Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.     

Myosins and pathology: genetics and biology

This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.     

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.     

Mosaic complementation demonstrates a regulatory role for myosin VIIa in actin dynamics of stereocilia

We have developed a bacterial artificial chromosome transgenesis approach that allowed the expression of myosin VIIa from the mouse X chromosome. We demonstrated the complementation of the Myo7a null mutant phenotype producing a fine mosaic of two types of sensory hair cells within inner ear epithelia of hemizygous transgenic females due to X inactivation. Direct comparisons between neighboring auditory hair cells that were different only with respect to myosin VIIa expression revealed that mutant stereocilia are significantly longer than those of their complemented counterparts. Myosin VIIa-deficient hair cells showed an abnormally persistent tip localization of whirlin, a protein directly linked to elongation of stereocilia, in stereocilia. Furthermore, myosin VIIa localized at the tips of all abnormally short stereocilia of mice deficient for either myosin XVa or whirlin. Our results strongly suggest that myosin VIIa regulates the establishment of a setpoint for stereocilium heights, and this novel role may influence their normal staircase-like arrangement within a bundle.     

Unconventional myosin VIIA is a novel A-kinase-anchoring protein

To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.     

Usher syndrome type I and the differentiation of inner ear sensory cells' hair bundles

Defects in myosin VIIa, the PDZ-domain-containing protein harmonin, cadherin 23, protocadherin 15, and the putative scaffolding protein sans, underlie five genetic forms of Usher syndrome type I (USH1), the most frequent cause of hereditary deafness-blindness in humans. Mice mutants defective for any of these proteins have a severe hearing impairment and display similar inner ear phenotypes characterized by the abnormal spreading of the sensory cells' stereocilia. These are highly specialized mechanoreceptive organelles derived from microvilli, that normally form a well-structured hair bundle at the apex of inner ear sensory cells. All the USH1 proteins, except sans, have been detected in the growing stereocilia. Moreover, biochemical studies have started to unravel the multiple direct molecular interactions between USH1 proteins. In particular, harmonin can bind to the other four USH1 proteins and to F-actin. Finally, cell biology studies have provided the first insights into the functions of these proteins, and revealed that cadherin 23, and probably protocadherin 15 also, are associated with transient lateral links that interconnect growing stereocilia. These connectors play a critical role in the differentiating hair bundle.     

Role of myosin VI in the differentiation of cochlear hair cells.

The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI (K. B. Avraham et al., 1995, Nat. Genet. 11, 369-375). Snell's waltzer mutants exhibit behavioural abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. We have investigated the effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the stereocilia. In the mutants, early stages of stereocilia development appear to proceed normally because at birth many stereocilia bundles have a normal appearance, but in places there are signs of disorganisation of the bundles. Over the next few days, the stereocilia become progressively more disorganised and fuse together. Practically all hair cells show fused stereocilia by 3 days after birth, and there is extensive stereocilia fusion by 7 days. By 20 days, giant stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between stereocilia, leading to fusion.     

Balanced levels of Espin are critical for stereociliary growth and length maintenance

Hearing and balance depend on microvilli-like actin-based projections of sensory hair cells called stereocilia. Their sensitivity to mechanical displacements on the nanometer scale requires a highly organized hair bundle in which the physical dimension of each stereocilium is tightly controlled. The length and diameter of each stereocilium are established during hair bundle maturation and maintained by life-long continuing dynamic regulation. Here, we studied the role of the actin-bundling protein Espin in stereociliary growth by examining the hair cell stereocilia of Espin-deficient jerker mice (Espn(je)), and the effects of transiently overexpressing Espin in the neuroepithelial cells of the organ of Corti cultures. Using fluorescence scanning confocal and electron microscopy, we found that a lack of Espin results in inhibition of stereociliary growth followed by progressive degeneration of the hair bundle. In contrast, overexpression of Espin induced lengthening of stereocilia and microvilli that mirrored the elongation of the actin filament bundle at their core. Interestingly, Espin deficiency also appeared to influence the localization of Myosin XVa, an unconventional myosin that is normally present at the stereocilia tip at levels proportional to stereocilia length. These results indicate that Espin is important for the growth and maintenance of the actin-based protrusions of inner ear neuroepithelial cells.     

Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish

Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the role of this protein in the sensory hair cells remains unclear. To investigate the function of myosin VI in zebrafish, we cloned and examined the expression pattern of myosin VI, which is duplicated in the zebrafish genome. One duplicate, myo6a, is expressed in a ubiquitous pattern during early development and at later stages, and is highly expressed in the brain, gut, and kidney. myo6b, on the other hand, is predominantly expressed in the sensory epithelium of the ear and lateral line at all developmental stages examined. Both molecules have different splice variants expressed in these tissues. Using a candidate gene approach, we show that myo6b is satellite, a gene responsible for auditory/vestibular defects in zebrafish larvae. Examination of hair cells in satellite mutants revealed that stereociliary bundles are irregular and disorganized. At the ultrastructural level, we observed that the apical surface of satellite mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised. At later stages, stereocilia fused together. We conclude that zebrafish myo6b is required for maintaining the integrity of the apical surface of hair cells, suggesting a conserved role for myosin VI in regulation of actin-based interactions with the plasma membrane.     

Expression of myosin VIIA in the developing chick inner ear neurons

The auditory-vestibular ganglion (AVG) is formed by the division of otic placode-derived neuroblasts, which then differentiate into auditory and vestibular afferent neurons. The developmental mechanisms that regulate neuronal cell fate determination, axonal pathfinding and innervation of otic neurons are poorly understood. The present study characterized the expression of myosin VIIA, along with the neuronal markers, Islet1, NeuroD1 and TuJ1, in the developing avian ear, during Hamburger–Hamilton (HH) stages 16–40. At early stages, when neuroblasts are delaminating from the otic epithelium, myosin VIIA expression was not observed. Myosin VIIA was initially detected in a subset of neurons during the early phase of neuronal differentiation (HH stage 20). As the AVG segregates into the auditory and vestibular portions, myosin VIIA was restricted to a subset of vestibular neurons, but was not present in auditory neurons. Myosin VIIA expression in the vestibular ganglion was maintained through HH stage 33 and was downregulated by stage 36. Myosin VIIA was also observed in the migrating processes of vestibular afferents as they begin to innervate the otic epithelium HH stage 22/23. Notably, afferents targeting hair cells of the cristae were positive for myosin VIIA while afferents targeting the utricular and saccular maculae were negative (HH stage 26–28). Although previous studies have reported that myosin VIIA is restricted to sensory hair cells, our data shows that myosin VIIA is also expressed in neurons of the developing chick ear. Our study suggests a possible role for myosin VIIA in axonal migration/pathfinding and/or innervation of vestibular afferents. In addition, myosin VIIA could be used as an early marker for vestibular neurons during the development of the avian AVG.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.     

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.     

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.     

Planar cell polarity in the inner ear: how do hair cells acquire their oriented structure?

Sensory hair cells in the ear and lateral line have an asymmetrical hair-bundle structure, essential for their function as directional mechanotransducers. We examine four questions: (1) how does the planar asymmetry of the individual hair cell originate? (2) How are the orientations of neighboring hair cells coordinated? (3) How is the orientation of a group of hair cells controlled in relation to the ear as a whole? (4) How does the initial cell asymmetry lead to creation of the asymmetrical hair bundle? Studies of the development of hairs and bristles in Drosophila, combined with genetic data from vertebrates, suggest that the answer to questions (1) and (2) lies in asymmetries that develop at the cell cortex and at cell-cell junctions, generated by products of a set of primary planar cell polarity genes, including the transmembrane receptor Frizzled. A separate and largely independent mechanism controls asymmmetric allocation of cell fate determinants such as Numb at mitosis, in Drosophila and possibly in the ear also. Little is known about long-range signals that might orient hair cells globally in the ear, but progress has been made in identifying a set of genes responsible for read-out of the primary polarity specification. These genes, in flies and vertebrates, provide a link to assembly of the polarized cytoskeleton; myosin VIIA appears to belong in this group. The mechanism creating the staircase pattern of stereocilium lengths is unknown, but could involve regulation of stereocilium growth by Ca(2+) ions entering via transduction channels.     

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.