Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

A balance of form and function: Planar polarity and development of the vestibular maculae

The mechanosensory hair cells of the inner ear have emerged as one of the primary models for studying the development of planar polarity in vertebrates. Planar polarity is the polarized organization of cells or cellular structures in the plane of an epithelium. For hair cells, planar polarity is manifest at the subcellular level in the polarized organization of the stereociliary bundle and at the cellular level in the coordinated orientation of stereociliary bundles between adjacent cells. This latter organization is commonly called Planar Cell Polarity and has been described in the greatest detail for auditory hair cells of the cochlea. A third level of planar polarity, referred to as tissue polarity, occurs in the utricular and saccular maculae; two inner ear sensory organs that use hair cells to detect linear acceleration and gravity. In the utricle and saccule hair cells are divided between two groups that have opposite stereociliary bundle polarities and, as a result, are able to detect movements in opposite directions. Thus vestibular hair cells are a unique model system for studying planar polarity because polarization develops at three different anatomical scales in the same sensory organ. Moreover the system has the potential to be used to dissect functional interactions between molecules regulating planar polarity at each of the three levels. Here the significance of planar polarity on vestibular system function will be discussed, and the molecular mechanisms associated with development of planar polarity at each anatomical level will be reviewed. Additional aspects of planar polarity that are unique to the vestibular maculae will also be introduced.     

Regulation of cochlear convergent extension by the vertebrate planar cell polarity pathway is dependent on p120-catenin

The vertebrate planar cell polarity (PCP) pathway consists of conserved PCP and ciliary genes. During development, the PCP pathway regulates convergent extension (CE) and uniform orientation of sensory hair cells in the cochlea. It is not clear how these diverse morphogenetic processes are regulated by a common set of PCP genes. Here, we show that cellular contacts and geometry change drastically and that the dynamic expression of N-cadherin and E-cadherin demarcates sharp boundaries during cochlear extension. The conditional knockout of a component of the adherens junctions, p120-catenin, leads to the reduction of E-cadherin and N-cadherin and to characteristic cochlear CE defects but not misorientation of hair cells. The specific CE defects in p120-catenin mutants are in contrast to associated CE and hair cell misorientation defects observed in common PCP gene mutants. Moreover, the loss-of-function of a conserved PCP gene, Vangl2, alters the dynamic distribution of N-cadherin and E-cadherin in the cochlea and causes similar abnormalities in cellular morphology to those found in p120-catenin mutants. Conversely, we found that Pcdh15 interacts genetically with PCP genes to regulate the formation of polar hair bundles, but not CE defects in the cochlea. Together, these results indicate that the vertebrate PCP pathway regulates CE and hair cell polarity independently and that a p120-catenin-dependent mechanism regulates CE of the cochlea.     

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.     

Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: reponse to loss of cell partners in the Bronx waltzer mouse

This report is concerned with control of cell shaping, positioning, and cytoskeletal integration in a highly ordered cochlear neuroepithelium. It is largely based on investigations of events that occur during abnormal morphogenesis of the organ of Corti in the Bronx waltzer (bv/bv) mutant mouse. The organ's sensory hair cells and adjacent supporting cells ordinarily construct a spatially elaborate and supracellularly integrated cytoskeletal framework. Large microtubule bundles are connected to cytoskeletal components in neighbouring cells by actin-containing meshworks that link them to substantial arrays of adherens junctions. In bv/bv mice, degeneration and loss of most inner hair cells and outer pillar cells occurs during organ development. These cells flank each side of a row of inner pillar cells that respond by upregulating assembly of their actin-containing meshworks. This only occurs in surface regions where they no longer contact cell types involved in construction of the cytoskeletal framework. The meshworks are larger and exhibit a more extensive sub-surface deployment than is normally the case. Hence, assembly of intercellular cytoskeletal connecting components can proceed without contact with appropriate cell neighbours but termination of assembly is apparently subject to a negative feedback control triggered by successful completion of intercellular connection with the correct cell neighbours. In addition, inner pillar cells compensate for loss of cell neighbours by interdigitating and overlapping each other more extensively than is usually the case to increase opportunities for generating adherens junctions. Certain adherens junctions in the organs of +/+ and bv/bv mice exhibit features that distinguish them from all previously described cell junctions. The dense plaques on their cytoplasmic faces are composed of aligned ridges. We suggest that they are called ribbed adherens junctions. Perturbations of cell shaping and positioning indicate that loss of inner hair cells is the primary consequence of the bv mutation. Most of the other abnormalities can be understood in terms of a secondary sequence of morphogenetic aberrations (precipitated by loss of inner hair cells). These aberrations provide new information about the ways in which supporting cells help to control hair cell positioning.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Who does the hair cell's 'do? Rho GTPases and hair-bundle morphogenesis.

The mechanosensitive hair bundles of vertebrate hair cells exhibit a remarkable variety of shapes. For a given location in a sensory epithelium, however, the shape and polarity of a hair bundle are specified precisely. Recent findings, in particular with analogous experimental systems of actin polymerization, suggest a model of hair-bundle morphogenesis whereby different Rho guanosine triphosphatases (GTPases) regulate the initiation phase and the elongation phase of local actin-filament assembly at the hair cell's apical membrane.     

A 90-degree rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells

In the neural plate and neural tube in the trunk region of the zebrafish embryo, dividing cells are oriented parallel to the plane of the neuroepithelium, while in neural keel/rod, cells divide perpendicular to it. This change in the orientation of mitosis is brought about by a 90 degrees rotation of the mitotic spindle. As the two halves of the neural primordium in keel/rod stage are in apposition, the perpendicular orientation of mitoses in this stage determines that daughter cells become allocated to both sides of the neural tube. To assess the role played by cell junctions in controlling the orientation of dividing cells, we studied the expression of components of adherens and tight junctions in the neuroepithelial cells. We find that these proteins are distributed irregularly at the neural plate stage and become polarised apically in the cell membrane only during the keel/rod stage. The stereotypic orientation of mitoses is perturbed only weakly upon loss of function of the cell junction components ASIP and aPKClambda, suggesting that mitotic orientation depends in part on the integrity of cell junctions and the polarity of the epithelium as a whole. However, the 90-degree rotation of the spindle does not require perfectly polarised cell junctions between the neuroepithelial cells.     

Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms

Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the hair cell-intrinsic polarity machinery that establishes the V-shape of the hair bundle is poorly understood. Here, we show that the microtubule motor subunit Kif3a regulates hair cell polarization through both ciliary and non-ciliary mechanisms. Disruption of Kif3a in the inner ear led to absence of the kinocilium, a shortened cochlear duct and flattened hair bundle morphology. Moreover, basal bodies are mispositioned along both the apicobasal and planar polarity axes of mutant hair cells, and hair bundle orientation was uncoupled from the basal body position. We show that a non-ciliary function of Kif3a regulates localized cortical activity of p21-activated kinases (PAK), which in turn controls basal body positioning in hair cells. Our results demonstrate that Kif3a-PAK signaling coordinates planar polarization of the hair bundle and the basal body in hair cells, and establish Kif3a as a key component of the hair cell-intrinsic polarity machinery, which acts in concert with the tissue polarity pathway.     

Deficiency of mDia, an actin nucleator, disrupts integrity of neuroepithelium and causes periventricular dysplasia

During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.     

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.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

Kinocilia mediate mechanosensitivity in developing zebrafish hair cells

Mechanosensitive cilia are vital to signaling and development across many species. In sensory hair cells, sound and movement are transduced by apical hair bundles. Each bundle is comprised of a single primary cilium (kinocilium) flanked by multiple rows of actin-filled projections (stereocilia). Extracellular tip links that interconnect stereocilia are thought to gate mechanosensitive channels. In contrast to stereocilia, kinocilia are not critical for hair-cell mechanotransduction. However, by sequentially imaging the structure of hair bundles and mechanosensitivity of individual lateral-line hair cells in?vivo, we uncovered a central role for kinocilia in mechanosensation during development. Our data demonstrate that nascent hair cells require kinocilia and kinocilial links for mechanosensitivity. Although nascent hair bundles have correct planar polarity, the polarity of their responses to mechanical stimuli is initially reversed. Later in development, a switch to correctly polarized mechanosensitivity coincides with the formation of tip links and the onset of tip-link-dependent mechanotransduction.     

Ultrastructural studies of the junctional complex in the musculature of the arrow-worm (Sagitta setosa) (Chaetognatha)

In the A fibres of the primary musculature of Sagitta, the junctional complex is made up of three kinds of junctions. From the apex to the base they occur in the following order: an apical zonula adherens, a columnar zonula then columnar maculae intermingled with gap junctions. Each columnar junction joins two intracellular filament networks in adjacent cells; this cytoskeleton is largely developed around the nucleus of the A fibres and in close relation with the contractile apparatus, especially at the I band level. The B fibres, which never reach the general cavity, lack zonula adherens and columnar zonula. The columnar junction constitutes a new type of junction which seems to belong to the adherens kind. At their level fibrous columns cross the extracellular space, joining the membranes. Each column faces two cytoplasmic densities localized against the cytoplasmic leaflets of the membranes. A cytoskeleton composed of bundles of cytoplasmic filaments is in close contact with these cytoplasmic densities. The great number of columnar junctions and associated cytoskeleton assure the cohesion of the tissue and the distribution of contractile forces in the absence of connective tissue. The abundance of gap junctions can account for the metabolic and ionic coupling of the fibres.     

DLG-1 Is a MAGUK Similar to SAP97 and Is Required for Adherens Junction Formation

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.