Hair Cell Stereocilia Research Articles

HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice.

Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS) the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL). After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action.

Inner ear hair cells deteriorate in mice engineered to have no or diminished innervation.

The innervation of the inner ear critically depends on the two neurotrophins Ntf3 and Bdnf. In contrast to this molecularly well-established dependency, evidence regarding the need of innervation for long-term maintenance of inner ear hair cells is inconclusive, due to experimental variability. Mutant mice that lack both neurotrophins could shed light on the long-term consequences of innervation loss on hair cells without introducing experimental variability, but do not survive after birth. Mutant mice with conditional deletion of both neurotrophins lose almost all innervation by postnatal day 10 and show an initially normal development of hair cells by this stage. No innervation remains after 3 weeks and complete loss of all innervation results in near complete loss of outer and many inner hair cells of the organ of Corti within 4 months. Mutants that retain one allele of either neurotrophin have only partial loss of innervation of the organ of Corti and show a longer viability of cochlear hair cells with more profound loss of inner hair cells. By 10 months, hair cells disappear with a base to apex progression, proportional to the residual density of innervation and similar to carboplatin ototoxicity. Similar to reports of hair cell loss after aminoglycoside treatment, blobbing of stereocilia of apparently dying hair cells protrude into the cochlear duct. Denervation of vestibular sensory epithelia for several months also resulted in variable results, ranging from unusual hair cells resembling the aberrations found in the organ of Corti, to near normal hair cells in the canal cristae. Fusion and/or resorption of stereocilia and loss of hair cells follows a pattern reminiscent of Myo6 and Cdc42 null mice. Our data support a role of innervation for long-term maintenance but with a remarkable local variation that needs to be taken into account when attempting regeneration of the organ of Corti.

XIRP2, an actin-binding protein essential for inner ear hair-cell stereocilia.

Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN) are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.

Anion exchanger 1b in stereocilia is required for the functioning of mechanotransducer channels in lateral-line hair cells of zebrafish.

The anion exchanger (AE) plays critical roles in physiological processes including CO2 transport and volume regulation in erythrocytes and acid-base regulation in renal tubules. Although expression of the AE in inner-ear hair cells was reported, its specific localization and function are still unclear. Using in situ hybridization, we found that the AE1b transcript is expressed in lateral-line hair cells of zebrafish larvae. An immunohistochemical analysis with a zebrafish-specific antibody localized AE1b to stereocilia of hair cells, and the expression was eliminated by morpholino knockdown of AE1b. A non-invasive, scanning ion-selective electrode technique was applied to analyze mechanotransducer (MET) channel-mediated Ca2+ influx at stereocilia of hair cells of intact fish. Ca2+ influx was effectively suppressed by AE1b morpholino knockdown and inhibitor (DIDS) treatment. Elevating external Ca2+ (0.2 to 2 mM) neutralized the inhibition of DIDS. Taken together, this study provides solid evidence to show that AE1b in stereocilia is required for the proper functioning of MET channels.

OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67).

BackgroundEarly-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family.MethodsA panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation.ResultsOnset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice.ConclusionThe function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0238-5) contains supplementary material, which is available to authorized users.

Ganglioside GM3 is essential for the structural integrity and function of cochlear hair cells.

GM3 synthase (ST3GAL5) is the first biosynthetic enzyme of a- and b-series gangliosides. Patients with GM3 synthase deficiency suffer severe neurological disability and deafness. Eight children (ages 4.1 ± 2.3 years) homozygous for ST3GAL5 c.694C>T had no detectable GM3 (a-series) or GD3 (b-series) in plasma. Their auditory function was characterized by the absence of middle ear muscle reflexes, distortion product otoacoustic emissions and cochlear microphonics, as well as abnormal auditory brainstem responses and cortical auditory-evoked potentials. In St3gal5(-/-) mice, stereocilia of outer hair cells showed signs of degeneration as early as postnatal Day 3 (P3); thereafter, blebs devoid of actin or tubulin appeared at the region of vestigial kinocilia, suggesting impaired vesicular trafficking. Stereocilia of St3gal5(-/-) inner hair cells were fused by P17, and protein tyrosine phosphatase receptor Q, normally linked to myosin VI at the tapered base of stereocilia, was maldistributed along the cell membrane. B4galnt1(-/-) (GM2 synthase-deficient) mice expressing only GM3 and GD3 gangliosides had normal auditory structure and function. Thus, GM3-dependent membrane microdomains might be essential for the proper organization and maintenance of stereocilia in auditory hair cells.

A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function.

Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.

Power Dissipation in the Subtectorial Space of the Mammalian Cochlea Is Modulated by Inner Hair Cell Stereocilia

The stereocilia bundle is the mechano-transduction apparatus of the inner ear. In the mammalian cochlea, the stereocilia bundles are situated in the subtectorial space (STS)—a micrometer-thick space between two flat surfaces vibrating relative to each other. Because microstructures vibrating in fluid are subject to high-viscous friction, previous studies considered the STS as the primary place of energy dissipation in the cochlea. Although there have been extensive studies on how metabolic energy is used to compensate the dissipation, much less attention has been paid to the mechanism of energy dissipation. Using a computational model, we investigated the power dissipation in the STS. The model simulates fluid flow around the inner hair cell (IHC) stereocilia bundle. The power dissipation in the STS because of the presence IHC stereocilia increased as the stimulating frequency decreased. Along the axis of the stimulating frequency, there were two asymptotic values of power dissipation. At high frequencies, the power dissipation was determined by the shear friction between the two flat surfaces of the STS. At low frequencies, the power dissipation was dominated by the viscous friction around the IHC stereocilia bundle—the IHC stereocilia increased the STS power dissipation by 50- to 100-fold. There exists a characteristic frequency for STS power dissipation, CFSTS, defined as the frequency where power dissipation drops to one-half of the low frequency value. The IHC stereocilia stiffness and the gap size between the IHC stereocilia and the tectorial membrane determine the characteristic frequency. In addition to the generally assumed shear flow, nonshear STS flow patterns were simulated. Different flow patterns have little effect on the CFSTS. When the mechano-transduction of the IHC was tuned near the vibrating frequency, the active motility of the IHC stereocilia bundle reduced the power dissipation in the STS.

Unconventional Mechanics of Lipid Membranes: A Potential Role for Mechanotransduction of Hair Cell Stereocilia

A force-conveying role of the lipid membrane across various mechanoreceptors is now an accepted hypothesis. However, such a mechanism is still not fully understood for mechanotransduction in the hair bundle of auditory sensory hair cells. A major goal of this theoretical assessment was to investigate the role of the lipid membrane in auditory mechanotransduction, especially in generating nonlinear bundle force versus displacement measurements, one of the main features of auditory mechanotransduction. To this end, a hair bundle model that generates lipid membrane tented deformation in the stereocilia was developed. A computational analysis of the model not only reproduced nonlinear bundle force measurements but also generated membrane energy that is potentially sufficient to activate the mechanosensitive ion channel of the hair cell. In addition, the model provides biophysical insight into 1) the likelihood that the channel must be linked in some way to the tip link; 2) how the interplay of the bending and stretching of the lipid bilayer may be responsible for the nonlinear force versus displacement response; 3) how measurements of negative stiffness may be a function of the rotational stiffness of the rootlets; and 4) how the standing tension of the tip link is required to interpret migration of the nonlinear force versus displacement and activation curves. These are all features of hair cell mechanotransduction, but the underlying biophysical mechanism has proved elusive for the last three decades.

Bistability in Ephaptic Coupling as a Possible Memory Element Explored through a Colloid Interptation of Stereocilia Bistability

When the processes of neurons are in close proximity they effectively couple to each other in a process termed Ephaptic coupling. This coupling occurs without the membranes touching when the space between them is some tens of nanometers. If the intra-membrane spacing has a mechanical bistability, which moves the membranes closer and further apart, the Ephaptic coupling will be turned on and off allowing one neuron to read, or not read the content of the other. This paper explores the possibilities of bistable ephaptic memory element by a study of an analogous system that operates at a much larger scale, the Stereocilia of the hair cell. Published measurement of hair cell Stereocilia force/displacement function shows both a negative slope region in the displacement function and tendency to express bistability. We show here how this negative slope region can arise through the exigency of colloid forces. An explanation of Colloid Theory, presented in a graphic form, shows how a colloid force function can be modified to match the measured hair cell cilia force function. The colloid force function is modified by a limiting function resulting from Stereocilia side links, structural details that tie together the hair cell Stereocilia clusters. Understanding how a limited, simple behavior such as Stereocilia bistability functions may point to a more general understanding of how bistability may underlie other areas of living organisms such as memory and computation.

Structural and Biophysical Characterization of Inner Ear Tip Link Variants

At the molecular level, human hearing relies upon the tip-to-tip interaction of two unique non-classical cadherins, protocadherin 15 (Pcdh15) and cadherin 23 (Cdh23). Together, these two proteins form a filament called the tip link that connects neighboring stereocilia of mechanosensitive hair cells. Sound waves cause the stereocilia to deflect applying a force to the tip link and opening a nearby transduction channel. Disruption of the tip link caused by loud sound or chemical treatments eliminates transduction currents and illustrates that tip link integrity is critical for mechanosensing. Recent studies have found that remodeling of the tip link after disruption is a dynamic process, which leads to the formation of atypical complexes that may incorporate alternatively spliced variants or isoforms of Pcdh15. Our current work focuses on understanding these unusual tip links and comparing them with the prototypical tip link. Here, we present the crystal structure of a new complex formed from Cdh23 and isoform 2 of Pcdh15 refined at 3.5 A resolution. While the molecular structure reveals subtle differences between the two complexes, the binding affinity between Cdh23 and isoform 2 of Pcdh15 is notably different from Cdh23 and isoform 1 of Pcdh15 as observed in analytical size exclusion chromatography. These results clearly demonstrate that alternative heterotypic tip link structures form stable protein-protein interactions in vitro and provide no evidence to support the existence of homotypic Pcdh15-Pcdh15 tip links. Additional studies will focus on other Pcdh15 isoforms and determine how changes in the tip link structure alter the mechanical properties of the filament.

Neurog1 can partially substitute for Atoh1 function in hair cell differentiation and maintenance during organ of Corti development.

Atoh1, a basic helix-loop-helix (bHLH) transcription factor (TF), is essential for the differentiation of hair cells (HCs), mechanotransducers that convert sound into auditory signals in the mammalian organ of Corti (OC). Previous work demonstrated that replacing mouse Atoh1 with the fly ortholog atonal rescues HC differentiation, indicating functional replacement by other bHLH genes. However, replacing Atoh1 with Neurog1 resulted in reduced HC differentiation compared with transient Atoh1 expression in a 'self-terminating' Atoh1 conditional null mouse (Atoh1-Cre; Atoh1(f/f)). We now show that combining Neurog1 in one allele with removal of floxed Atoh1 in a self-terminating conditional mutant (Atoh1-Cre; Atoh1(f/kiNeurog1)) mouse results in significantly more differentiated inner HCs and outer HCs that have a prolonged longevity of 9 months compared with Atoh1 self-terminating littermates. Stereocilia bundles are partially disorganized, disoriented and not HC type specific. Replacement of Atoh1 with Neurog1 maintains limited expression of Pou4f3 and Barhl1 and rescues HCs quantitatively, but not qualitatively. OC patterning and supporting cell differentiation are also partially disrupted. Diffusible factors involved in patterning are reduced (Fgf8) and factors involved in cell-cell interactions are affected (Jag1, Hes5). Despite the presence of many HCs with stereocilia these mice are deaf, possibly owing to HC and OC patterning defects. This study provides a novel approach to disrupt OC development through modulating the HC-specific intracellular TF network. The resulting disorganized OC indicates that normally differentiated HCs act as 'self-organizers' for OC development and that Atoh1 plays a crucial role to initiate HC stereocilia differentiation independently of HC viability.

Single-Molecule Force-Spectroscopy of Inner Ear Proteins

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature allows us to identify single-molecule rupture events in pulling experiments.

Subunit determination of the conductance of hair-cell mechanotransducer channels

Cochlear hair cells convert sound stimuli into electrical signals by gating of mechanically sensitive ion channels in their stereociliary (hair) bundle. The molecular identity of this ion channel is still unclear, but its properties are modulated by accessory proteins. Two such proteins are transmembrane channel-like protein isoform 1 (TMC1) and tetraspan membrane protein of hair cell stereocilia (TMHS, also known as lipoma HMGIC fusion partner-like 5, LHFPL5), both thought to be integral components of the mechanotransduction machinery. Here we show that, in mice harboring an Lhfpl5 null mutation, the unitary conductance of outer hair cell mechanotransducer (MT) channels was reduced relative to wild type, and the tonotopic gradient in conductance, where channels from the cochlear base are nearly twice as conducting as those at the apex, was almost absent. The macroscopic MT current in these mutants was attenuated and the tonotopic gradient in amplitude was also lost, although the current was not completely extinguished. The consequences of Lhfpl5 mutation mirror those due to Tmc1 mutation, suggesting a part of the MT-channel conferring a large and tonotopically variable conductance is similarly disrupted in the absence of Lhfpl5 or Tmc1. Immunolabelling demonstrated TMC1 throughout the stereociliary bundles in wild type but not in Lhfpl5 mutants, implying the channel effect of Lhfpl5 mutations stems from down-regulation of TMC1. Both LHFPL5 and TMC1 were shown to interact with protocadherin-15, a component of the tip link, which applies force to the MT channel. We propose that titration of the TMC1 content of the MT channel sets the gradient in unitary conductance along the cochlea.

Methionine Sulfoxide Reductase A, B1 and B2 Are Likely to Be Involved in the Protection against Oxidative Stress in the Inner Ear

The methionine sulfoxide reductase (Msr) family of proteins is a class of repair enzymes that reduce methionine-S (MsrA) or methionine-R (MsrB) sulfoxide to methionine. Recent studies have reported that mutations in the MSRB3 gene cause autosomal recessive hearing loss in humans, and in mice MsrB3 deficiency leads to profound hearing loss due to hair cell apoptosis and stereocilia degeneration. However, apart from MsrB3, studies on Msr proteins in the inner ear have not yet been reported. In this study, we identified and characterized Msr expression in the cochlea and vestibule. First, we confirmed RNA expression levels of Msr family members in the cochlea and vestibule using reverse transcription PCR and detected Msr family members in both tissues. We also conducted immunohistochemical staining to localize Msr family members within the cochlea and vestibule. In the cochlea, MsrA was detected in supporting cells, spiral ligament, spiral limbus, Reissner's membrane and the spiral ganglion. MsrB1 was specifically expressed in hair cells and the spiral ganglion. MsrB2 was noted in the spiral ganglion, tectorial membrane and stria vascularis. In the vestibule, MsrA and MsrB1 were detected in hair cells and the vestibular ganglion, while MsrB2 was restricted to the vestibular ganglion. In this study, we identified distinct distributions of Msr family members in the organ of Corti and hypothesized that MsrA, MsrB1 and MsrB2 protect proteins in the organ of Corti from oxidative stress.

Revealing the structure of stereociliary actin by X-ray nanoimaging.

Hair cell stereocilia are crucial for hearing and the sense of balance. They include an array of accurately packed, parallel actin filaments and act as levers, which transform mechanical deformation into neuronal signals. The length of vestibular stereocilia reaches several micrometers, whereas, for individual microfilaments, the diameter and therefore the characteristic length scale in the lateral direction is on the order of a few nanometers. These orders of magnitude render X-rays an ideal tool for investigating actin packing, and numerous studies on reconstituted in vitro systems have revealed important information. Here we report on the characterization of intact stereocilia using two nanoscale X-ray techniques. We use X-ray ptychography to image stereocilia with quantitative phase contrast and high dose efficiency, showing stereocilia with diameters and lengths in the expected range. We further employ X-ray nanodiffraction using a nanofocused X-ray beam on the same order of magnitude as the width of a stereocilium. Despite the small probe volume we can clearly visualize the stereocilia bundles. From the individual diffraction patterns we determine the local orientation of the actin structures and can clearly correlate them with the corresponding visible-light fluorescence images. Furthermore, azimuthal integration of individual diffraction patterns reveals distinct intensity curves, showing modulations of the signal, which reflect the relevant length scales and pronounced order in the biological system. The applied techniques are not limited to the studies on stereocilia but have the potential of being applied to many biological and soft-matter systems, in particular if a pronounced degree of order is present.

Effects of salicylate on sound-evoked outer hair cell stereocilia deflections.

Hearing depends on sound-evoked deflections of the stereocilia that protrude from the sensory hair cells in the inner ear. Although sound provides an important force driving stereocilia, forces generated through mechanically sensitive ion channels and through the motor protein prestin have been shown to influence stereocilia motion in solitary hair cells. While a possible influence of prestin on mechanically sensitive ion channels has not been systematically investigated, a decrease in transducer currents is evident in solitary hair cells when prestin is blocked with salicylate, raising the question of whether a reduced prestin activity or salicylate itself affected the mechanotransduction apparatus. We used two- and three-dimensional time-resolved confocal imaging to visualize outer hair cell stereocilia during sound stimulation in the apical turn of cochlear explant preparations from the guinea pig. Surprisingly, following application of salicylate, outer hair cell stereocilia deflections increased, while cochlear microphonic potentials decreased. However, when prestin activity was altered with the chloride ionophore tributyltin, both the cochlear microphonic potential and the stereocilia deflection amplitude decreased. Neither positive nor negative current stimulation abolished the bundle movements in the presence of salicylate, indicating that the observed effects did not depend on the endocochlear potential. These data suggest that salicylate may alter the mechanical properties of stereocilia, decreasing their bending stiffness.

Ultrastructure of the Odontocete Organ of Corti: Scanning and transmission electron microscopy

The morphological study of the Odontocete organ of Corti, together with possible alterations associated with damage from sound exposure, represents a key conservation approach to assess the effects of acoustic pollution on marine ecosystems. By collaborating with stranding networks from several European countries, 150 ears from 13 species of Odontocetes were collected and analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Based on our analyses, we first describe and compare Odontocete cochlear structures and then propose a diagnostic method to identify inner ear alterations in stranded individuals. The two species analyzed by TEM (Phocoena phocoena and Stenella coeruleoalba) showed morphological characteristics in the lower basal turn of high-frequency hearing species. Among other striking features, outer hair cell bodies were extremely small and were strongly attached to Deiters cells. Such morphological characteristics, shared with horseshoe bats, suggest that there has been convergent evolution of sound reception mechanisms among echolocating species. Despite possible autolytic artifacts due to technical and experimental constraints, the SEM analysis allowed us to detect the presence of scarring processes resulting from the disappearance of outer hair cells from the epithelium. In addition, in contrast to the rapid decomposition process of the sensory epithelium after death (especially of the inner hair cells), the tectorial membrane appeared to be more resistant to postmortem autolysis effects. Analysis of the stereocilia imprint pattern at the undersurface of the tectorial membrane may provide a way to detect possible ultrastructural alterations of the hair cell stereocilia by mirroring them on the tectorial membrane.

The collagen receptor DDR1 co-localizes with the non-muscle myosin IIA in mice inner ear and contributes to the cytoarchitecture and stability of motile cells.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor activated by native collagen. DDRs regulate cell adhesion, migration and various other cell functions. Deletion of the DDR1 gene in mice is associated with a severe decrease in auditory function and substantial structural alterations in a heterogeneous group of cells, including cells containing actin/myosin contractile elements, e.g., outer hair cells (OHCs) (Meyer zum Gottesberge et al. Lab Invest, 88: 27-37, 2008). The non-muscle myosin heavy chain isoform IIA (NM-IIA), encoded by MYH9, is implicated in the regulation of cell spreading, cellular reshaping and movement and cell migration and adhesion. In this study, we identify DDR1 and NM-IIA co-localization in the type III fibrocytes (tension fibrocytes) of the spiral ligament, the OHCs and the stereocilia of both OHCs and inner hair cells. We show for the first time that DDR1 malfunction causes OHC deformation and the separation of the lateral wall, the location of the cellular motor responsible for the electromotile property, explicitly in those regions showing DDR1 and NM-IIA co-localization. On the basis of our results, we propose that DDR1 acts in concert with proteins of the actin/myosin complex to maintain mechanical forces in the inner ear and to stabilize OHC cellular shape for proper auditory signal transduction.

Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a.

ABSTRACTPlanar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six “core PCP” components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. We recently demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, we generated three mutant alleles in mice. We show that the complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting us to explore an association of Prickle1 with the Wnt pathway. We show that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. Our studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome.