Clarin-2 gene supplementation durably preserves hearing in a model of progressive hearing loss.

Hearing loss is a major health concern affecting millions of people worldwide with currently limited treatment options. In clarin-2-deficient Clrn2-/- mice, used here as a model of progressive hearing loss, we report synaptic auditory abnormalities in addition to the previously demonstrated defects of hair bundle structure and mechanoelectrical transduction. We sought an in-depth evaluation of viral-mediated gene delivery as a therapy for these hearing-impaired mice. Supplementation with either the murine Clrn2 or human CLRN2 genes preserved normal hearing in treated Clrn2-/- mice. Conversely, mutated forms of CLRN2, identified in patients with post-lingual moderate to severe hearing loss, failed to prevent hearing loss. The ectopic expression of clarin-2 successfully prevented the loss of stereocilia, maintained normal mechanoelectrical transduction, preserved inner hair cell synaptic function, and ensured near-normal hearing thresholds over time. Maximal hearing preservation was observed when Clrn2 was delivered prior to the loss of transducing stereocilia. Our findings demonstrate that gene therapy is effective for the treatment of post-lingual hearing impairment and age-related deafness associated with CLRN2 patient mutations.

Vestibular Deficits in Deafness: Clinical Presentation, Animal Modeling, and Treatment Solutions.

The inner ear is responsible for both hearing and balance. These functions are dependent on the correct functioning of mechanosensitive hair cells, which convert sound- and motion-induced stimuli into electrical signals conveyed to the brain. During evolution of the inner ear, the major changes occurred in the hearing organ, whereas the structure of the vestibular organs remained constant in all vertebrates over the same period. Vestibular deficits are highly prevalent in humans, due to multiple intersecting causes: genetics, environmental factors, ototoxic drugs, infections and aging. Studies of deafness genes associated with balance deficits and their corresponding animal models have shed light on the development and function of these two sensory systems. Bilateral vestibular deficits often impair individual postural control, gaze stabilization, locomotion and spatial orientation. The resulting dizziness, vertigo, and/or falls (frequent in elderly populations) greatly affect patient quality of life. In the absence of treatment, prosthetic devices, such as vestibular implants, providing information about the direction, amplitude and velocity of body movements, are being developed and have given promising results in animal models and humans. Novel methods and techniques have led to major progress in gene therapies targeting the inner ear (gene supplementation and gene editing), 3D inner ear organoids and reprograming protocols for generating hair cell-like cells. These rapid advances in multiscale approaches covering basic research, clinical diagnostics and therapies are fostering interdisciplinary research to develop personalized treatments for vestibular disorders.

The genetic and phenotypic landscapes of Usher syndrome: from disease mechanisms to a new classification.

Usher syndrome (USH) is the most common cause of deaf-blindness in humans, with a prevalence of about 1/10,000 (~ 400,000 people worldwide). Cochlear implants are currently used to reduce the burden of hearing loss in severe-to-profoundly deaf patients, but many promising treatments including gene, cell, and drug therapies to restore the native function of the inner ear and retinal sensory cells are under investigation. The traditional clinical classification of Usher syndrome defines three major subtypes-USH1, 2 and 3-according to hearing loss severity and onset, the presence or absence of vestibular dysfunction, and age at onset of retinitis pigmentosa. Pathogenic variants of nine USH genes have been initially reported: MYO7A, USH1C, PCDH15, CDH23, and USH1G for USH1, USH2A, ADGRV1, and WHRN for USH2, and CLRN1 for USH3. Based on the co-occurrence of hearing and vision deficits, the list of USH genes has been extended to few other genes, but with limited supporting information. A consensus on combined criteria for Usher syndrome is crucial for the development of accurate diagnosis and to improve patient management. In recent years, a wealth of information has been obtained concerning the properties of the Usher proteins, related molecular networks, potential genotype-phenotype correlations, and the pathogenic mechanisms underlying the impairment or loss of hearing, balance and vision. The advent of precision medicine calls for a clear and more precise diagnosis of Usher syndrome, exploiting all the existing data to develop a combined clinical/genetic/network/functional classification for Usher syndrome.

Review of Genotype-Phenotype Correlations in Usher Syndrome.

Usher syndrome (USH) encompasses a group of clinically and genetically heterogenous disorders defined by the triad of sensorineural hearing loss (SNHL), vestibular dysfunction, and vision loss. USH is the most common cause of deaf blindness. USH is divided clinically into three subtypes-USH1, USH2, and USH3-based on symptom severity, progression, and age of onset. The underlying genetics of these USH forms are, however, significantly more complex, with over a dozen genes linked to the three primary clinical subtypes and other atypical USH phenotypes. Several of these genes are associated with other deaf-blindness syndromes that share significant clinical overlap with USH, pointing to the limits of a clinically based classification system. The genotype-phenotype relationships among USH forms also may vary significantly based on the location and type of mutation in the gene of interest. Understanding these genotype-phenotype relationships and associated natural disease histories is necessary for the successful development and application of gene-based therapies and precision medicine approaches to USH. Currently, the state of knowledge varies widely depending on the gene of interest. Recent studies utilizing next-generation sequencing technology have expanded the list of known pathogenic mutations in USH genes, identified new genes associated with USH-like phenotypes, and proposed algorithms to predict the phenotypic effects of specific categories of allelic variants. Further work is required to validate USH gene causality, and better define USH genotype-phenotype relationships and disease natural histories-particularly for rare mutations-to lay the groundwork for the future of USH treatment.

Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss.

Blindness and deafness are the most frequent sensory disorders in humans. Whatever their cause - genetic, environmental, or due to toxic agents, or aging - the deterioration of these senses is often linked to irreversible damage to the light-sensing photoreceptor cells (blindness) and/or the mechanosensitive hair cells (deafness). Efforts are increasingly focused on preventing disease progression by correcting or replacing the blindness and deafness-causal pathogenic alleles. In recent years, gene replacement therapies for rare monogenic disorders of the retina have given positive results, leading to the marketing of the first gene therapy product for a form of childhood hereditary blindness. Promising results, with a partial restoration of auditory function, have also been reported in preclinical models of human deafness. Silencing approaches, including antisense oligonucleotides, adeno-associated virus (AAV)-mediated microRNA delivery, and genome-editing approaches have also been applied to various genetic forms of blindness and deafness The discovery of new DNA- and RNA-based CRISPR/Cas nucleases, and the new generations of base, prime, and RNA editors offers new possibilities for directly repairing point mutations and therapeutically restoring gene function. Thanks to easy access and immune-privilege status of self-contained compartments, the eye and the ear continue to be at the forefront of developing therapies for genetic diseases. Here, we review the ongoing applications and achievements of this new class of emerging therapeutics in the sensory organs of vision and hearing, highlighting the challenges ahead and the solutions to be overcome for their successful therapeutic application in vivo.

Stem Cells and Gene Therapy in Progressive Hearing Loss: the State of the Art.

Progressive non-syndromic sensorineural hearing loss (PNSHL) is the most common cause of sensory impairment, affecting more than a third of individuals over the age of 65. PNSHL includes noise-induced hearing loss (NIHL) and inherited forms of deafness, among which is delayed-onset autosomal dominant hearing loss (AD PNSHL). PNSHL is a prime candidate for genetic therapies due to the fact that PNSHL has been studied extensively, and there is a potentially wide window between identification of the disorder and the onset of hearing loss. Several gene therapy strategies exist that show potential for targeting PNSHL, including viral and non-viral approaches, and gene editing versus gene-modulating approaches. To fully explore the potential of these therapy strategies, a faithful in vitro model of the human inner ear is needed. Such models may come from induced pluripotent stem cells (iPSCs). The development of new treatment modalities by combining iPSC modeling with novel and innovative gene therapy approaches will pave the way for future applications leading to improved quality of life for many affected individuals and their families.

A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans.

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Inner Ear Gene Therapies Take Off: Current Promises and Future Challenges.

Hearing impairment is the most frequent sensory deficit in humans of all age groups, from children (1/500) to the elderly (more than 50% of the over-75 s). Over 50% of congenital deafness are hereditary in nature. The other major causes of deafness, which also may have genetic predisposition, are aging, acoustic trauma, ototoxic drugs such as aminoglycosides, and noise exposure. Over the last two decades, the study of inherited deafness forms and related animal models has been instrumental in deciphering the molecular, cellular, and physiological mechanisms of disease. However, there is still no curative treatment for sensorineural deafness. Hearing loss is currently palliated by rehabilitation methods: conventional hearing aids, and for more severe forms, cochlear implants. Efforts are continuing to improve these devices to help users to understand speech in noisy environments and to appreciate music. However, neither approach can mediate a full recovery of hearing sensitivity and/or restoration of the native inner ear sensory epithelia. New therapeutic approaches based on gene transfer and gene editing tools are being developed in animal models. In this review, we focus on the successful restoration of auditory and vestibular functions in certain inner ear conditions, paving the way for future clinical applications.

Disease mechanisms and gene therapy for Usher syndrome.

Usher syndrome (USH) is a major cause of deaf-blindness in humans, affecting ∼400 000 patients worldwide. Three clinical subtypes, USH1-3, have been defined, with 10 USH genes identified so far. In recent years, in addition to identification of new Usher genes and diagnostic tools, major progress has been made in understanding the role of Usher proteins and how they cooperate through interaction networks to ensure proper development, architecture and function of the stereociliary bundle at the apex of sensory hair cells in the inner ear. Several Usher mouse models of known human Usher genes have been characterized. These mice faithfully reproduce the auditory phenotype associated with Usher syndrome and the vestibular phenotype associated with some mutations in USH genes, particularly USH1. Interestingly, very few mouse models of Usher syndrome recapitulate the retinal phenotype associated with the disease in human. Usher patients can benefit from hearing aids or cochlear implants, which partially alleviate auditory sensory deprivation. However, there are currently no biological treatments available for auditory or visual dysfunction in Usher patients. Development of novel therapies for Usher syndrome has sprouted over the past decade, building on recent progress in gene transfer and new gene editing tools. Promising success demonstrating recovery of hearing and balance functions have been obtained via distinct therapeutic strategies in animal models. Clinical translation to Usher patients, however, calls for further improvements and concerted efforts to overcome the challenges ahead.

Identification of a CDH12 potential candidate genetic variant for an autosomal dominant form of transgrediens and progrediens palmoplantar keratoderma in a Tunisian family.

Molecular diagnosis of rare inherited palmoplantar keratoderma (PPK) is still challenging. We investigated at the clinical and genetic level a consanguineous Tunisian family presenting an autosomal dominant atypical form of transgrediens and progrediens PPK to better characterize this ultrarare disease and to identify its molecular etiology. Whole-exome sequencing (WES), filtering strategies, and bioinformatics analysis have been achieved. Clinical investigation and follow up over 13 years of this Tunisian family with three siblings formerly diagnosed as an autosomal recessive form of Mal de Melela-like conducted us to reconsider its initial phenotype. Indeed, the three patients presented clinical features that overlap both Mal de Meleda and progressive symmetric erythrokeratoderma (PSEK). The mode of inheritance was also reconsidered, since the mother, initially classified as unaffected, exhibited a similar expression of the disease. WES analysis showed the absence of potentially functional rare variants in known PPKs or PSEK-related genes. Results revealed a novel heterozygous nonsynonymous variant in cadherin-12 gene (CDH12, NM_004061, c.1655C > A, p.Thr552Asn) in all affected family members. This variant is absent in dbSNP and in 50 in-house control exomes. In addition, in silico analysis of the mutated 3D domain structure predicted that this variant would result in cadherin-12 protein destabilization and thermal instability. Functional annotation and biological network construction data provide further supporting evidence for the potential role of CDH12 in the maintenance of skin integrity. Taken together, these results suggest that CDH12 gene is a potential candidate gene for an atypical presentation of an autosomal dominant form of transgrediens and progrediens PPK.

Clarin-2 is essential for hearing by maintaining stereocilia integrity and function.

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome.

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4-/-) and postnatal hair cell-specific conditional (Clrn1ex4fl/fl Myo15-Cre+/-) knockout mice. Clrn1ex4-/- mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/- mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavß2 auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/- mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

Cone degeneration is triggered by the absence of USH1 proteins but prevented by antioxidant treatments.

Usher syndrome type 1 (USH1) is a major cause of inherited deafness and blindness in humans. The eye disorder is often referred to as retinitis pigmentosa, which is characterized by a secondary cone degeneration following the rod loss. The development of treatments to prevent retinal degeneration has been hampered by the lack of clear evidence for retinal degeneration in mutant mice deficient for the Ush1 genes, which instead faithfully mimic the hearing deficit. We show that, under normal housing conditions, Ush1g-/- and Ush1c-/- albino mice have dysfunctional cone photoreceptors whereas pigmented knockout animals have normal photoreceptors. The key involvement of oxidative stress in photoreceptor apoptosis and the ensued retinal gliosis were further confirmed by their prevention when the mutant mice are reared under darkness and/or supplemented with antioxidants. The primary degeneration of cone photoreceptors contrasts with the typical forms of retinitis pigmentosa. Altogether, we propose that oxidative stress probably accounts for the high clinical heterogeneity among USH1 siblings, which also unveils potential targets for blindness prevention.

CIB2, defective in isolated deafness, is key for auditory hair cell mechanotransduction and survival.

Defects of CIB2, calcium- and integrin-binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type-IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2-/- mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2-/- mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair-cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2-/- mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.

Conformational switch of harmonin, a submembrane scaffold protein of the hair cell mechanoelectrical transduction machinery.

Mutations in the gene encoding harmonin, a multi-PDZ domain-containing submembrane protein, cause Usher syndrome type 1 (congenital deafness and balance disorder, and early-onset sight loss). The structure of the protein and biological activities of its three different classes of splice isoforms (a, b, and c) remain poorly understood. Combining biochemical and biophysical analyses, we show that harmonin-a1 can switch between open and closed conformations through intramolecular binding of its C-terminal PDZ-binding motif to its N-terminal supramodule NTD-PDZ1 and through a flexible PDZ2-PDZ3 linker. This conformational switch presumably extends to most harmonin isoforms, and it is expected to have an impact on the interaction with some binding partners, as shown here for cadherin-related 23, another component of the hair cell mechanoelectrical transduction machinery.

Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal.

Spectrin ßV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians.

The remarkable hearing capacities of mammals arise from various evolutionary innovations. These include the cochlear outer hair cells and their singular feature, somatic electromotility, i.e., the ability of their cylindrical cell body to shorten and elongate upon cell depolarization and hyperpolarization, respectively. To shed light on the processes underlying the emergence of electromotility, we focused on the ßV giant spectrin, a major component of the outer hair cells' cortical cytoskeleton. We identified strong signatures of adaptive evolution at multiple sites along the spectrin-ßV amino acid sequence in the lineage leading to mammals, together with substantial differences in the subcellular location of this protein between the frog and the mouse inner ear hair cells. In frog hair cells, spectrin ßV was invariably detected near the apical junctional complex and above the cuticular plate, a dense F-actin meshwork located underneath the apical plasma membrane. In the mouse, the protein had a broad punctate cytoplasmic distribution in the vestibular hair cells, whereas it was detected in the entire lateral wall of cochlear outer hair cells and had an intermediary distribution (both cytoplasmic and cortical, but restricted to the cell apical region) in cochlear inner hair cells. Our results support a scenario where the singular organization of the outer hair cells' cortical cytoskeleton may have emerged from molecular networks initially involved in membrane trafficking, which were present near the apical junctional complex in the hair cells of mammalian ancestors and would have subsequently expanded to the entire lateral wall in outer hair cells.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients.

Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.