us to investigate whether the capacity to activate YAP, an effector in the mechanosensitive Hippo pathway, correlates with regenerative capacity in acceleration-sensing utricles of chickens and mice of both sexes. After hair cell ablation, YAP accumulated in supporting cell nuclei in chicken utricles and promoted regenerative proliferation, but YAP remained cytoplasmic and little proliferation occurred in mouse utricles. YAP localization in supporting cells was also more sensitive to shape change and inhibition of MST1/2 in chicken utricles than in mouse utricles. Genetic manipulations showed that in vivo expression of the YAP-S127A variant caused robust proliferation of neonatal mouse supporting cells, which produced progeny that expressed hair cell markers, but proliferative responses declined postnatally. Expression of YAP-5SA, which more effectively evades inhibitory phosphorylation, resulted in TEAD-dependent proliferation of striolar supporting cells, even in adult utricles. Conditional deletion of LATS1/2 kinases abolished the inhibitory phosphorylation of endogenous YAP and led to striolar proliferation in adult mouse utricles. The findings suggest that damage overcomes inhibitory Hippo signaling and facilitates regenerative proliferation in nonmammalian utricles, whereas constitutive LATS1/2 kinase activity suppresses YAP-TEAD signaling in mammalian utricles and contributes to maintaining the proliferative quiescence that appears to underlie the permanence of sensory deficits.SIGNIFICANCE STATEMENT Loud sounds, ototoxic drugs, infections, and aging kill sensory hair cells in the ear, causing irreversible hearing loss and balance deficits for millions. In nonmammals, damage evokes shape changes in supporting cells, which can divide and regenerate hair cells. Such shape changes are limited in mammalian ears, where supporting cells develop E-cadherin-rich apical junctions reinforced by robust F-actin bands, and the cells fail to divide. Here, we find that damage readily activates YAP in supporting cells within balance epithelia of chickens, but not mice. Deleting LATS kinases or expressing YAP variants that evade LATS-mediated inhibitory phosphorylation induces proliferation in supporting cells of adult mice. YAP signaling eventually may be harnessed to overcome proliferative quiescence that limits regeneration in mammalian ears.

Identification and characterization of key long non-coding RNAs in the mouse cochlea.

The auditory system is a complex sensory network with an orchestrated multilayer regulatory programme governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope®  analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

Pou4f1 Defines a Subgroup of Type I Spiral Ganglion Neurons and Is Necessary for Normal Inner Hair Cell Presynaptic Ca2+ Signaling.

Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells (IHCs) and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the IHC. More recently, single-cell profiling has identified multiple groups of SGNs based on transcriptional profiling; however, correlations between any of these groups and distinct neuronal physiology have not been determined. In this study, we show that expression of the POU (Pit-Oct-Unc) transcription factor Pou4f1 in type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early path-finding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca2+ channels in IHCs primarily by increasing their voltage sensitivity. Moreover, the modiolar to pillar gradient of active zone Ca2+ influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca2+ signaling in IHCs.SIGNIFICANCE STATEMENT Physiologically distinct classes of type I spiral ganglion neurons (SGNs) are necessary to encode sound intensities spanning the audible range. Although anatomical studies have demonstrated structural correlates for some physiologically defined classes of type I SGNs, an understanding of the molecular pathways that specify each type is only now emerging. Here, we demonstrate that expression of the transcription factor Pou4f1 corresponds to a distinct subgroup of type I SGNs that synapse on the modiolar side of inner hair cells. The conditional deletion of Pou4f1 after SGN formation does not disrupt ganglion size or morphology, change the distribution of IHC synaptic locations, or affect the creation of synapses, but it does influence the voltage dependence and strength of Ca2+ influx at presynaptic active zones in inner hair cells.

GSK3 regulates hair cell fate in the developing mammalian cochlea.

The development of asymmetric patterns along biologically relevant axes is a hallmark of many vertebrate organs or structures. One example is the sensory epithelium of the mammalian auditory system. Two distinct types of mechanosensory hair cells (inner and outer) and at least six types of associated supporting cells are precisely and asymmetrically arrayed along the radial (medial-lateral) axis of the cochlear spiral. Immunolabeling of developing cochleae indicates differential expression of Glycogen synthase kinase 3ß (GSK3ß) along the same axis. To determine whether GSK3ß plays a role in specification of cell fates along the medial-lateral axis, GSK3 activity was blocked pharmacologically in cochlear explants. Results indicate significant changes in both the number of hair cells and in the specification of hair cell phenotypes. The overall number of inner hair cells increased as a result of both a shift in the medial boundary between sensory and non-sensory regions of the cochlea and a change in the specification of inner and outer hair cell phenotypes. Previous studies have inhibited GSK3 as a method to examine effects of canonical Wnt signaling. However, quantification of changes in Wnt pathway target genes in GSK3-inhibited cochleae, and treatment with more specific Wnt agonists, indicated that the Wnt pathway is not activated. Instead, expression of Bmp4 in a population of GSK3ß-expressing cells was shown to be down-regulated. Finally, addition of BMP4 to GSK3-inhibited cochleae achieved a partial rescue of the hair cell phenotype. These results demonstrate a role for GSK3ß in the specification of cellular identities along the medial-lateral axis of the cochlea and provide evidence for a positive role for GSK3ß in the expression of Bmp4.

Cell migration, intercalation and growth regulate mammalian cochlear extension.

Developmental remodeling of the sensory epithelium of the cochlea is required for the formation of an elongated, tonotopically organized auditory organ, but the cellular processes that mediate these events are largely unknown. We used both morphological assessments of cellular rearrangements and time-lapse imaging to visualize cochlear remodeling in mouse. Analysis of cell redistribution showed that the cochlea extends through a combination of radial intercalation and cell growth. Live imaging demonstrated that concomitant cellular intercalation results in a brief period of epithelial convergence, although subsequent changes in cell size lead to medial-lateral spreading. Supporting cells, which retain contact with the basement membrane, exhibit biased protrusive activity and directed movement along the axis of extension. By contrast, hair cells lose contact with the basement membrane, but contribute to continued outgrowth through increased cell size. Regulation of cellular protrusions, movement and intercalation within the cochlea all require myosin II. These results establish, for the first time, many of the cellular processes that drive the distribution of sensory cells along the tonotopic axis of the cochlea.

An optimized protocol for retina single-cell RNA sequencing.

Purpose: Single-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis. Methods: We modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq. Results: We report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq. Conclusions: We provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.

Spectrum of genes for inherited hearing loss in the Israeli Jewish population, including the novel human deafness gene ATOH1.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss: ATOH1 (Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results show that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.

Making connections in the inner ear: recent insights into the development of spiral ganglion neurons and their connectivity with sensory hair cells.

In mammals, auditory information is processed by the hair cells (HCs) located in the cochlea and then rapidly transmitted to the CNS via a specialized cluster of bipolar afferent connections known as the spiral ganglion neurons (SGNs). Although many anatomical aspects of SGNs are well described, the molecular and cellular mechanisms underlying their genesis, how they are precisely arranged along the cochlear duct, and the guidance mechanisms that promote the innervation of their hair cell targets are only now being understood. Building upon foundational studies of neurogenesis and neurotrophins, we review here new concepts and technologies that are helping to enrich our understanding of the development of the nervous system within the inner ear.

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea.

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.

A dual function for canonical Wnt/ß-catenin signaling in the developing mammalian cochlea.

The canonical Wnt/ß-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/ß-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/ß-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/ß-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.

Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear.

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.