One of the striking messages from the revolutions of molecular genetics and “evo-devo” (Duboule,2010) is the notion that in highly different phyla, analogous functions are often fulfilled by families of homologous genes. While anteroposterior patterning by Hox genes throughout the animal kingdom may be the most celebrated example of this phenomenon, the directing of cell polarity in the plane of epithelia by a common set of genes is equally striking. Proper development and functioning of tissues require among many other things the establishment of common orientation in a sheet of cells. Planar cell polarity (PCP) signaling refers to the mechanism or mechanisms responsible for providing the cells with the information on this polarity. An illustrious case in point is seen when mutations affect the genes involved in this PCP signaling pathway: In insects, a disrupted orientation of wing hairs and body bristles is seen, while mammals present with a seemingly similar disorganization of the far from homologous epidermal hairs (Devenport and Fuchs,2008). It is now clear that PCP signaling appears to be widespread in the animal kingdom.
In 1944, Bridges and Brehme described the first mutant allele of lethal giant larvae (lgl), an apicobasal polarity gene recently shown to be involved in PCP as well (Bridges and Brehme,1944; Kaplan and Tolwinski,2010). In 2000, still only three Drosophila mutants of this class were known, as reviewed by Bilder (Stewart et al.,1972; Bilder and Perrimon,2000; Bilder,2004). The field of PCP signaling was actually opened with the work of Lawrence and Shelton and others (Lawrence and Shelton,1975; Vinson and Adler,1987), who introduced the notion of planar polarity and its transmission from cell to cell. See for comprehensive reviews, (Bilder,2004; Klein and Mlodzik,2005). Further work in the fruit fly resulted in the identification of a set of “core” PCP factors at the center of this signaling mechanism, including Diego (Dgo), Van Gogh (Vang), Flamingo (Fmi), Prickle (Pk), Frizzled (Fz), and Disheveled (Dsh). These core factors typically become localized in opposite subcellular regions, generating molecular polarization and establishing morphological and functional polarization).
The molecular events underlying planar polarity have been studied initially in Drosophila. These analyses revealed that the first directional cue for localization of the core proteins is thought to be derived from the atypical cadherins Fat (Ft) and Dachsous (Ds), that act in concert with the Golgi protein Four-jointed. An alternative theory holds that an unidentified morphogen, acting in parallel with Fj, Ds, and Fat provides this critical early cue (see for references Chen et al.,2008). Ft and Ds localization appears to induce alignment of microtubules at the apical side of the cell and in a proximodistal orientation.
Importantly, asymmetric distribution of core factors is often transient and precedes crucial morphological events such as prehair growth in fly wing, and similarly analogous events in vertebrate tissues. In this case, a Vang-Pk complex localizes at the proximal side of the cell and a complex containing Fz, Dsh, and the ankyrin repeat protein Diego (Dgo) distally. Signaling between cells is an essential factor in the stabilization of polarization; it involves factors such as Flamingo (Fmi), fz, and vang. The seven-pass transmembrane protein Fmi mediates cell interactions through homophilic interactions and is localized both distally and proximally; Vang heterophilically interacts with the extracellular domain of fz (Wu and Mlodzik,2008). In those cases where vertebrate orthologs of these genes have been studied, they have analogous roles, interactions and subcellular locations; see Figure 1.
Relation to Other Pathways
Both in Drosophila and vertebrates frizzled and disheveled orthologs mediate PCP signaling. In vertebrates, Wnt factors have been shown to be ligands of a subset of the frizzled proteins involved in “noncanonical Wnt signaling.” Despite the lack of evidence that in Drosophila wingless is involved, this term is sometimes also used to refer to PCP signaling in Drosophila. Fat signaling may be considered a separate pathway in addition to apical basal and “core PCP” signaling (Lawrence et al.,2007; Goodrich,2008).
The multidomain protein Dsh (Dvl in vertebrates) is the common mediator of the canonical and noncanonical Wnt signaling. The distinctive difference between these pathways is that downstream from Dvl/Dsh, activation of the canonical Wnt pathway prevents degradation of cytoplasmic β-Catenin, leading to activation of β-catenin mediated transcription, whereas in the noncanonical pathway a Jun kinase/Rac/Rho pathway is activated. This kinase is a key regulator of cytoskeletal dynamics, which is likely to be essential for the mechanism by which PCP signaling orients cells (Strutt et al.,1997; Habas et al.,2001; Marlow et al.,2002). Apicobasal polarity and PCP are interlinked by the core PCP proteins that colocalize with adherens junctions. Direct physical interactions between PCP proteins and apicobasal determinants have been reported; see (Axelrod,2009).
In this review, we attempt to provide an overview of this broad field, focusing on vertebrate studies and including data from Drosophila mainly as illustration of its historical basis, and referring where possible to existing reviews.
INVERTEBRATES VERSUS VERTEBRATES
PCP regulates the ordered orientation of external structures such as epidermal hairs in mammals as well as internal structures such as the hair cells in the organ of Corti (see Wu and Mlodzik,2009). At a molecular level, it is clear that many of the proteins that have been linked to PCP in the fly, including the core proteins vang, fz, Dsh, and fmi, have been evolutionary conserved and have analogous roles in vertebrates. Single or double mutants of mouse Van Gogh-Like1 (Vangl1), Vangl2, Fzd3, Fzd6, Dvl1, Dvl2, and the fmi ortholog Celsr1 have been shown to cause distinguishing phenotypes and combinations of phenotypes that can be explained, directly or indirectly, by disturbed planar polarity. As often when comparing the Drosophila and mammalian model systems, complexity is increased by duplication events of many of the genes involved. Consequently, studying PCP-related phenotypes in vertebrates often requires generating complex genotypes.
In vertebrates additional proteins have been identified that have a function in PCP (Wang and Nathans,2007). Scribble (Scrib) and Ptk7 mutants exhibit classic PCP phenotypes and they show genetic interaction with Vangl2 (Murdoch et al.,2001; Lu et al.,2004), and the frizzled ligands Wnt11 (Zhou et al.,2007), Wnt5a (Qian et al.,2007), and possibly Wnt9b (Karner et al.,2009) function in the regulation of PCP whereas a Wnt ligand has not been shown to act in PCP signaling to date in Drosophila. See for recent reviews also Axelrod (2009) and McNeill (2010).
The Early Embryo
Wnt/PCP signaling is an essential player in the processes that control convergence and extension (CE) and other cell migration during gastrulation. It provides directional cues that are necessary for the orchestration of these migrations and at least some of these cues involve regulated planar polarity. Evidence comes from studies of phenotypes of mutants and morphants of known PCP genes include gastrulation phenotypes. We refer for this evidence to the reviews of (Tada et al.,2002; Roszko et al.,2009). Much of the original work has been done in zebrafish and Xenopus, attractive models for studying cell migration in early vertebrate development, but evidence from the mouse also abounds. Mouse mutant embryos carrying two or more loss of function alleles of PCP genes also have been reported to be abnormally short and broader (Wang et al.,2006a; Ybot-Gonzalez et al.,2007; Yen et al.,2009) confirming disrupted CE. Vertebrate orthologs of Drosophila genes that had been linked previously to PCP, including Vangl and Fzd genes, as well as Wnt5a and Wnt11, were thus implicated in CE.
A possible explanation at the cellular level for these and other migration defects in PCP mutants is that lamellipodia on migrating cells are polarized these actin-rich cell “feet” being responsible for the traction necessary for cell movement. In Xenopus cells deficient in Dvl, these lamellipodia are not polarized and randomized, resulting in a CE defect (Wallingford,2006).
Dact genes constitute an example of PCP related function with an impact on gastrulation. Dact proteins were discovered in a search for proteins interacting with the Dvl protein. In mouse and man they are encoded by a family of three paralogous genes. Probably these genes are vertebrate-specific. It has been shown that they modulate Wnt signaling through their interaction with the PDZ domain of Dvl. Comprehension of their roles through mutant studies is complicated by the fact that they modulate both Wnt pathways. Dact1 mutants have neural tube closure defects as well as posterior truncation defects including short tail and lack of anus or urinary outlet. These phenotypes have an early basis during gastrulation in disturbed germ layer formation. Posterior truncation is commonly associated with disrupted Wnt/β-Catenin signaling, but in the case of the Dact mutants the earliest defect could be coupled directly with PCP signaling. Suggestive of a CE defect, at four- to seven-somite stages mutant embryos were abnormally wide posteriorly, with a broader presumptive notochord region than wild-types. Moreover, known targets of canonical Wnt signaling were normally expressed, whereas Rho kinase activity was decreased. Several observations strongly suggest that Dact1 regulates Vangl2 during gastrulation: Vangl2 protein levels are elevated in the primitive streak of Dact1 mutants, and removal of one functional Vangl2 allele rescued much of the phenotype of homozygous Dact1 defects (Suriben et al.,2009). Finally, further molecular studies showed that Dact1 regulation of Vangl2 involves direct physical interaction of the two proteins.
Neurulation and PCP
During primary neurulation, the edges of the neural plate fold up and then inward until they make contact and fuse, thus transforming the neural plate in a neural tube. In the mouse tail, secondary neurulation takes place involving cavitating from a solid rod.
The larger width of early embryos with deficient PCP signaling may preclude the fusion of the neural folds, thus causing the neural tube closure defects seen in many mouse PCP mutants. Both in frog and mouse the failure of the neural tube to close has been linked to disrupted PCP signaling. Craniorachischisis, an extreme phenotype consisting of failure of the neural tube to close between midbrain and tail, plausibly results from the abnormal large distance of the neural tube rims in PCP mutants (Copp et al.,2003a). It is caused by a neural tube “closure 1 defect,” referring to the most posterior of three or two sites from which closure initiates sequentially in the mouse (Copp et al.,2003b). Significantly, most or all mutants displaying craniorachischisis have been placed into the PCP pathway. Examples include the Vangl2, Celsr1, Scrib, and Ptk7 mutants where a single homozygous mutation causes this phenotype. Double homozygous mutants such as Dvl1/2, Dvl2/3, and Fz3/6 also show craniorachischisis, while single mutants for these genes do not exhibit this phenotype, presumably due to redundancy between these paralogous genes. Several other Wnt-signaling related genes have been implicated in neural tube closure and PCP by genetic interaction with Vangl2. The Vangl2LP/+; Wnt5a−/−, Vangl2LP/+; Cthrc1LacZ/LacZ, Vangl2LP/+; Grhl3CT/CT, Vangl2LP/+; CoblC101/C101, Vangl2LP/+; Sfrp1−/−; Sfrp2−/−; Sfrp5+/−, Vangl2LP/+; Sfrp1−/−; Sfrp2+/−; Sfrp5−/− double, triple, or quadruple mutants all show variable degrees of neural tube closure defects in the form of craniorachischisis, exencephaly or spina bifida (Greene et al.,2009). All of these genes have been linked to PCP signaling (Carroll et al.,2003; Stiefel et al.,2003; Qian et al.,2007; Satoh et al.,2008; Yamamoto et al.,2008; Misra and Matise,2010).
Oriented Cell Divisions
A cellular process that often depends on PCP signaling and that conceivably contributes to the neural tube defects in PCP mutants is oriented cell division. A link between oriented cell division and noncanonical Wnt signaling has been demonstrated in Drosophila (Gho and Schweisguth,1998) and zebrafish (Gong et al.,2004). Given the highly ordered directional cell divisions in the developing neural tube it is plausible that disruption of this process may interfere with closure of the neural tube in animals with primary neurulation such as mouse and frog. In zebrafish, in which secondary neurulation occurs throughout the spinal cord, lumina are formed along the medullary cord, which will eventually fuse to form the neural tube. The initiation of this process requires a precisely regulated rearrangement of cells around the midline followed by a unique cell division that produces daughter cells, with apicobasal polarity being mirrored. Planar polarity is involved in these events, as in zebrafish PCP mutants these polarized divisions take place ectopically, away from the midline, leading to multiple ectopic neural tubes (Lowery and Sive,2004; Tawk et al.,2007; Clarke,2009; Roszko et al.,2009). Work of Ciruna and coworkers (2006) convincingly pinned down this role of PCP. Their work was building upon earlier observations showing that in zebrafish, division of a neural progenitor in the neural keel leads to bilateral distribution of the daughter cells as the apical daughter cell crosses the midline into the contralateral side of the neural tube (Kimmel et al.,1994; Concha and Adams,1998). Detailed analysis of this cellular behavior in Vangl2 (trilobite) mutants showed that following initially normal behavior of the apical and basal mutant daughter cells, subsequently the apical cell did not intercalate in the contralateral neuroepithelial layer (Ciruna et al.,2006). This effect was shown to be cell autonomous by means of grafting of mutant and wild-type cells into wild-type hosts. A further observation in this study firmly linking this process to cellular polarization signaling is the asymmetrical cellular localization of a Pk fusion protein in wild-type cells that is disturbed in the Vangl2 mutant. Mitotic inhibition rescued the neurulation defection Vangl2 mutants, establishing with a high degree of certainty the link between cell division and morphogenesis (Ciruna et al.,2006). More recently, Quesada-Hernandez and co-workers reported impaired CE movements during gastrulation in double mutants for the zebrafish fzd7 paralogs fz7a and fz7b, which could be rescued by overexpression of fzd7, but not the PCP gene Wnt11. By directly manipulating spindle orientation by means of antibody-mediated blockage of Dynein, they were able to demonstrate that disruption of spindle orientation by itself interferes with midline formation, but not with extension of the body-axis. While it had been previously suggested that the axis extension defects that accompany midline formation defects in PCP mutants could be causally linked, these observations separate these two processes (Quesada-Hernandez et al.,2010).
Two further different contexts in which PCP has been linked to oriented cell division are the kidney and the gastrointestinal tract: (i) disruption of PCP leads to partial randomization of oriented cell divisions in polycystic kidney disease (Fischer et al.,2006) and (ii) mice in which either Wnt5a, or Vangl2 or specific combinations of the PCP regulating Sfrp genes have been inactivated display disruption of oriented cell divisions in the developing fore-stomach (Matsuyama et al.,2009).
Recently, Segalen and coworkers, using a cell system in which polarity can be manipulated, presented convincing evidence that spindle orientation is dependent on Dvl. More specifically, the effect is mediated through an interaction of the DEP domain of Dvl with a protein named Mud in zebrafish and NuMA in Drosophila (Segalen et al.,2010). Even more recently Solnica-Krezel et al. addressed the link between centrosome position and the PCP pathway in zebrafish gastrulation. They showed that the position of MTOCs (centrosome/microtubule organizing centre) is polarized both in apicobasal and planar directions. Polarization (relative to nucleus and body axis) in the plane of both ectoderm and mesoderm of gastrulae was shown to depend on PCP signaling. Moreover, upsetting microtubule structure by using the inhibitor of microtubule nocodazole led to deficient anterior localization of Pk, although only the initial generation and not the maintenance of polarization. This report establishes a link between molecular and cell biological aspects of the PCP pathway (Sepich et al.,2011).
Cilia are organelles that project from almost all animal cells and most other eukaryotic cells. Known functions of cilia include mechanosensation linked to cell signaling in primary cilia, and the mechanical functions of motile cilia in the directing of neuronal cell migration and fluid flow, which includes the determination of laterality by the a-typical nodal cilia.
An increasing number of long-known human diseases have recently turned out to be ciliopathies. They include polycystic kidney disease as well as several less common diseases such as Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome, orofaciodigital syndrome, and nephronophthisis. Cilium structure, its role in development and its connections with Hedgehog and other signaling pathways have recently been reviewed (Goetz and Anderson,2010). From different experimental approaches in Xenopus, zebrafish, and mouse an essential function of PCP has emerged in the last few years, in the determination of cilia orientation and its consequences for mechanical fluid dynamics. The PCP signaling has been implicated in apical actin organization, explaining the functional link at a molecular level.
Among the earliest evidence for a link between PCP and cilia was the observation that mice with mutations in genes linked to the human BBS, a disorder associated with ciliary dysfunction, show defects such as failed neural tube closure, open eyelids at birth and misalignment of hair cells in the cochlea. The link with the PCP pathway suggested by these observations was confirmed when analysis of the cochlear phenotypes in double-heterozygous and other compound mutants involving Vangl2 showed synergism and therefore genetic interaction (Ross et al.,2005; Rida and Chen,2009). The necessity of Dvl for ciliogenesis in Xenopus was demonstrated by Park and coworkers who knocked down Dvl1-3 using morpholinos and showed that size and number of cilia were decreased in the multiciliated epidermal cells (Park et al.,2008). While this demonstrates a role of PCP upstream from ciliogenesis, other evidence indicates a role in the mechanism underlying orientation of cilia, governing for instance the directional fluid flow brought on by motile cilia.
A relation between planar polarity and cilia orientation has been studied in several groups. It appears that in Xenopus larvae skin, PCP-dependent signals from nonciliated cells induce orientation of cilia in epidermal cells. Subsequently, a cilia-generated flow feeds back to further establish and fine tune the direction of the flow, PCP signaling being a part of this feedback mechanism (Mitchell et al.,2007,2009). Guirao et al. (2010) more recently studied the mechanism regulating cerebrospinal fluid (CSF) flow in the developing mouse brain. These authors stress the importance of the impact of flow on orientation, both flow and PCP by itself not being sufficient. In contrast with the Dvl morphants mentioned above (Park et al.,2008), beating cilia did develop in an ex vivo culture system in Vangl2 mutant tissue, however generating a randomly directed flow. A response to hydrodynamic forces of artificial flow seen in wild-type cultures was not seen in mutant cultures; therefore PCP is essential for the response to hydrodynamic forces (Guirao et al.,2010). Another link between CSF circulation and PCP signaling was reported by Tissir et al. (2010). Mouse mutants for Celsr2, like Celsr3 a vertebrate paralog of the Drosophila PCP core gene Fmi, develop hydrocephalus because of disrupted CSF. A similar but stronger phenotype in Celsr2/Celsr3 double mutants showed that both genes have overlapping functions. The Celsr genes act clearly upstream from ciliogenesis as is evident from affected ciliogenesis and abnormal subcellular location of Vangl2 and Fzd3.
Left Right Determination
Embryonic laterality refers to the establishment of the left–right axis. It is created by a fluid flow depending on ciliary movements in a key region of the vertebrate gastrula. In the mouse, this region is the ventral node; in Xenopus, the gastrocoel roofplate; and in zebrafish, Kuppfer's vesicle. To achieve a correct ciliary movement that results in a leftward fluid flow, it is necessary that the basal body of the nodal cilia shifts from a central position to the posterior side of the cells and acquires a tilted position as shown for mouse (Nonaka et al.,2005; Okada et al.,2005), rabbit, and Medaka (Okada et al.,2005). This process has been shown to be disrupted in mouse mutants lacking five of six Dvl alleles, but not in Wnt3a mutants, showing that PCP signaling and not canonical Wnt signaling is involved (Hashimoto et al.,2010). Essentially equivalent results were reported for Xenopus and zebrafish embryos in which Vangl2 was knocked down using morpholino technology (Antic et al.,2010; Borovina et al.,2010) and for mouse Vangl1 heterozygotes (Antic et al.,2010). Furthermore, mutants for Bicc1 and the genetically interacting genes Inversin and irrc6 lead to early (i.e., before left Nodal expression) laterality defects. All these genes have been linked to ciliary functions, left–right determination and PCP signaling.
Inversin is a modulator of Dvl genes and thought to act as a switch between the canonical and noncanonical Wnt pathways (Simons et al.,2005). The Drosophila gene Bicaudal-C encodes an RNA-binding protein that regulates genes at a translational level. By inhibiting Oscar translation, it is thus involved in anteroposterior development. The vertebrate Bicaudal-C ortholog Bcc1 has been linked to cystic kidney in zebrafish (Bouvrette et al.,2010) and cilia function in mouse and Xenopus (Maisonneuve et al.,2009) by acting as a modulator of Dvl signaling (Maisonneuve et al.,2009). Lrrc6 is the gene mutated in the seahorse zebrafish cystic kidney mutant. It encodes the leucine rich repeat protein seahorse, which physically interacts with dvl and is thought to modulate both canonical and noncanonical Wnt pathways (Kishimoto et al.,2008).
Recently, a strong laterality phenotype was reported in mouse mutant embryos lacking both Vangl1 and Vangl2. The embryos did not turn and no heart folding occurred (Song et al.,2010). Analysis of the node of embryonic day (E) 8.0 embryos demonstrated that cilia were morphologically normal, but did not have their normal posterior localization, confirming that in this context PCP signaling is downstream from ciliogenesis and upstream from ciliary orientation.
Several of the further defects seen in PCP mutants and discussed below may also have, with various degrees of likeliness, a basis in ciliary defects.
Cardiac Defects in PCP Mutants
A role for PCP signaling in cardiogenesis was suggested by expression of the core factors Vangl1, Vangl2, and Scrib in the developing mouse heart and confirmed in mutant studies. Cardiac abnormalities are seen in Vangl2LP/LP, Vangl1GT/+;Vangl2LP/+, ScrbCRC/CRC, Dvl2−/− and Dvl3−/− mutants, including affected heart looping, smaller ventricles, ventricular septal defects, double outlet right ventricles and an abnormal arrangement of outflow arteries known as retro-esophageal subclavian artery (Phillips et al.,2007,2008; Etheridge et al.,2008; Torban et al.,2008). Significantly, similar phenotypes have been found in mutants for the noncanonical Wnt11 gene (Zhou et al.,2007), suggesting that this gene is an upstream regulator of heart development through the PCP pathway (Flaherty and Dawn,2008). Possible explanations for the cardiac phenotypes seen in PCP mutants were put forward by Philips et al. (2007) who suggested a link with heart looping. The formation of the four chambered heart in mammals from a linear heart tube includes the rightward looping of the tube that takes place between E8.5 and E10.5 (Buckingham et al.,2005; Manner,2009). Studies have shown that defects in heart looping may ultimately result in cardiac septation and alignment defects that include abnormalities in the outflow region of the heart (Phillips et al.,2007). Weak expression of looping defects could also conceivably result from early disrupted left–right determination. Philips et al. proposed that that polarity-dependent changes in the cytoskeletal network result in cell shape changes that are essential for looping. In addition, it now seems plausible that weak expression of the laterality defects discussed above contribute to some of the late appearing cardiac defects that have been described in PCP mutants. Finally these authors speculated that the abnormalities of the myocardializing cells of the outflow tract cushions (from which the valves develop) in Scrib mutants might be caused by defective PCP signaling, as these cells are an example of polarized, migrating cells (Phillips et al.,2007).
The coronary vasculature has been shown to be defective in Vangl2 mutant embryos (Phillips et al.,2008). RhoA/Rho kinase activity is decreased in the ventricular cells indicating that this is a true PCP defect. Vangl2 is not expressed in the epicardium, the progenitors of the coronary vessels, but migration of epicardially derived cells was impaired. It therefore seems that signaling from the myocardium to the epicardium, previously known to be an essential part of coronary development, is disrupted in these mutants. If confirmed, this would represent a rare example of a non–cell-autonomous PCP defect.
The Inner Ear
Mouse ear development starts around E8.0 with the appearance of the otic placodes, which invaginate to form the otic pit. This epithelial sac is reshaped to form the endolymphatic sac and duct, the presumptive utricular and saccular portions of the inner ear (see Fig. 2A). In the adult inner ear, six different structures contain sensory epithelia: the organ of Corti situated along the cochlear duct is responsible for the recognition and transmission of auditory signals, the sensory epithelia in the saccula and utricle are involved in perception of linear and angular acceleration and the sensory patches in the three cristae associated to the semicircular channels of the vestibule are responsible for maintaining balance (Fig. 2B). These sensory epithelia consist of nonsensory support cells and sensory hair cells. The mechanosensory hair cells are characterized by a bundle of stereocilia protruding from the luminal surface of the cell. Stereociliary bundles are organized and polarized in such a way that all the hair cells are aligned and oriented in one direction (Barald and Kelley,2004). The sensory epithelium in the inner ear with the highest degree of organization is the organ of Corti located in the cochlea. The hair cells are aligned in four rows along the cochlear duct, three rows of outer hair cells, and one row of inner hair cells (Fig. 2C). These hair cells have a characteristic V-shaped bundle of stereocilia all individually aligned and pointing in the same direction toward the lateral side of the organ of Corti (Fig. 2D; Rida and Chen,2009). Mutant phenotypes of the hair cell arrangement can be divided into two subgroups: (i) defects in proliferation and differentiation leading to extra or fewer rows of hair cells and (ii) defects in hair cell polarity, where hair cell alignment and orientation are affected (Fig. 2E).
While disrupted Notch/Delta signaling can lead to supernumerary, subnumerary, and/or ectopic hair cells (Hawkins and Lovett,2004; Brooker et al.,2006), a distinct type of defects is caused by disruption of PCP signaling.
In mutants linked to the PCP pathway, the cochlea has sometimes been described to be short and abnormally wide, which suggests an extension convergent defect (Wang et al.,2005). In addition, in many cases arrangement of the normally very regular rows of hair cells is disturbed (Fig. 2E).
This loss of an organized alignment of the stereocilia bundles in the organ of Corti is an easily perceptible read out and is frequently used to study PCP phenotypes in mice. The organ of Corti is one of a limited number of tissues in the mouse where the asymmetric localization of PCP proteins has convincingly been demonstrated. Vangl1,2 and Fzd3,6 localize to the medial (center of the cochlear spiral) side of the cells (Wang et al.,2006b) and Dvl1,2,3 proteins (Etheridge et al.,2008) to the lateral side. Loss of the polarized localization of these proteins leads to defects in hair cell orientation.
In the Vangl2LP/LP organ of Corti, the hair cell orientation is completely randomized and Fzd3 seems to be completely absent from the cell membrane. In the Celsr1Crash mutant, loss of polarized localization of Vangl2 leads to a similar phenotype (Montcouquiol et al.,2006). This indicates that these PCP proteins depend on each other to become asymmetrically localized.
Rather more difficult to study, an analogous role for PCP signaling has also been reported in the stereocilia of the vestibular sensory epithelia. The presumptive PCP protein Prickle-like 2 (Pk2) was seen to be medially localized in epithelial cells of utricle and saccula, before morphological polarization, while Fzd6 was localized at the lateral side of vestibular support cells (Deans et al.,2007). The expression of Pk2 in the context of the Vangl2 hypomorph mutant Looptail was qualitatively and quantitatively affected, demonstrating a functional role of Vangl2 and confirming that Pk2 is actually behaving as a PCP protein in vestibular epithelium.
Hair cell organization starts with the emergence of a single microtubule-rich primary cilium on the apical cell surface. A link between this primary cilium and the PCP pathway is expected based on (i) the position of the cilium being linked to the position of the stereocilia and (ii) the polarization of a row of stereocilia (the stereociliary bundle) being preceded by the polarization of the kinocilium, an (often transient) specialized primary cilium in the middle of this stereociliary bundle; Rida and Chen have recently reviewed the role of PCP in the inner ear (Rida and Chen,2009).
As mentioned above, mice with mutations in genes involved in BBS, show affected phenotypes similar to those in PCP mutants. These include neural tube defects, open eyelids at birth, and disruption of the hair cell orientation in the organ of Corti. In the hair cells, the kinocilium has lost its close relation with the stereocilia bundle. Double-heterozygous mutants with Vangl2 show an increased disruption of the hair cell orientation, indicating a link with the PCP pathway (Ross et al.,2005; Rida and Chen,2009).
In a different cilia mutant, deficient for the gene encoding Intraflagellar Transport Protein 88 (Ift88), the hair cell orientation is affected and the cochlea is shorter and wider, indicating a CE defect. Furthermore, the primary cilium is much shorter if present at all, and no longer closely associated with the stereocilia bundle. This leads to circular stereocilia bundles in addition to misorientation of the “V-shaped” bundles. The polarized localization of the core PCP protein Vangl2 is, however, not altered. The cochlear phenotype deteriorates further in Ift88CKO/CKO;Vangl2Lp/+ mutants, again indicating a link between PCP signaling and the position of the primary cilium. The data on both the BBS and Ift88 mutants suggest that the ciliary basal body, by means of the polarization of the centrioles, directs the intrinsic polarity of the hair cells (Jones et al.,2008; Rida and Chen,2009).
The significance of PCP signaling for the development of tubular organs has recently become evident. As cited above, inactivation involving Wnt5a, Vangl2, or a combination of Sfrp1/2/5 alleles leads to morphological defects in the developing forestomach of mutant mice (Matsuyama et al.,2009).
In the kidney, in addition to cilia-based and PCP-linked defects mentioned above, Karner et al. (2009) demonstrated that, at postnatal stages, Wnt9b has a role in renal tubule morphogenesis. Previously, inactivation of this gene had been shown to disrupt epitheliomesenchymal transitions that are essential for renal vesicle induction, which appeared to be mediated mainly or completely through canonical Wnt signaling (Carroll et al.,2005; Park et al.,2007). Using a conditional knockout approach to circumvent early lethality, the later effect was proposed to be linked to disrupted planar polarity. In these Wnt9b mutants kidney tubules with abnormally large diameter, as well as eventually cysts develop, not as a consequence of overproliferation but presumably due to disturbed planar polarity of the tubule epithelium, in which cells are abnormally elongated (Karner et al.,2009).
The uterus and vagina of mice homozygous for the LoopTail allele of Vangl2 have been shown to be morphologically highly abnormal. These defects include inperforate or septate vagina, lack of oviduct coiling, short uterine horns, and failure of the uterine horns to fuse at the cervix (Strong and Hollander,1949; Vandenberg and Sassoon,2009). Wnt7a mutants have a comparable phenotype and Wnt7a is expressed at lower levels in the uteri of Vangl2 mutants. Moreover, Vangl2 and Wnt7a interact genetically with each other. Together with abnormal cellular morphology and localization of the PCP factor Scrib, these observations suggest that the abnormal morphology of the female reproductive tract in these mutants is another expression of disrupted PCP signaling (Vandenberg and Sassoon,2009).
Recently a function in lung development was revealed by a study that showed that all three modes of branching that occur in mouse lung buds (Metzger et al.,2008) require the function of Celsr1 and Vangl2 (Yates et al.,2010). In an ex vivo culture system, inhibition of Rho kinase mimicked the phenotype and the genetic defect could be rescued by Rho overexpression. Similar defects occur in Scrib and Ptk7 mutant longs (cited in Yates et al.,2010).
Upstream from the PCP Signaling Pathway
The disruption of regulatory processes upstream from factors involved in PCP can lead to their misexpression, and thus produce phenotypes characteristic of disturbed PCP signaling itself. An example is already given in the discussion of the Dact genes in the section Early Embryo.
The Secretory Pathway
Almost one-third of all proteins are formed in association with the membranes of the endoplasmatic reticulum (ER). These extracellular and membrane proteins need to be actively transported from the ER to the Golgi apparatus. Small peptide sequences on secretory proteins are a signal for COPII-coated vesicle mediated ER-to-Golgi transport at the ER exit sites. Work in yeast identified a series of proteins that form the COPII-coated vesicles, including Sar1, Sec23, Sec24, Sec13, and Sec31. The genes encoding these proteins have been conserved in vertebrates and they in many cases duplicated (see Farhan et al.,2007; Sato and Nakano,2007).
Quite unexpectedly, mutations in the Sec24b gene, one of four genes encoding a family of Sec24 proteins in mice, have been linked to typical PCP phenotypes including craniorachischisis, disorganized hair cell arrangement in the organ of Corti (Merte et al.,2010; Wansleeben et al.,2010) as well as the cardiac outflow tract abnormality retro-esophageal subclavian artery (Wansleeben et al.,2010). Abnormal subcellular localization of the core PCP factor Vangl2, confirming a link with PCP signaling, was demonstrated by both groups of authors.
These results appear to reveal an unexpected level of specificity of the intracellular trafficking system. Intriguingly, it was recently reported that in Drosophila apicobasal polarity defects arise because of mutations in genes encoding components of COPII coated vesicles. Among these genes is the haunted gene encoding a factor that is structurally related to Sec24C and Sec24D (Norum et al.,2010). While this concerns a different type of polarity, it might be another example of cell polarity being sensitive to generalized cellular abnormalities caused by defective trafficking. Alternatively, later research might demonstrate a more closely related nature of both Sec24 phenotypes given the overlap in canonical and noncanonical Wnt signaling. Furthermore, in several instances, the two types of polarity have been interlinked. For instance, Drosophila Scribble was originally described as an apicobasal determinant, whereas its vertebrate ortholog is a typical PCP gene; however, recently Scribble was found to be an effector of the core PCP gene vang (Courbard et al.,2009).
Another example of the importance of regulated transport of PCP proteins having an impact on planar polarity was described by Shimada et al. (2006). These authors addressed the mechanism underlying the polarized localization of one of the core factors, fz. They followed a green fluorescent protein (GFP) -tagged fz protein in Drosophila wing epithelial cells by in vivo time-lapse imaging as it localized to the membrane just before prehair formation. It appeared that the tagged fz protein was transported in vesicles by means of proximodistally oriented microtubules at the level of the adherens junctions, and preferentially toward the distal cortex. As predicted by this hypothesis, disruption of microtubules perturbed the localization of fz and, moreover, caused mislocalization of prehair formation as would be the case in fz mutants. Furthermore, the localization was confirmed by transmission electron microscopy. It is not known whether this mechanism is also active in vertebrate cells.
Role of Ubiquitination
Ubiquitination forms a striking example of regulated protein degradation as a modulator of PCP signaling. Previously ubiquitination has been shown to be involved in canonical Wnt signaling as β-catenin is a target of ubiquitination (e.g., Miyazaki et al.,2004). Lee and coworkers (2007) found that in Xenopus the ras-related Xrab40 protein interacts with the hydrophobic Xcullin5 protein to form a ubiquitin ligase at the Golgi apparatus. Knocking down the activity of the components of this complex interferes with membrane localization of Dvl. These authors proposed that this affects the noncanonical pathway. More recently, Narimatsu and coworkers demonstrated a typical PCP phenotype including affected CE, neural tube closure defects and disorganization of the organ of Corti in mouse mutants for the genes encoding the Smurf1 and -2 ubiquitin ligases (Narimatsu et al.,2009). The phenotype could be linked with loss of the asymmetric subcellular localization of Pk1, which appears to be caused by this protein being targeted for degradation by complex containing in addition to Smurf, Par6, and Dvl proteins. Interestingly, Par6 had been previously linked to apicobasal polarity and asymmetric cell divisions, while Smurf was originally known as a modulator of transforming growth factor-beta (TGFβ) signaling.
Planar cell polarity signaling represents a fundamental mechanism that informs a cell in a multicellular organism on its position in a three-dimensional world. Given that all or most animals probably use a similar molecular pathway, it must be evolutionary very old.
Within the organism, PCP seems to participate in many different essential processes. While originally its biological function in epithelia was the main target of research, it becomes increasingly clear that it can have a role in other tissues as well.
Presumably many aspects of the functions of PCP signaling have not yet been uncovered due to early lethality of mutants involved, as well as to some extent, due to functional redundancy between genes. Not surprisingly, there is a strong tendency to re-evaluate PCP-linked phenotypes using conditional mutagenesis strategies, which will also make it more feasible to generate complex genotypes. Undoubtedly this will reveal in the next few years even more widespread roles for this pathway in particular at later stages, including processes in adult organisms and more connections with disease are to be expected. Much of the work on the mechanosensory cells of the organ of Corti, for instance, has been done in mouse mutants that die at stages that precede full development of these hair cells. Examples of PCP-associated defects that have not been explained beyond speculation include the cardiac defects. The hypothetical contribution of small laterality defects to cardiac outflow tract defects should also emerge from appropriate tissue specific knockouts. The role of PCP in tubular organs has only recently become evident and this subfield is expected to expand. It will be interesting to see the involvement of PCP in gut development and homeostasis. Another theme might be the importance of PCP for neural crest cell migration, for which there is evidence in the Xenopus model (De Calisto et al.,2005; Matthews et al.,2008; Shnitsar and Borchers,2008), but so far barely in mammals.
Nearly all of the knowledge about PCP derives from fundamental scientific research, but links with medically relevant issues have become increasingly important. In addition to the diseases linked to dysfunctional cilia, evidence has been found for a connection between epithelial polarity and carcinogenesis and possibly metastasis (Zhan et al.,2008; Cantrell and Jessen,2010). Longstanding speculations were confirmed that defective PCP might accompany the loss of cell polarity seen when healthy epithelial cells turn into carcinoma or carcinomas become metastatic. This might be seen as redeeming a promise, given the fact that the field started with observations on fly mutants that displayed tumorous growth (Schneiderman and Gateff,1967).
We thank Jacqueline Deschamps for reading an early version of the manuscript. Research of the authors was funded by a grant from the Netherlands government (Stem Cells in Development and Disease).