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

Planar Cell Polarity in the Inner Ear: How Do HairCells Acquire Their Oriented Structure?

Julian Lewis, Alex Davies

Vertebrate Development Laboratory, Cancer Research UK, 44 Lincoln’s Inn Fields,London WC2A 3PX, United Kingdom

Received 3 June 2002; accepted 16 July 2002

ABSTRACT: Sensory hair cells in the ear and lat-eral line have an asymmetrical hair-bundle structure,essential for their function as directional mechanotrans-ducers. We examine four questions: (1) how does theplanar asymmetry of the individual hair cell originate?(2) How are the orientations of neighboring hair cellscoordinated? (3) How is the orientation of a group ofhair cells controlled in relation to the ear as a whole? (4)How does the initial cell asymmetry lead to creation ofthe asymmetrical hair bundle? Studies of the develop-ment of hairs and bristles in Drosophila, combined withgenetic data from vertebrates, suggest that the answer toquestions (1) and (2) lies in asymmetries that develop atthe cell cortex and at cell–cell junctions, generated byproducts of a set of primary planar cell polarity genes,including the transmembrane receptor Frizzled. A sep-

arate and largely independent mechanism controlsasymmmetric allocation of cell fate determinants such asNumb at mitosis, in Drosophila and possibly in the earalso. Little is known about long-range signals that mightorient hair cells globally in the ear, but progress hasbeen made in identifying a set of genes responsible forread-out of the primary polarity specification. Thesegenes, in flies and vertebrates, provide a link to assemblyof the polarized cytoskeleton; myosin VIIA appears tobelong in this group. The mechanism creating the stair-case pattern of stereocilium lengths is unknown, butcould involve regulation of stereocilium growth by Ca2�

ions entering via transduction channels. © 2002 Wiley

Periodicals, Inc. J Neurobiol 53: 190–201, 2002

Keywords: planar cell polarity; hair cell; Frizzled; ear;stereocilia

INTRODUCTION

Almost all epithelial cells—which is to say most ofthe cell types in the body—have a well-defined apico-basal polarity: the top and the bottom of the cell havedifferent characteristics. More rarely, epithelial cellsalso have a well-defined planar cell polarity, that is, anasymmetry that can be represented by an arrow ori-ented in the plane of the epithelium. Planar cell po-larity is important in the respiratory tract, for example,where every ciliated cell must orient its beating so asto sweep mucus up out of the lungs, and not down into

them, nor round and round the circumference of theairway; likewise in the oviduct, the ciliated cells musthave the correct planar cell polarity to sweep theovum towards the uterus.

The most striking example of planar cell polarity inthe vertebrate body is provided, however, by the sen-sory hair cells of the inner ear and lateral line. Each ofthese has a bundle of stereocilia protruding from itsapical surface to detect mechanical displacements,and the bundle is asymmetric in its structure andfunction. The stereocilia are arranged in rows gradedin height, forming a staircase pattern, and defining a“forward” direction—a vector pointing from theshortest stereocilia towards the longest—on the cellsurface. The cell is electrically excited by a shearforce that tilts the mechanosensory bundle forwards,inhibited by one that tilts it backwards, and unrespon-

Correspondence to: J. Lewis ([email protected]).Contract grant sponsor: Cancer Research UK.

© 2002 Wiley Periodicals, Inc.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/neu.10124

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sive to a tilt sideways. Thus, correct perception ofsound, gravity, acceleration, and (for fish and amphib-ians) of movement of water over the body surface,requires that hair cells be oriented with a specific,correctly coordinated planar cell polarity. In this re-view, we consider what is known about the mecha-nisms by which hair cell planar polarity is set up.

THE NATURE OF THE PROBLEM ANDITS CONTEXT IN THE EAR

Three Tiers of Control in Planar CellPolarity

To clarify the conceptual framework, it is helpful tocontrast two sorts of polarized system. The first isexemplified by a field of wheat when the wind blows:all stalks bend in the same direction, and this reflectsthe operation of the wind on each stalk individually,conferring on it an asymmetry that it would lack if nowind blew. At the opposite extreme, consider theferromagnet. Here, each atom has an intrinsic mag-netic polarity, reflecting its internal dynamics; neigh-boring atoms are strongly correlated in orientationthrough short-range forces, creating magnetic do-mains within which all the atoms point the same way;and long-range magnetic forces orient the domainsglobally in relation to the surroundings. In this case,there are three distinct but coupled mechanisms—onethat confers the asymmetry on the individual atom,another that aligns close neighbors in relation to oneanother, and a third that aligns the groups of atoms inrelation to their distant surroundings. The atoms indi-vidually possess a polarity even when the alignmentmechanisms fail (as in a ferromagnet heated above theCurie temperature); and they can be aligned locally inrelation to their immediate neighbors, creating ori-ented magnetic domains, even when there is no long-range magnetic force to align one domain with an-other (as in a piece of iron that has never beenmagnetized by exposure to a magnetic field).

The hair cells in the ear, we would suggest, aremore like atoms in a ferromagnet than like wheat-stalks in a wind-blown field. Each individual cell hasa well-defined planar polarity that is the product of itsown internal cytoskeletal symmetry-breaking mecha-nisms and exists regardless of how the cell is orientedin relation to its surroundings. The orientations ofneighboring hair cells, moreover, are usually stronglycorrelated, even when the orientation pattern is non-uniform on a global scale and includes complexwhorls and abrupt reversals at domain boundaries(Platt, 1977; Denman-Johnson and Forge, 1999). The

global pattern, nevertheless, is normally well definedand predictable—and has to be, if the system is toperform its sensory function properly. According tothis view of hair-cell planar polarity, therefore, we areconfronted by three distinct questions, correspondingto three potentially different mechanisms of control.First, how is the internal asymmetry of the hair cellgenerated? Second, how is the orientation of the axisof asymmetry (the polarity vector) of a given cellcoupled to that of its neighbors? Third, how is theorientation of the local group of hair cells controlledglobally by influences from more distant structures,according to situation in the body as a whole? Thesequestions are inescapably linked to a fourth, which weshall also briefly discuss: how does the cell use itsbasic polarity specification to guide development ofthe asymmetrical structures on which mechanosen-sory transduction depends?

We begin by considering the developmental mi-croanatomy of planar cell polarity in the ear and itsrelation to sensory function, before turning to molec-ular genetics.

Genesis of the Polarized Hair Bundle:Relations between Structure andFunction

The mechanosensory bundle on the apex of each haircell consists of the set of stereocilia, each with a stiffcore of bundled actin filaments, and a single kinoci-lium, containing microtubules. In some species, insome regions of the inner ear—for example, in thecochlea of mammals—the kinocilium regresses as thehair cell matures, but during the early, critical phaseof hair cell development it is always present. Thewhole structure manifests its planar polarity in twoobvious ways: the kinocilium always lies to one sideof the bundle of stereocilia, and the stereociliary bun-dle itself always has a well-defined orientation, withthe tallest stereocilia on the side next to the kinociliumand the shortest on the side away from it. Structuralpolarity is related to function in a straightforward way(Hudspeth, 1997). An extracellular filament, called atip link, runs from the tip of each shorter stereociliumup to an insertion point on the side of the next tallerstereocilium. When the bundle of stereocilia is tiltedin the forward direction by a mechanical stimulus, thetip links are stretched, exerting tension on their at-tachment points in the stereociliary plasma mem-brane. Ion channels located at these attachment pointsare thought to be pulled open by the tension in the tiplinks, allowing an influx of positive ions that depo-larizes the cell. The tip links are kept taut by amyosin-1c–based adaptation motor that tends to tow

Planar Cell Polarity in the Inner Ear 191

the attachment points of the tip links upwards, to-wards the apex of each stereocilium, presumablythrough an interaction with the actin filaments in thecore (Holt et al., 2002). Thus, the ordered arrange-ment of stereocilia, graded from short to tall, andhence the planar polarity of the hair cell, is absolutelyessential for the mechanism of transduction. Con-versely, according to at least one theory, as we explainbelow, the mechanism of transduction may itself benecessary for development of the staircase pattern ofstereocilia.

The development of this structure has been de-scribed most carefully for the basilar papilla of thechick (Tilney et al., 1992), but the process is similar inother sensory patches (Forge et al., 1997; Denman-Johnson and Forge, 1999). The kinocilium is alreadyvisible on the nascent hair cell as soon as it hascompleted its final mitosis and is centrally placed onthe cell apex. At first, the surrounding regions of theapical surface are populated by microvilli of uniformsize. Soon, however, microvilli in the central regionbegin to enlarge to become stereocilia, and the kino-cilium becomes displaced to one side of the cell apex.Length differences among the nascent stereocilia thenbegin to appear: those closest to the kinocilium are thefirst to rise above their neighbors; once the stereociliaof this first row have gained a lead, those in the nextrow begin to elongate also, then the next, and so on,creating the stepped sequence of stereociliary lengths.

The pattern of events suggests that each stereoci-lium may elongate in response to an upward tug froma taller neighbor: the idea would be that the kinoci-lium, as the protrusion that initially towers above therest, initiates the process, and the effect then propa-gates across the bundle as each row of stereocilia isdrawn upwards in turn. Tip links are present from anearly stage (Pickles et al., 1991), and this has sug-gested a way in which such a mechanism might beimplemented. Specifically, it has been proposed thatthe elongation of each stereocilium—a process ofactin assembly—might be controlled by entry of Ca2�

through the transduction channels (Tilney et al.,1988). If these are towed towards the tips of thestereocilia, and linked by tip links that go from onestereocilium to the next, they will be placed undertension by the adaptation motor, and thereby to someextent opened, whenever one stereocilium of a linkedpair becomes longer than its neighbor. If Ca2� some-how promotes actin assembly and stereocilium elon-gation, this will give rise to a positive feedback,tending to maintain the length difference between theshorter and the longer stereocilium and to cause bothto grow in length. The heights of stereocilia within asingle bundle will be coordinated in a regular graded

series as a result of the mechanical linkages betweenthem, and the orientation of the bundle—that is, theposition of the tallest stereocilia relative to the short-est—will be determined by the site where stereociliafirst begin to lengthen at an accelerated rate, which inturn is dictated by the site of the kinocilium.

This remains a highly speculative model. It tallieswith the geometry of vestibular hair cells, but it doesnot quite so easily explain the geometry of hair cellsin the cochlea, where the stereocilia are arranged in astepped series of rows, but all the stereocilia within agiven row have the same height. Nevertheless, themodel is at least consistent with observations on mu-tant mice that lack functional myosin VIIA (Self et al.,1998). In these animals, several separate small clumpsof stereocilia develop on a typical single hair cell.Within each clump the stereocilia are regularly gradedin height, but the orientations of the separate clumpsare random and uncorrelated, as might be expected ifthey are not linked to one another or to the kinocilium.The model is also consistent with the timing of de-velopment of tip links, which begin to be visible justbefore the stereocilia begin to elongate differentially(Pickles et al., 1991), and with the drastic disorgani-zation of the hair bundles that is seen in embryostreated with streptomycin (Pickles and Rouse, 1991),which blocks the transduction channels.

Whatever the mechanism that generates the stair-case pattern of stereocilia, it is clear that the hair cellplanar polarity vector becomes defined before thispattern is mature, and is apparent already in the earlydisplacement of the kinocilium to one side of the cellapex. We can, therefore, discuss the problem of howthe initial cellular asymmetry is created and oriented,independently of questions about how it guides sub-sequent development of polarized cell architecture.

Hair-Cell Orientation Patterns in theDeveloping Ear and Lateral Line

The planar polarities of the hair cells in different kindsof sensory patch are coordinated in different patterns.Hair cells of the basilar papilla (or Organ of Corti) areall oriented in the same direction, pointing away fromthe neural (or modiolar) side of the structure. Bycontrast, in a lateral line organ of a fish or amphibian,hair cells of contrary polarity are mixed together, allpointing in one or the other of two precisely oppositedirections, the members of one set being innervatedby branches of one nerve fiber, those of the other setby branches of another (Winklbauer, 1989; Rouse andPickles, 1991) (Fig. 1). Hair cells of the maculae showstill more complex planar polarity patterns (Platt,1977; Denman-Johnson and Forge, 1999). Through-

192 Lewis and Davies

out most of the area of a typical macula, the polarityvector of each hair cell is aligned with that of itsimmediate neighbors, but there are also fault lines,called striolar regions, where abrupt reversals of po-larity occur. Moreover, in nonstriolar regions, thepolarity vectors may gradually fan out or veer fromone direction to another. Despite all these variations,however, two general principles seem to apply. First,each hair cell individually has a well-defined planarasymmetry, with kinocilium and stepped array of ste-reocilia as described above, regardless of its place inthe pattern. Second, away from the fault lines, theorientations of neighboring hair cells are closely cor-related—either parallel, as in the ear’s sensorypatches, or antiparallel, as in lateral line organs.

If we are to understand how the orientation pat-terns arise, a first question is whether each hair cell is

generated with its polarity vector correctly orientedfrom the outset, or whether the final orientation isachieved by remodeling or rotating the cell after it hasalready developed its own internal asymmetry.

Some observations in the basilar papilla of thechick suggested the latter possibility. Newborn haircells appear erratic in their orientation, and it wassuggested that they are towed into their precise finalalignment by an interaction with the overlying tecto-rial membrane, which rests on the tips of the nascenthair bundles and undergoes a shear movement relativeto the sensory epithelium as the structure grows (Co-tanche and Corwin, 1991). But it is hard to see howthis mechanism could operate in the lateral line organsor in the vestibular patches, with their complex pat-terns of hair-cell orientation and very different rela-tionship to overlying matrix (Denman-Johnson andForge, 1999). In any case, even in the basilar papilla,the initial orientations, though imprecise, are far fromrandom (Stone and Cotanche, 1991). To a first ap-proximation, therefore, it appears that hair cell planarpolarity vectors are generated ab initio in the correctorientation—which is to say, in a specific relationshipto their environment. How, then, does a hair cellacquire its planar polarity, and what mechanisms con-trol its alignment? For clues to an answer, we turnnow to Drosophila. In the light of the Drosophilafindings, it becomes easier to interpret the vertebratedata, or at least to frame specific hypotheses about themechanisms at work.

EVIDENCE FROM DROSOPHILA

Planar Cell Polarity Mechanisms inDrosophila: Role of the Primary PCPGenes

Planar cell polarity has been mainly investigated inthree types of tissue in Drosophila: (1) the epitheliumof the wing and various other body surfaces, whereeach cell carries an oriented protrusion called a winghair or trichome; (2) the sensory bristles on the thoraxand elsewhere, consisting typically of four cells, oneof which forms a bristle shaft, oriented in the sameway as the trichomes on neighboring epithelial cells;and (3) the retina, where assemblies of eight photo-receptors plus ancillary cells form oriented omma-tidia. In cases (2) and (3), there is a well-definedplanar polarity both in the arrangement of the differ-ent cells relative to one another in the group, and inthe intrinsic structure of the individual cells. The twolevels of organization are linked, as we shall see, andthis makes these systems more complex than (1) as

Figure 1 Hair-cell polarities in a lateral line organ of a5-day zebrafish larva. The actin-rich stereociliary bundlesare stained green with fluorescent phalloidin, while themicrotubules of the kinocilia are stained red with an anti-acetylated-tubulin antibody. As shown diagrammatically inthe inset, 7 of the 14 hair cells are oriented in one direction,while the other 7 are oriented in the diametrically oppositedirection. (Courtesy of Tanya Whitfield; adapted from theZebrafish Information Network Anatomical Dictionary,http://zfin.org/cgi-bin/ZFIN_jump?record�ZDB-IMAGE-001220-8.)


examples of planar cell polarity. Nevertheless, there isa core set of genes that are crucial for the creation andcoordination of planar cell polarity in all three sys-tems (Adler, 2002). We shall call these the primaryPCP genes.

Wing hairs and sensory bristles are most directlyrelevant to our vertebrate theme (Eaton, 1997). Boththese types of structure develop as oriented protru-sions containing both microtubules and actin filamentbundles. In this respect they show some resemblanceto the hair bundles of vertebrate hair cells, eventhough the actin filaments and microtubules are con-tained in a single protrusion instead of being parcelledout in separate stereocilia and kinocilium. In fact, onthe basis of similarities in function, structure, devel-opmental origins, and molecular mechanisms of de-velopment, it has been argued that the bristle shaftcells of Drosophila may be regarded as homologs ofvertebrate hair cells (Lewis, 1991; Whitfield et al.,1997; Adam et al., 1998; Eddison et al., 2000; Mullerand Littlewood-Evans, 2001).

The set of primary PCP genes responsible forplanar cell polarity in Drosophila includes frizzled(fz), dishevelled (dsh), flamingo (fmi, also known asstarry night), dachsous (ds), strabismus (stbm, alsoknown as van Gogh), and prickle (pk5, also known asspiny legs) (see Adler, 2002, for a review). Theirfunctions are most simply displayed in the develop-ment of wing hairs. In a normal wing, each epidermalcell has a hair located at the distal side of its apex, andthis hair points towards the distal margin of the wing.Mutation of any of the genes in the list above disruptsthis uniform planar polarity, creating patterns inwhich, depending on the severity of the mutation andthe region of the body, the cells either individuallylack polarity, possessing hairs that arise from thecenter of the cell apex and point apically instead ofdistally, or are individually polarized but randomlyoriented, or are coordinated with their immediateneighbors but globally misdirected, so that instead ofall pointing distally they form complex swirling pat-terns. The varied degree of disruption implies thatthere is some redundancy in the system of polaritycontrol, but the mutant phenotypes, nevertheless, givean indication of how the system works.

The protein products of all the primary PCP geneslisted above are either transmembrane or membrane-associated.

1. Frizzled is a seven-pass transmembrane recep-tor belonging to the family of receptors forWingless/Wnt proteins.

2. Dishevelled is an intracellular PDZ-domain-

containing protein that is recruited to the mem-brane by Frizzled.

3. Flamingo is giant transmembrane protein—acurious hybrid between the protocadherin fam-ily and the G-protein–coupled receptor super-family: it has nine cadherin repeats in its extra-cellular domain, plus four EGF-like motifs andtwo laminin G motifs, linked to a seven-passtransmembrane region.

4. Dachsous is also a giant member of the cadherinsuperfamily, with 27 cadherin repeats in itsextracellular domain, but with only a singletransmembrane domain, and an intracellular do-main that resembles the beta-catenin bindingintracellular domain of typical vertebrate cad-herins.

5. Strabismus is a protein with four putative trans-membrane domains, and a PDZ domain-bindingmotif; it may interact with Dishevelled.

6. Prickle contains three LIM protein interactiondomains.

All six proteins of the set listed above are concen-trated at cell–cell junctions where the distal end ofone cell (the tip of its planar-polarity vector) contactsthe proximal end of another. At least three proteins ofthe core set are not only necessary for correct polarity,but also behave as markers of polarity: Frizzled andDishevelled are concentrated at the distal surface ofeach wing cell, while Prickle (together with Strabis-mus, in the eye at least) is concentrated at the proxi-mal surface (Strutt, 2001; Tree et al., 2002). When aclone of mutant cells is created lacking Frizzled, cellpolarity is disrupted not only inside the mutant patch,but also in a small region distal to it (Vinson andAdler, 1987). Conversely, if a clone is created lackingPrickle, polarity is disrupted inside the mutant patchand in a small region proximal to it (Gubb et al.,1999). Evidently, the coordination of the planar po-larity of neighboring cells depends on an orientinginfluence that propagates from cell to cell via cell–cellcontacts. In fact, presence of Frizzled at one face of acell (giving it a distal character) forces the adjacentface of the next cell to recruit Prickle (giving it aproximal character), and vice versa. There appears tobe a positive feedback in the assembly of this polar-ized interface, producing an all-or-none outcome inwhich the cell–cell junction adopts either the onepolarity or the other (Tree et al., 2002). Inside eachcell, moreover, it seems that a mechanism exists toensure that if Prickled becomes concentrated at onepole, Frizzled and its partner Dishevelled will becomerestricted to the opposite pole. In this way, each cell


acquires a definite internal polarity, and transmits itsown polarity to the next cell in line.

The details of the intracellular mechanism thatprevents development of cells with a double-distal ordouble-proximal pattern are not yet clear. It is possi-ble that a positive feedback may operate here, too,capable of amplifying any initial weak asymmetryinto a well-defined cell polarity even without benefitof influences from neighboring cells. There is a sug-gestion that mutually antagonistic interactions ofPrickle and Frizzled with Dishevelled are critical(Tree et al., 2002). Other essential planar cell polaritycomponents such as the cell adhesion molecules Fla-mingo (Usui et al., 1999) and Dachsous (Adler et al.,1998) are presumed to be required for assembly of thesignaling interfaces at the proximal and distal poles ofthe cell, where these proteins are concentrated; theymay also play some part in the intracellular mecha-nism that forces opposite ends of the cell to adoptopposite characters.

Planar Cell Polarity Control inDrosophila: Global Orienting Signals

These studies of genes and cell biology in Drosophilagive us a glimpse of machinery responsible for thefirst two levels of polarity organization outlined in ouranalogy with ferromagnetism: the machinery, that is,that gives each cell its intrinsic polarity and coordi-nates the polarity vectors of neighbors within eachlocal domain. In Drosophila epidermis, these twoaspects of polarization appear to be closely interde-pendent. But what mechanism governs the globalorganization of the polarity field, ensuring a uniformorientation over the whole wing surface, for example,with the cells in every domain pointing toward thedistal margin? Here one might expect to find a depen-dence on some secreted signaling molecule, capableof acting at long range to influence the orientation ofthe cells. A concentration gradient of the signalingmolecule could serve as the vector field that organizesthe global pattern. In fact, a large series of experi-ments on the insect thorax and abdomen has beeninterpreted in terms of concentration gradients servingto define even the local details of the polarity pattern(Lawrence et al., 2002). The authors of this workpostulate a two-tier mechanism, in which a concen-tration gradient of a primary signaling molecule—Hedgehog protein in some regions, an unknown factorelsewhere—indirectly provides long-range control bygoverning production of an unknown second factor X,whose concentration gradient directly defines the lo-cal details. A contrary interpretation, closer to theview we take in this essay, might be that there is no

factor X, but that local details are governed by theFrizzled-dependent mechanism for propagating polar-ity from cell to cell via cell–cell junctions. But in anycase, it seems that a long-range signal has to beinvoked at some level in the control system. AlthoughHedgehog appears to have this role in some regions,in other places, such as the wing, it clearly does not,because it is not expressed in the required pattern.

An obvious suspect for the global signaling func-tion would be Wingless or some other member of theDrosophila Wnt family, because these molecules areknown to be extracellular ligands for Frizzled pro-teins. Indeed, the Wnt homolog Lin-44 controls cellpolarity in Caenorhabditis elegans (Herman et al.,1995; Herman, 2001). Moreover, in vertebrate em-bryos, the cell movements of convergent extensionduring gastrulation, which depend on the same mo-lecular machinery as planar polarity in Drosophila,including homologs of Frizzled, Dishevelled, andStrabismus, are governed by a Wnt family member(Heisenberg et al., 2000; Park and Moon, 2002).However, no planar-polarity role for Wingless or anyother Wnt has been demonstrated in Drosophila, andin experiments where the pattern of Wingless expres-sion is altered, hairs and bristles still point in thenormal direction (Adler, 2002; Lawrence et al., 2002).Another candidate is the protein Four-jointed. Muta-tions in the four-jointed gene disturb planar polarity,and clonal analysis shows that the effect is felt forsome distance outside the mutant clone. From itssequence, the gene product appears to be a transmem-brane molecule whose extracellular domain could becleaved off to act as a diffusible signal, and geneexpression data suggest that it is present in a proxi-mal-to-distal gradient in the developing wing (Zeidleret al., 2000).

Downstream from Planar Cell PolarityDetermination in Drosophila: (I) PCPRead-out Genes and the Genesis of theOriented Cytoskeletal Structure

Once Frizzled and Prickle and their respective com-panions have become localized to opposite poles ofthe cell, defining a planar polarity vector in accor-dance with local and global cues, the cell begins togenerate its oriented cytoskeletal structures. The sub-cellular location of Frizzled predicts the site and di-rection of outgrowth of the hair; and in mutant cellslacking Frizzled the hair projects from the center ofthe apical surface instead of the distal margin (Wongand Adler, 1993). A further set of gene products,downstream from those we have just discussed, makethe link between Frizzled polarity and hair polarity


and bring about the assembly of the hair cytoskeleton.Mutations in these PCP read-out genes interfere withthe location or structure of the hair, but do not alterthe primary cell polarity as defined by the asymmetricdistribution of Frizzled protein (Adler, 2002).

The products of the PCP read-out genes includevarious proteins of unknown biochemical function,such as Inturned and Multiple wing hairs, as well asknown regulators of actin filament assembly such asDrosophila Rho-associated kinase (Drok), RhoA, andDiaphanous. Myosin VIIA (also known as Crinkled)also belongs in this class (Kollmar, 1999; Winter etal., 2001; Adler, 2002). Mutations in these compo-nents may cause the hair to be misplaced on the cellapex; or several hairs may form on a single cellinstead of the usual single one; or the hairs may beindividually deformed or branched. Mutations in my-osin VIIA, for example, as well as mutations in Drokand RhoA, cause each Drosophila wing cell to de-velop multiple wing hairs instead of one; but thesehairs are still correctly oriented and located at thedistal edge of the cell. Similar disturbances can beproduced by treatment of the developing tissue withtoxins such as cytochalasin D that interfere with actinfilament assembly (Turner and Adler, 1998). Microtu-bules also seem to play an essential part in organisinghair formation, because treatment with microtubule-dis-rupting agents such as vinblastine also causes a multiple-wing-hair phenotype (Turner and Adler, 1998).

Downstream from Planar Cell PolarityDetermination in Drosophila: (II)Orientation of Asymmetric Cell Division

Planar cell polarity in Drosophila (and in Caenorha-biditis elegans (Thorpe et al., 2000)) is important inan additional way that is relevant to problems ofvertebrate ear development. Besides organizing cy-toskeletal asymmetries of the polarized cells, it helpsto govern the asymmetric distribution of cell fatedeterminants in the cells when they divide. It thusplays a part in controlling the production of differentspecialized cell types. This has been studied espe-cially in the development of sensory bristles.

The four cells of a typical sensory bristle all arisefrom a single sense-organ precursor (SOP) in thedeveloping epidermis. Although its neighbors developas ordinary epidermal cells, the SOP cell dividesasymmetrically to produce two daughters committedto different fates. One, called pIIa, is determined asprogenitor of the bristle shaft and socket cells—anal-ogous (and perhaps homologous) to the vertebratesensory hair cell and supporting cell. The other, calledpIIb, is determined as progenitor of the sensory neu-

ron and neural sheath cell plus an additional cell thatmigrates away. The difference of fate results frominheritance of different amounts of the cellular deter-minant Numb, which acts by blocking receptivity toNotch signaling (Rhyu et al., 1994; Knoblich, 2001).At mitosis, the dividing SOP cell can be seen to havea crescent of Numb protein in its anterior cortex (withregard to planar polarity, the antero-posterior axis inthe thorax, where bristle development has beenmainly studied, corresponds to the proximo-distal axisin the wing). The mitotic spindle is aligned along thesame antero-posterior axis in such a way that only onedaughter—the anterior daughter—inherits Numb pro-tein. Correct positioning of the Numb crescent relieson Frizzled: in mutants lacking Frizzled, both theNumb crescent and the mitotic spindle orientation arerandomly oriented (Gho and Schweisguth, 1998; Bel-laiche et al., 2001). As a result, the fates of thedaughters are often mis-specified. In such mutants,however, the Numb crescent, though randomly lo-cated, still develops on one side of the SOP cell: thecell machinery for generating asymmetry in the dis-tribution of Numb still operates. This machinery hasbeen well reviewed elsewhere, and many of its com-ponents have been identified (Jan and Jan, 2001;Knoblich, 2001).

An intracellular signal critical for genesis of theNumb asymmetry is provided by the G-protein sub-unit G�i. When this is put out of action by mutation,Numb fails to become restricted to just one part of thecell cortex, but the distribution of Frizzled is notdisturbed, and the orientation of hairs in the mutantepithelium remains normal (Schaefer et al., 2001).Thus, the cells have at least two mechanisms forgenerating internal asymmetry, each capable of oper-ating independently of the other; but the correct ori-entation of the mitotic spindle and of the Numb asym-metry that governs cell fate determination depends onan influence from the Frizzled system that governs thecytoskeletal polarity (Bellaiche et al., 2001). In fact,asymmetry in the intracellular distribution of Numb isrequired again when the pIIa and pIIb cells divide, tocontrol the fates of their daughters; but this is orientedby mechanisms independent of the Frizzled planarpolarity pathway (Gho and Schweisguth, 1998). Formost purposes, therefore, we can regard the determi-nation of cytoskeletal cell polarity in Drosophila, asmanifest in the orientation of hairs or bristles, aslargely independent and separate from the determina-tion of cell fate decisions such as that between bristleshaft cell and supporting socket cell. The two pro-cesses are governed by distinct sets of genes—theprimary PCP genes for the first, the asymmetric celldivision genes for the second.


MOLECULAR MECHANISMS IN THEVERTEBRATE EAR

Genetics of Hair-Cell Polarity Control inthe Vertebrate Ear: (I) Primary PCPGenes

The studies in Drosophila give us a conceptual frame-work and some specific indications of how hair-cellpolarity might be controlled at a molecular level in thevertebrate ear, where much less is understood. Wemust now examine the direct molecular and geneticevidence from vertebrates. How far are the parallelswith Drosophila likely to extend, and what new ordifferent mechanisms are likely to operate?

There are, first of all, encouraging indications fromgene expression patterns that the role of the primaryPCP genes may be conserved from invertebrates tovertebrates. At least four different frizzled homologs—c-Frizzled-1, -7, and –10—are expressed in theotocyst of the chick, for example, and all of these areexpressed in the developing sensory patches (Stark etal., 2000; and our own unpublished observations)(Fig. 2). In the mouse, frizzled-4 has been found to beexpressed in cochlear inner hair cells; curiously, mu-tants with this gene knocked out go deaf progres-sively, but without any loss of hair cells or auditoryneurons (Wang et al., 2001). Studies in the chickshow that at least two Wnt genes (Wnt2b and Wnt3A)

are expressed in the otocyst (Hollyday et al., 1995;Jasoni et al., 1999). Moreover, mice express all oftheir three flamingo homologs (called Celsr1-3) in theotocyst (Shima et al., 2002). The other Drosophilaprotocadherin gene essential for planar cell polarity,dachsous, has a mammalian ortholog called Pro-tocadherin 16 (PCDH16), and several other mamma-lian homologs, not quite so closely related, includingProtocadherin 15 (PCDH15) and Cadherin 23(CDH23, also called Otocadherin). No data on ex-pression or function are available for PCDH16, butPCDH15 (Alagramam et al., 2001) and CDH23 (DiPalma et al., 2001) are both required in the ear forcorrect hair-bundle formation. Mutations in these lat-ter two genes are responsible, respectively, for Ushersyndrome types 1F and 1D—two similar disorders,characterized by severe congenital deafness and ves-tibular malfunction, combined with a progressive ret-initis pigmentosa that has a later onset (Petit, 2001).Both these human syndromes have counterparts in themouse, where the ear pathology can be investigated indetail. PCDH15 mutations are the underlying abnor-mality in the Ames waltzer mouse (Alagramam et al.,2001), while mutations in CDH23 are the underlyinglesion in the waltzer mouse (Di Palma et al., 2001)and in the sputnik zebrafish mutant (Nicolson et al.,1998; Whitfield et al., 2002). In both cases, the haircells are abnormal, with disorganized stereocilia, amisplaced kinocilium, and, often, a polarity that is notproperly coordinated with that of neighbors. Othermembers of the Drosophila primary PCP gene setbesides those mentioned above also have homologs invertebrates, although little is known about their ex-pression or function in the developing inner ear. Thereare, therefore, many hints that hair-cell polarity in theear might be established by a system of genes homol-ogous to the primary PCP genes of the fly; but we arefar from having a proof.

Interestingly, studies of normal vestibular hair celldevelopment in the mouse ear have revealed a struc-ture that could be the link coupling asymmetric cellsurface features to asymmetric positioning of the ki-nocilium: the basal body of the kinocilium has astriated rootlet that becomes transiently aligned par-allel to the cell apex and appears to tether the basalbody to an adherens junction on one side of the cell(Denman-Johnson and Forge, 1999).

Genetics of Hair-Cell Polarity Control inthe Vertebrate Ear: (II) PCP Read-outGenes

Turning from the primary PCP genes to the PCPread-out genes, the evidence for homology between

Figure 2 Expression of a frizzled gene in vestibular haircells of an 8-day chick embryo. Red staining shows thepattern of in situ hybridization with a probe for c-frizzled-5,which at this stage is restricted to the hair cells. The sup-porting cells are stained green with an antibody against theNotch ligand c-Serrate1.


the vertebrate system and the fly system is perhaps alittle stronger. As has been well argued elsewhere(Kollmar, 1999), morphogenesis of the hair bundledoes indeed seem to depend on the control of actinassembly in much the same way as in fly bristles andhairs, with the small GTPase RhoA providing a link tothe Frizzled PCP pathway. In both systems, for ex-ample, an important part is played by the proteinDiaphanous, which serves to couple activated RhoAto the proteins that directly regulate polymerization ofactin. Dominant mutations in a human Diaphanoushomolog are responsible for a form of progressivehearing loss, DFNA1 (Lynch et al., 1997). (The effectof homozygous loss of this gene in the ear has notbeen reported, and would presumably be more severe.As is often the case, early lethality may prevent usfrom seeing the full ear phenotype.)

The role of myosin VIIA in both insect and verte-brate systems is particularly striking. As we havealready discussed, myosin VIIA in the fly also actsdownstream from RhoA to control cytoskeletal devel-opment, and mutations in the myosin VIIA gene leadto malformed bristles and hairs. In humans, mutationsin this gene are responsible for the Usher 1B syn-drome (Weil et al., 1995), in mice for the shakerdeafness phenotype (Gibson et al., 1995), and in ze-brafish for the mariner phenotype, where the inner earis again nonfunctional (Ernest et al., 2000). MyosinVIIA in the ear is restricted to hair cells, where it isseen throughout the cytoplasm and in the stereocilia(Hasson et al., 1995; Kussel-Andermann et al., 2000).In the absence of myosin VIIA, the stereociliary bun-dles become grossly disorganized, although the haircells initially display a polarity that seems at leastapproximately correct (Self et al., 1998, their Fig. 2).

Hair-Cell Determination, AsymmetricCell Division, and Hair-Cell Polarity inthe Vertebrate Ear

In Drosophila, as we have seen, planar cell polarityduring sensory bristle development—and in someother contexts—is associated with asymmetric celldivision, generating different cell types by differentialsegregation of cell fate determinants at mitosis. Thetwo processes are coupled in the sense that the orien-tation of the two types of asymmetry is coordinated,but independent in the sense that either type of asym-metry can be generated in the absence of the other. Inthe vertebrate ear, it is not clear whether asymmetriccell division plays a part in the production of haircells. A homolog of Numb is expressed in the cells ofthe developing sensory patch, and is localized basallyin the precursor cells (Eddison et al., 2000), but there

is no evidence as yet that it controls cell fate throughasymmetric divisions. Although hair cells can arise assisters of supporting cells (Fekete et al., 1998), theycan also arise just as commonly as sisters of hair cells,at least during regeneration in the chick basilar papilla(Stone and Rubel, 2000).

An extreme test of the relationship between haircell determination and hair cell planar polarity isprovided by the zebrafish mind bomb (mib) mutation.mib mutants, by several criteria, have a defect in theNotch signaling pathway: for example, excessivenumbers of primary neurons are generated in theCNS, reflecting a failure of the lateral inhibitionmechanism that would normally enable each nascentneuron to prevent its neighbors from differentiating asneurons also. In a mib inner ear, all cells of a sensorypatch differentiate prematurely as hair cells, againpresumably because of a failure of lateral inhibition:cells adopting a hair cell fate fail to inhibit theirneighbors from doing the same, thereby allowingthem all to become hair cells also (Haddon et al.,1998). The mutant patches, which consist entirely ofhair cells without supporting cells, are eventually ex-cluded from the epithelium and the hair cells disap-pear, but before this occurs the hair cells producemorphologically normal polarized bundles of stereo-cilia with a normal kinocilium (Haddon et al., 1999).Neighboring hair cells align their bundles in a similardirection, much as in a wild-type macula. Thus, (1)the polarization of hair cells individually, and (2) thecoordination of the orientations of neighbors appear tobe independent of Notch signaling, independent ofsupporting cells, and independent of asymmetric celldivision. The third tier of polarity control—the orien-tation of hair cells in relation to the ear as a whole—ishard to assess in these mutants, because the timing ofhair-cell differentiation and the dimensions of thehair-cell patches are so severely abnormal.

Notch pathway mutations have also been studied inthe mouse, although none of those analyzed show earphenotypes as severe as that of mind bomb in the fish.Thus, in mice homozygous for a knock-out mutationof Jagged2, coding for one of the three Notch ligandsexpressed in the developing sensory patches of theear, there is an overproduction of hair cells by about20% (Lanford et al., 1999). Thanks to the extremeprecision of the normal structure of the organ of Corti,it can be seen that the hair cell orientations in themutant are less well coordinated than in the wild-type.It remains to be seen whether this reflects a realdisturbance of planar polarity mechanisms, or merelya mechanical effect of the abnormal packing of haircells and supporting cells.


CONCLUSION

In this essay, we have focused on the mechanisms thatcreate and orient planar cell polarity in hair cells, andwe have argued that clues from Drosophila may helpus to identify the relevant molecules and discover howthey work. Downstream from planar polarity determi-nation, there are, of course, many other proteins in-volved in hair-bundle morphogenesis, some of themimplicated in hereditary deafness, and we havetouched on these only briefly. Further details can befound in several excellent recent reviews (Holme andSteel, 1999; Kollmar, 1999; Muller and Littlewood-Evans, 2001; Petit, 2001; Steel and Kros, 2001; Whit-field et al., 2002).

Although studies in Drosophila should help us tounderstand some of the basic principles, there arecertainly important aspects of planar polarity in ver-tebrate hair cells that have no direct counterpart in thefly. For example, the mechanism of transduction in avertebrate hair cell, involving separate stereocilia con-taining transduction channels coupled by tip links, hasno parallel in the insect bristle. The bristle shaft cell isthought to serve merely for passive relay of the me-chanical stimulus to the associated neuron, in whosedendrite transduction occurs (Chung et al., 2001).Thus analogies with Drosophila have nothing to tellus about the speculative hypothesis we outlined ear-lier, suggesting that creation of the staircase pattern ofthe stereociliary bundle could itself be controlled bythe transduction apparatus. Indeed, although a re-markable amount of new molecular information onear function and development has been gathered in thelast 10 years, through studies both in vertebrates andin invertebrates, it remains as much of a challenge asever to explain how all the components function to-gether to produce the marvellously precise and com-plex architecture of the hair cell (Tilney et al., 1992).But if we can fathom the origins of planar cell polarityin the ear, we shall at least be able to see the founda-tions on which that architecture is built.

We thank Tanya Whitfield for contributing a photo-graph, and Helen McNeill, Manolis Fanto, and the membersof the Vertebrate Development Lab at Cancer Research UKfor comments and discussions.

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