Shaping the mammalian auditory sensory organ by the planar cell polarity pathway

The human ear is capable of processing sound with a remarkable resolution over a wide range of intensity and frequency. This ability depends largely on the extraordinary feats of the hearing organ, the organ of Corti and its sensory hair cells. The organ of Corti consists of precisely patterned rows of sensory hair cells and supporting cells along the length of the snail-shaped cochlear duct. On the apical surface of each hair cell, several rows of actin-containing protrusions, known as stereocilia, form a "V"-shaped staircase. The vertices of all the "V"-shaped stereocilia point away from the center of the cochlea. The uniform orientation of stereocilia in the organ of Corti manifests a distinctive form of polarity known as planar cell polarity (PCP). Functionally, the direction of stereociliary bundle deflection controls the mechanical channels located in the stereocilia for auditory transduction. In addition, hair cells are tonotopically organized along the length of the cochlea. Thus, the uniform orientation of stereociliary bundles along the length of the cochlea is critical for effective mechanotransduction and for frequency selection. Here we summarize the morphological and molecular events that bestow the structural characteristics of the mammalian hearing organ, the growth of the snail-shaped cochlear duct and the establishment of PCP in the organ of Corti. The PCP of the sensory organs in the vestibule of the inner ear will also be described briefly.     

Ankrd6 is a mammalian functional homolog of Drosophila planar cell polarity gene diego and regulates coordinated cellular orientation in the mouse inner ear

The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. Here we report that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling.     

Breaking cellular symmetry along planar axes in Drosophila and vertebrates

In many organs, epithelial cells are polarized not only along the apicobasal axis, but also along a second axis within a plane. Acquisition of the latter polarity, known as planar cell polarity (PCP) or tissue polarity, is crucial for specialized cellular functions. Genetic programming of PCP has been most thoroughly studied in Drosophila, which has allowed identification of a number of regulatory molecules that are evolutionally conserved. One group of the regulators is responsible for interpreting a hypothetical polarity cue and directing local cytoskeletal reorganization. This group includes a seven-pass transmembrane cadherin known as Flamingo (also known as Starry night), other receptors, and downstream components; and many of those molecules are redistributed to restricted subcellular compartments. Recent studies on a trio of cell-surface molecules challenge a previous hypothesis about the identity of the polarity cue and prompt a novel hypothesis about a global input. Studies on vertebrate systems support the notion that the molecular mechanisms demonstrated in Drosophila are applicable to at least two classes of polarized behaviors of vertebrate cells: sensory hair morphogenesis in the inner ear epithelium, and convergent extension movements during gastrulation.     

Revisiting planar cell polarity in the inner ear

Since the first implication of the core planar cell polarity (PCP) pathway in stereocilia orientation of sensory hair cells in the mammalian cochlea, much has been written about this subject, in terms of understanding how this pathway can shape the mammalian hair cells and using the inner ear as a model system to understand mammalian PCP signaling. However, many conflicting results have arisen, leading to puzzling questions regarding the actual mechanism and roles of core PCP signaling in mammals and invertebrates. In this review, we summarize our current knowledge on the establishment of PCP during inner ear development and revisit the contrast between wing epithelial cells in Drosophila melanogaster and sensory epithelia in the mammalian cochlea. Notably, we focus on similarities and differences in the asymmetric distribution of core PCP proteins in the context of cell autonomous versus non-autonomous role of PCP signaling in the two systems. Additionally, we address the relationship between the kinocilium position and PCP in cochlear hair cells and increasing results suggest an alternate cell autonomous pathway in regulating PCP in sensory hair cells.     

Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation?

Many types of cell show different aspects of polarization. Epithelial cells display a ubiquitous apical-basolateral polarity but often are also polarized in the plane of the epithelium - a feature referred to as 'planar cell polarity' (PCP). In Drosophila all adult epithelial cuticular structures are polarized within the plane, whereas in vertebrates examples of PCP include aspects of skin development, features of the inner ear epithelium, and the morphology and behavior of mesenchymal cells undergoing the morphogenetic movement called 'convergent extension'. Recent advances in the study of PCP establishment are beginning to unravel the molecular mechanisms that underlie this aspect of cell and tissue differentiation. Here I discuss new developments in our molecular understanding of PCP in Drosophila and compare them towhat is known about the regulation of convergent extension in vertebrates.     

Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding

Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in D. melanogaster. Here we provide mechanistic insight into Frizzled/Dishevelled signalling regulation. We show that the ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling.     

Planar cell polarity and a potential role for a Wnt morphogen gradient in stereociliary bundle orientation in the mammalian inner ear

The planar cell polarity (PCP) pathway, a noncanonical Wnt signaling pathway, is crucial for embryonic development in all animals as it is responsible for the regulation of coordinated orientation of structures within the plane of the various epithelia. In the mammalian cochlea, one of the best examples of planar polarity in vertebrates, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are all uniformly polarized. Generation of this polarity is important for hair cell mechanotransduction and auditory perception as stereociliary bundles are only sensitive to vibrations in their single plane of polarization. We describe the two step developmental process that results in the generation of planar polarity in the mammalian inner ear. Furthermore, we review evidence for the role of Wnt signaling, and the possible generation of a Wnt gradient, in planar polarity.     

Origins of inner ear sensory organs revealed by fate map and time-lapse analyses

The inner ear develops from a simple ectodermal thickening called the otic placode into a labyrinth of chambers which house sensory organs that sense sound and are used to maintain balance. Although the morphology and function of the sensory organs are well characterized, their origins and lineage relationships are virtually unknown. In this study, we generated a fate map of Xenopus laevis inner ear at otic placode and otocyst stages to determine the developmental origins of the sensory organs. Our lineage analysis shows that all regions of the otic placode and otocyst can give rise to the sensory organs of the inner ear, though there were differences between labeled quadrants in the range of derivatives formed. A given region often gives rise to cells in multiple sensory organs, including cells that apparently dispersed from anterior to posterior poles and vice versa. These results suggest that a single sensory organ arises from cells in different parts of the placode or otocyst and that cell mixing plays a large role in ear development. Time-lapse videomicroscopy provides further evidence that cells from opposite regions of the inner ear mix during the development of the inner ear, and this mixing begins at placode stages. Lastly, bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta (TGF-beta) family, is expressed in all sensory organs of the frog inner ear, as it is in the developing chicken ear. Inner ear fate maps provide a context for interpreting gene expression patterns and embryological manipulations.     

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.     

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.     

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.     

Shaping sound in space: the regulation of inner ear patterning

The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development - ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells - are orchestrated by gradients of signaling molecules.     

Planar cell polarity in Drosophila

In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified.     

Ectopic noggin blocks sensory and nonsensory organ morphogenesis in the chicken inner ear

Bone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Planar cell polarity in Drosophila

In all multicellular organisms, epithelial cells are not only polarized along the apical-basal axis, but also within the epithelial plane, giving cells a sense of direction. Planar cell polarity (PCP) signaling regulates establishment of polarity within the plane of an epithelium. The outcomes of PCP signaling are diverse and include the determination of cell fates, the generation of asymmetric but highly aligned structures, such as the stereocilia in the human inner ear or the hairs on a fly wing, or the directional migration of cells during convergence and extension during vertebrate gastrulation. In humans, aberrant PCP signaling can result in severe developmental defects, such as open neural tubes (spina bifida), and can cause cystic kidneys. In this review, we discuss the basic mechanism and more recent findings of PCP signaling focusing on Drosophila melanogaster, the model organism in which most key PCP components were initially identified.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Pluripotent stem cells from the adult mouse inner ear

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.     

Identification and characterization of a novel chemically induced allele at the planar cell polarity gene Vangl2

Planar cell polarity (PCP) signaling controls a number of morphogenetic processes including convergent extension during gastrulation and neural tube formation. Defects in this pathway cause neural tube defects (NTD), the most common malformations of the central nervous system. The Looptail (Lp) mutant mouse was the first mammalian mutant implicating a PCP gene (Vangl2) in the pathogenesis of NTD. We report on a novel chemically induced mutant allele at Vangl2 called Curly Bob that causes a missense mutation p.Ile268Asn (I268N) in the Vangl2 protein. This mutant segregates in a semi-dominant fashion with heterozygote mice displaying a looped tail appearance, bobbing head, and a circling behavior. Homozygote mutant embryos suffer from a severe form of NTD called craniorachischisis, severe PCP defects in the inner hair cells of the cochlea and posterior cristae, and display a distinct defect in retinal axon guidance. This mutant genetically interacts with the Lp allele (Vangl2 ^S464N) in neural tube development and inner ear hair cell polarity. The Vangl2^I268N protein variant is expressed at very low levels in affected neural and retinal tissues of mutant homozygote embryos. Biochemical studies show that Vangl2^I268N exhibits impaired targeting to the plasma membrane and accumulates in the endoplasmic reticulum. The Vangl2^I268N variant no longer physically interacts with its PCP partner DVL3 and has a reduced protein half-life. This mutant provides an important model for dissecting the role of Vangl2 in the development of the neural tube, establishment of polarity of sensory cells of the auditory and vestibular systems, and retinal axon guidance.