REVIEW ARTICLE Diego and friends play again Old planar cell polarity players in new positions ´ ´ ´ Jozsef Mihaly, Tamas Matusek and Csilla Pataki Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary Keywords Diego; Drosophila; Four-jointed; inturned; tissue polarity Correspondence ´ J Mihaly, Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvari krt 62, Hungary Fax: +36 62 433503 Tel: +36 62 599687 E-mail: mihaly@brc.hu (Received 21 February 2005, accepted 27 April 2005) The formation of properly differentiated organs often requires the planar coordination of cell polarization within the tissues Such planar cell polarization (PCP) events are best studied in Drosophila, where many of the key players, known as PCP genes, have already been identified Genetic analysis of the PCP genes suggests that the establishment of polarity consists of three major steps The first step involves the generation of a global polarity cue; this in turn promotes the second step, the redistribution of the core PCP proteins, leading to the formation of asymmetrically localized signaling centers During the third step, these complexes control tissue-specific cellular responses through the activation of cell type specific effector genes Here we discuss some of the most recent advances that have provided valuable new insight into each of the three major steps of planar cell polarization doi:10.1111/j.1742-4658.2005.04758.x Functional tissues are comprised of polarized cell types Cellular polarization can be manifested in many different ways, depending on the orientation and axis of polarity Well known examples include the Drosophila ovary and embryo, where all major body axes are determined in a single cell; neuronal cells that typically exhibit axonal-dendritic polarity and epithelial cells that are characterized by apicalbasal polarity In many instances, however, tissue differentiation also requires the coordination of cell polarity within the plane of a tissue – a feature referred to as planar cell polarization (PCP) or tissue polarity for short Although PCP can be observed throughout the animal kingdom (vertebrate examples include fish scales, bird feathers and hairs in mammals, or the neurosensory epithelium in the inner ear), the regulation of such coordinated cell polarization events has been best studied in the fruitfly, Drosophila melanogaster PCP in flies is most evident in the wing, which is covered by uniformly polarized, distally pointing hairs, in the epidermis, where sensory bristles and trichomes point to the posterior, and in the eye, where PCP results in a mirror symmetry arrangement of the ommatidia or unit eyes Polarization in these tissues is controlled by the gene products of the PCP genes, mutants of which impair planar organization Some of the PCP genes, which have been placed into the core group, appear to affect polarity in all of the tissues, whereas others function in a tissue-specific way The core group includes the seven-pass transmembrane receptor frizzled (fz), the cytoplasmic signal transducer dishevelled (dsh), the cytoplasmic LIM domain protein prickle (pk), the atypical cadherin flamingo (fmi), the four-pass transmembrane protein strabismus (stbm) and the ankyrin repeat protein diego (dgo) [1–9] Genetic analysis of the PCP genes indicates that polarity establishment can be subdivided into three major steps Abbreviations MF, morphogenetic furrow; PCP, planar cell polarization FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS 3241 Planar cell polarity in Drosophila First, a long-range polarity signal is set up At present, the molecular nature of this signal is unclear, but it is believed that the atypical cadherins fat (ft) and dachsous (ds), the type II transmembrane protein four-jointed (fj) and the transcriptional repressor atrophin (atro) are all involved in the generation or the modulation of this long-range positional cue [10–20] In the second major step, the core PCP proteins redistribute and build up asymmetrically localized multiprotein complexes Finally, these apical membrane-associated signaling centers control the tissue-specific cellular responses through the activation of cell-type specific effectors While this remains a very general model that ignores important details, many such details, together with exciting new findings suggesting that the core mechanisms of PCP regulation are conserved from flies to human, have recently been summarized in excellent reviews [21–25] Thus, in this review we focus only on a selected set of unexpected recent findings including (a) the discovery of a new role for dgo in the apical recruitment and ⁄ or maintenance of PCP complexes [26]; (b) the demonstration that secretion is not required for Fj function, but instead it acts intracellularly in the Golgi apparatus [27]; and (c) the finding that the inturned (in) gene product is localized proximally in the wing cells [28], although it was previously considered to be a PCP effector directly regulating wing hair outgrowth at the distal vertex PCP in the Drosophila wing and eye In Drosophila, tissue polarity has been studied in a number of different body regions but it is been best understood in the wing and eye In the wing, each cell produces a single distally oriented hair at its distal vertex (Fig 1A) These structures are apical membrane outgrowths that are stiffened by actin and microtubule elements Mutations in PCP genes disrupt wing hair polarity in several different ways (Fig 1B) Some of them, such as fz, alter hair orientation and also in this case hairs often form in the center of the cell instead of the most distal part [29] Certain other mutations, however, such as in, multiple wing hair (mwh) and fuzzy (fy) not affect hair orientation, but result in the formation of multiple hairs from a single cell [29,30] Thus, PCP genes appear to regulate three major aspects of wing hair development: they restrict wing hair outgrowth to the distalmost part (distal vertex) of the cell, they control hair orientation and they determine the number of hairs produced 3242 ´ J Mihaly et al In contrast to the wing, where each individual cell normally becomes polarized, PCP in the eye is reflected in the arrangement of a group of cells corresponding to the unit eyes called ommatidia (Fig 1C) Each ommatidium consists of 20 cells including eight photoreceptor cells and 12 supporting cells Sectioning of the adult eye reveals that the ommatidia are chiral structures as photoreceptor cells aquire an asymmetrical trapezoidal shape within each of these multicellular units [31] Interestingly, the ommatidia in the dorsal half of the eye all adopt the same chirality, but this is opposite to that adopted in the ventral half resulting in a mirror symmetry arrangement (Fig 1D) The line where the dorsal and ventral ommatidia meet corresponds to the dorsal–ventral midline, often called the equator This spectacular planar organization is settled during imaginal disc development, a few hours after the photoreceptor preclusters emerge from the morphogenetic furrow (MF) of the eye-antennal disc The preclusters first become asymmetric and adopt dorsal or ventral chirality; subsequently they rotate accordingly, i.e 90° clockwise in the dorsal clusters and 90° counterclockwise in the ventral clusters (Fig 1C) It has recently become clear that the key to PCP generation in the eye is the step deciding the fate of the R3 ⁄ R4 photoreceptor cells It has been demonstrated that an fz PCP pathway dependent cell fate specification in the R3 ⁄ R4 pair is required for correct chirality choice and rotation of the whole ommatidial cluster [32,33] In agreement with this, PCP mutations can alter the chirality choice, resulting in chirality flips or symmetric ommatidia, and can also lead to various rotation defects (Fig 1D,E) Asymmetric localization of the core PCP proteins A major breakthrough towards an understanding of the molecular mechanisms controlling PCP came with the discovery that the core PCP proteins accumulate asymmetrically in cells [7,9,16,34–42] The first set of key observations established that, although the core PCP proteins in the wing cells are initially found in uniformly distributed complexes, a few hours before prehair formation they undergo relocalization and become differentially enriched along the proximal– distal axis, displaying a peculiar zigzag pattern Fz and Dsh become localized to distal cell membranes, whereas Stbm and Pk localize to the proximal side, while Fmi is found on both sides of the apical membrane (Fig 2A) Asymmetric PCP protein distribution can also be observed in the developing eye disc [16,37,38,40], although it is only in the precursor cells FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS ´ J Mihaly et al Planar cell polarity in Drosophila B A Proximal Distal C D Dorsal MF 7/8 Equator 7/8 Ventral E Normal R3/R3 R4/R4 Dorsal-Ventral inversion Misrotation Chirality flip Fig Planar polarity and PCP phenotypes in the wing and eye (A) The establishment of PCP in the wing begins with actin accumulation at the distal vertex (middle cartoon) that will subsequently lead to the formation of a distally pointing hair (shown in green) (B) The absence of PCP genes can affect hair formation in different ways Hairs are sometimes disoriented, and the site of hair outgrowth is often not restricted to the distal most part of the cell, or multiple hairs form in a single cell (mutant forms are indicated in red) (C) Ommatidial preclusters emerge from the morphogenetic furrow (MF) of the eye disc and initially form symmetric structures As eye development proceeds preclusters rotate 90° towards the equator, i.e dorsal clusters rotate clockwise, while ventral ones rotate counterclockwise At the end of this process the R3 ⁄ R4 cell pair acquires an asymmetric position within the cluster, and thus chirality also becomes established (R3 cells are highlighted in green, R4 cells in red) (D) The mirror symmetric structure of an adult eye can be disrupted by PCP mutations that can cause rotation defects, dorsal-ventral inversions, and loss of chirality resulting in symmetrical ommatidia with either R3 ⁄ R3 or R4 ⁄ R4 cell pairs (see enlarged on E) of the R3 ⁄ R4 photoreceptors where protein relocalization takes place leading to a transiently asymmetric localization Interestingly, Fz and Dsh become localized on the R3 side, Stbm and Pk on the R4 side, and Fmi on both sides of the R3 ⁄ R4 boundary (Fig 2B) Thus, the PCP protein distribution at the R3 ⁄ R4 boundary in the eye displays striking similarities to that of the distal ⁄ proximal cell border in the wing and hence the R3 ⁄ R4 cell boundary appears to be functionally equivalent to the distal ⁄ proximal cell boundary in the wing Consistent with the protein distribution in the eye, for fz and dsh there are genetic requirements in R3 [32], for stbm and pk in R4 [5,41], and for fmi in both R3 and R4 [37] It has been demonstrated that all the PCP proteins are required for the correct localization of each of the others, suggesting that these molecules might act FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS together in a multiprotein complex However, detailed phenotypic analysis in the wing and eye has revealed that the different proteins might play different roles in the process of PCP protein localization In the wing, this is suggested by the fact that, while some PCP mutations (e.g fmi) impair the apical localization of the other proteins, others (e.g dsh) merely affect the asymmetric enrichment of PCP proteins without disrupting their apical localization In the eye, different PCP mutations affect the localization of the other PCP proteins in markedly different ways: (a) the apical protein localization is compromised; (b) the asymmetric pattern is lost, but the apical localization remains unaffected; (c) asymmetric enrichment occurs, but in random orientation with respect to the equator (resulting in chirality flips) Together, these observations suggest that PCP protein localization can be divided into 3243 ´ J Mihaly et al Planar cell polarity in Drosophila A 24h APF Proximal Distal Proximal Apical Distal Apical Basal B Apical localization requires Diego 32h APF Basal Polar Fmi Dgo Fz Stbm Dsh Pk Row4 Row7 3 Equatorial Fig Core PCP protein localization in the developing wing and eye (A) During the initial phase of pupal wing development [up to  24 h after prepupa formation (APF)] the protein products of the core PCP genes are found in apically localized symmetric complexes (shown on the left) However, at  24 h APF they redistribute into asymmetric complexes that are present transiently until actin accumulation begins at  32 h APF Between 24 and 32 h APF Fz, Dsh and Dgo are enriched on distal cell membranes, Stbm and Pk accumulate on proximal membranes, while Fmi is found on both sides (right panel) (B) Although core PCP protein localization in the eye is somewhat more complicated than in the wing, it appears that PCP protein distribution across the R3 ⁄ R4 cell boundary is remarkably similar to that of the distal–proximal cell boundaries in the wing Notably, after the initial phases of ommatidia differentiation when PCP proteins not show polarized accumulation, five or six rows behind the morphogenetic furrow Fz, Dsh and Dgo begin to preferentially accumulate on the R3 side, whereas Stbm and Pk accumulate on the R4 side, and Fmi becomes enriched on both sides of the R3 ⁄ R4 interface Developing ommatidia are shown in five-cell precluster stages before and after asymmetric redistribution takes place, Row and Row 7, respectively Color code of the PCP proteins is identical in both (A) and (B) Numbers on (B) indicate the identity of the photoreceptor precursor cells two main phases: proteins first become localized to adherens junctions in the apicolateral membrane, and in the second stage they become asymmetrically distributed along the proximal–distal axis in the case of the wing, or on the R3 ⁄ R4 cell boundary in the eye Additionally, the asymmetric distribution in the eye must be coordinated with respect to the dorso–ventral midline 3244 What is the molecular mechanism that ensures the apical localization of the PCP protein complex, and how is membrane recruitment achieved for the predicted cytoplasmic proteins Dsh, Pk and Dgo? It was recently proposed by Bastock et al [42] and subsequently reviewed in detail by Strutt [24] that the PCP proteins might act in a hierarchy to generate asymmetrically localized apicolateral complexes (Fig 3A) This model postulates that Fmi acts at the top of the hierarchy and is responsible for recruiting the other transmembrane proteins, Fz and Stbm This is supported by the finding that in the absence of fmi negligible amounts of any other PCP, protein can be detected in the apicolateral region (Table 1) In the simplest case, Fmi would recruit Fz and Stbm by direct protein–protein interactions, although no direct binding partner has so far been found for Fmi Despite this discrepancy, it is now well established that Fmi, Fz and Stbm are certainly required for the membrane recruitment of the putative cytoplasmic proteins, Dsh, Pk and Dgo (Table 1) In accord with this, Fz has been shown to bind Dsh [43] and is able to recruit Dsh to membranes in heterologous assays [44] Furthermore, physical interactions have been reported between Stbm and Dsh, and between Stbm and Pk [41,42], suggesting a model in which at least Dsh and Pk become apicolaterally localized due to direct binding to Fz and Stbm Because in the absence of Dsh, Pk or Dgo, apicolateral recruitment of the other PCP proteins is not affected, but their asymmetric redistribution does not take place [9,42] (Table 1), it seemed reasonable to assume that, although Dsh, Pk and Dgo play negligible roles in apicolateral recruitment, they are required to promote the assembly and ⁄ or the maintenance of asymmetric PCP complexes An interesting recent paper [26] has now questioned this simple interpretation and presented new insight into the mechanisms regulating the initial apical localization and subsequent maintenance of PCP complexes The research in this paper is focused on dgo, the least well characterized core PCP gene Previous work has shown that Dgo is colocalized with Fz and Fmi during polarity establishment in the wing, and apical Dgo localization depends on these proteins [9] At that time, however, it was not possible to determine the precise subcellular localization of the dgo gene product Das et al have now reported that Dgo accumulates on the distal side of the wing cells Not surprisingly, in the eye Dgo becomes enriched on the R3 side of the R3 ⁄ R4 cell boundary, and it follows that, just like fz and dsh, dgo is genetically required in R3 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS ´ J Mihaly et al Planar cell polarity in Drosophila B A Stbm Fmi Fz Fmi Pk Dgo Dsh Stbm Fz Dsh Fmi Dgo Pk Fmi Stbm Pk Fz Dsh Dgo Fmi Stbm Pk Fz Dsh Fmi Dgo Fig Two possible models of apical PCP protein recruitment (A) One model, mainly based on data in the wing, proposed that Fmi lies on the top of the hierarchy of apical recruitment, and it is responsible for recruiting Fz and Stbm (top panel) Subsequently, these membrane proteins would recruit the putative cytoplasmic proteins, Dsh, Dgo and Pk (bottom panel) (B) The second model, based on eye data, suggests that Fz and Stbm would be the initial membrane recruiters of Dsh, Dgo and Pk (top panel), and these proteins would then be required to maintain Fmi apically (blue arrows, bottom panel) In turn, Fmi would promote the maintenance of the whole core PCP complex at adjacent cell membranes Black arrows represent the genetic requirements for apical recruitment, grey ovals indicate the nuclei (A) and (B) are modified figures after Bastock et al [42], and Das et al [26], respectively The absence of dgo does not affect the apical localization or the asymmetric enrichment of PCP proteins in the eye, but the asymmetric accumulation is apparently randomized with respect to the equator In contrast, the apical localization of Dgo is completely abolished in an fz mutant tissue, and strongly reduced in fmi clones, while the absence of stbm or pk although induces a short delay in Dgo localization, the overall pattern remains largely normal, albeit randomized Strikingly, however, in dgo, pk or dgo, stbm double mutant clones, the apical localization of both Fz and Fmi is strongly reduced Additionally, in the dgo, pk combination, Stbm and Dsh also fail to form apically localized complexes, although this might not reflect a direct requirement as Fmi localization is compromised as well The situation with pk, stbm double mutants is more complex because the apical localization of Fmi and Fz in the eye is lost anterior to the MF, whereas the apical localization is hardly affected posterior to the furrow even if the asymmetric distribution is perturbed [26] In contrast to the eye, Fmi and Fz localization is severely reduced in pk, stbm double mutant wing cells [42] Finally, despite the fact that single mutants of pk and stbm not significantly affect apical Dgo localization in the eye, in pk, stbm double mutant clones Dgo is much reduced at the apical cortex Significantly, it has also been revealed by yeast two-hybrid and GST pull-down assays that Dgo interacts physically with Pk and Stbm FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS These results suggest that, opposite to what might be expected from single mutant analysis, Pk and Dgo are also required for membrane localization of the PCP factors, though this is a redundant requirement between Dgo, Pk and Stbm These data led Das et al [26] to outline a model to explain how PCP complexes might be formed and maintained during the early phases of PCP establishment (Fig 3B) They propose that the cytoplasmic PCP proteins (Dsh, Dgo and Pk) initially recruited to the membrane by Fz and Stbm form a protein complex that is required to maintain Fmi apically In turn, apical Fmi promotes the maintenance of the PCP complex at adjacent cell membranes and can also facilitate their signaling specific interactions This model is consistent with the available data and also supported by the protein–protein interaction results However, in so far as Fmi is concerned, Das et al came to just the opposite conclusion to that of Bastock et al who suggested that Fmi lies at the top of the hierarchy of apical PCP protein recruitment [42] In that view, Fmi is required for the initial membrane recruitment of Fz and Stbm (Fig 3A) While these opposing views might simply reflect tissue-specific differences between the eye and the wing, an fz, stbm double mutant analysis could be informative in respect of the order of initial membrane recruitment If Fmi is at the top, in such fz, stbm double mutants Fmi localization should not be significantly affected, whereas if Fz and Stbm were the initial recruiters (as suggested 3245 ´ J Mihaly et al Planar cell polarity in Drosophila Table Core PCP protein localization in the wing and eye in PCP mutant backgrounds This table summarizes the relevant aspects of protein localization in the wing and the eye [7,9,26,34–42] Api., apical localization; Asy., asymmetric redistribution; D, normal but delayed localization; ND, not determined The ratio between filled and empty circles indicates the amount of properly localized proteins compared with wild-type level: ddd, wild type level; sss, complete loss of localization a Reduced Pk level at the apical membrane, but increased level in the cytoplasm [40–42] tion behind the MF, but loss of apical localization anterior to the MF [26] by Das et al.), apical Fmi should be lost in the fz, stbm double mutant clone In fact, in the wing, unlike the situation in the eye, stbm moderately reduces the level of apically localized Fmi and fz also has a weak effect, which could be used as an argument in favor of Fmi localization being dependent on Fz and Stbm Such simple assumptions, however, must be treated with caution because one of the limitations of the genetic approaches used during these experiments is that they not clearly distinguish between initial apical recruitment and maintenance At present therefore it is not possible to distinguish between the two alternatives that have been put forward to explain the apical recruitment and maintenance of PCP complexes Moreover, as we know very little about the molecular composition of the PCP complexes formed in vivo and 3246 b Data not shown in [38] c Normal apical localiza- the feedback mechanisms that might help to stabilize them, it is clear that other models are also possible Nevertheless, the employment of double mutant analysis has proved to be a very useful tool to discover new aspects of PCP establishment in the Drosophila wing and eye It seems likely that the examination of further double mutant combinations will promote a deeper understanding of this process It would be interesting to examine double mutant combinations between the fz, dsh and the pk, stbm, dgo groups as single mutants of these in most cases have either no effect or only a weak effect on apical localization Finally, it would also be of interest to compare the results of double mutant analyses in the wing and eye as this could yield further hints concerning the tissue specific differences already revealed by single mutant analysis FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS ´ J Mihaly et al Long-range patterning and the Golgiassociated protein, Four-jointed A few hours after PCP proteins have been recruited to the apicolateral regions, they become asymmetrically distributed How does this happen? Although the answer to this question is largely unclear, it is believed that redistribution occurs in response to a directional signal that coordinates polarity with the axis of the tissue It is also generally thought that the polarity signal induces a bias in Fz activity along the proximal ⁄ distal axis of the wing and the equatorial ⁄ polar axis of the eye [16,18,36] Subsequently, the initially created subtle difference in Fz activity on the opposite sides of the cells would be amplified by intercellular feedback mechanisms, leading to high Fz signaling on the distal (i.e in the wing) and equatorial (i.e in the eye) sides, and to low level signaling on the opposite sides [39] While this is an attractive model, there are several important points that need to be verified It is not yet clear what the link is between differential Fz activation and asymmetric redistribution What is the cause and what is the consequence here, if there is a direct casual relationship at all? Another important problem is that the source and nature of the polarity signal remain elusive, including the important question of whether it is a long-range or a short-acting signal Models based on the former possibility propose that polarity is established as a result of interpreting the concentration of a long-range signal (most probably a secreted factor) present in a concentration gradient across the tissue Alternatively, a locally acting short-range signal could be used to polarize one cell, which would in turn generate a signal to polarize its neighbors via a signal relay mechanism Finally, we note that there are profound tissue-specific differences between the wing, eye and abdomen, and thus the in vivo mechanism could change from tissue to tissue, including a combination of the long- and short-range models Despite the fact that the molecular nature of the mysterious polarity cue (often called factor X) is not known, several genes have recently been implicated in long-range signaling acting upstream of asymmetric PCP protein redistribution A great body of work on the developing wing, eye and abdomen has led to a model in which the activity gradients of the atypical cadherins ft and ds and the type II transmembrane protein Fj generate or modulate the activity of a longrange polarity signal [11–20] A feature almost certainly relevant to this issue is that the same Ft ⁄ Ds ⁄ Fj module is involved in the proximal-distal patterning of the wing and leg [12,45–48], and hence it is tempting to speculate that planar polarity establishment (at least FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS Planar cell polarity in Drosophila in the wing) is directly coupled to growth and patterning of the tissue (see also [23]) Direct evidence in support of this view is missing, however, indicating that further work will be required to clarify the link between PCP and tissue patterning Whether the activity of the Ft ⁄ Ds ⁄ Fj module will ultimately lead to the secretion of an Fz ligand or coordinate PCP in a different way is also an open question It appears that to resolve these problems we need a better understanding of the signaling events between these proteins and, potentially, other pathways and proteins; and there is a need to learn more about the molecular biology and biochemical properties of these proteins Some pioneering experiments have already provided evidence that ft acts through the transcriptional corepressor Atro [17], support for the in vivo existence of a Ft–Ds heterophilic interaction [19], and last but not least, revealed that Fj is a Golgi-associated protein [27] Former studies demonstrated that fj is expressed in a gradient in the developing wing and eye [11,12], and that clones of cells which either lack or ectopically express Fj cause both autonomous and nonautonomous PCP defects in these tissues [11,12] Additionally, an in vitro analysis has indicated that the extracellular C-term of the Fj protein can be cleaved, resulting in a secreted form [47] Together, these results strongly suggested that Fj functions as a secreted signaling molecule present in a gradient on the proximo–distal axis of the wing and the dorso-ventral axis of the eye Strutt et al have now tested this idea directly by comparing the signaling abilities of modified Fj forms that are either poorly cleaved, constitutively secreted or anchored to the Golgi [27] During this elegant set of experiments, they used overexpression and rescue assays to test the in vivo activities of the different Fj forms and concluded that secreted Fj is not the active form, but, unexpectedly, Fj acts intracellularly This is consistent with their antibody staining result that most Fj is localized to discrete spots inside the cells, the majority of which correspond to the Golgi apparatus Significantly, they also demonstrated that, although the Golgi-tethered Fj is not secreted, when overexpressed it is still able to produce nonautonomous polarity phenotypes To explain this finding, they propose that Fj most likely acts by modulating the activity of other proteins involved in intercellular signaling The best candidates as targets of Fj are the atypical cadherins Ds and Ft, which have been shown to act downstream of fj [16] The analysis of fj mutant clones revealed that Fj is clearly involved in the control of cell adhesion, and it regulates the intracellular distribution of Ds and Ft, which are likely to bind to each other in vivo [15,18,19] This set of results has led to 3247 Planar cell polarity in Drosophila the proposal that Fj may regulate cell adhesion by modulating Ds ⁄ Ft heterophilic interactions [15,18,19] Although the molecular mechanism of this modulation remains to be discovered, it seems conceivable that Fj may have an enzymatic activity that is involved in the post-translational modification of Ft and ⁄ or Ds (or an unidentified protein), in much the same way as Fringe (Fng) regulates Notch (N) activity [49,50] The idea that Fj mediates the post-translational modification of Ft and ⁄ or Ds has interesting implications as regards the potential redundancy at the level of Fj function As ft and ds exhibit strong planar polarity phenotypes when homozygous [10,14,16], while fj displays polarity phenotypes almost only on the boundaries of mutant clones [12], the weak fj phenotype was explained by redundancy However, the origin and nature of this redundancy remain uncertain, largely because of the lack of information on the molecular function of fj In the light of recent data, it may be speculated that, while Fj modulates Ft ⁄ Ds activity, for example, by adding certain type of post-translational modifications on it, full Ft ⁄ Ds activity would require additional upstream inputs (e.g additional types of post-translational modifications) Thus, Fj activity would contribute to the activation of Ft ⁄ Ds, but alone it would not be sufficient to create a fully functional form As a consequence, the absence of Fj would be compensated by additional, an as yet unidentified factors also involved in Ft ⁄ Ds modulation Although the precise mechanisms by which these elements contribute to the formation of global polarity cues have not been fully clarified, experiments of this type offer promising new developments in the PCP field, and underline the importance of biochemical approaches in elucidating further details of PCP establishment Inturned: a new turn in the game While much attention has recently been paid to the asymmetric localization of the PCP proteins and to the mechanisms of action of the potential upstream elements, novel studies on inturned (in), a planar polarity effector gene, have led to the discovery of a new level of complexity among the downstream PCP elements as well Earlier experiments revealed that, in the absence of in or fy, wing cells fail to restrict hair outgrowth to the distal vertex; instead, they form multiple hairs at ectopic locations [29] It has also been shown that the in and fy loss-of-function mutations are epistatic to fz, dsh and pk, suggesting that they function downstream of the core PCP genes [29] A simple interpretation of these results is that in and 3248 ´ J Mihaly et al fy act as inhibitors of hair formation, while one possible function of fz would be to inhibit in and fy locally to allow prehair initiation at the distal vertex [29] Although this hypothesis is consistent with the observations that the asymmetric accumulation of Fz and Dsh is not altered in fy or in mutants [34–36], the molecular details of the potential inhibitory mechanisms were entirely missing It has now been reported that the In protein becomes preferentially accumulated at the proximal edge of the pupal wing cells under the instruction of the core PCP genes [28] This pattern which is apparent several hours before prehair initiation, closely resembles the zigzag pattern typical for core PCP proteins; indeed, the In staining largely overlaps with that of Fz At present, it is not clear how In is recruited to the proximal side of the wing cells One tempting possibility is that In is recruited by Pk or Stbm as these proteins also accumulate there [39,42]; however, it was not possible to detect a direct interaction between In and Pk or Stbm with the yeast two hybrid system [28] Although such an interaction is still possible, another alternative would be that Fuzzy (Fy) or Fritz (Frtz) or both are involved in In recruitment and localization This would be consistent with several different findings First, asymmetric In accumulation and In protein stability have both been shown to depend on fy and frtz [28] Second, although it has not been studied in detail, fy appears to encode a transmembrane protein that could recruit In directly [51] Third, single or double mutants of fy, frtz and in display almost identical wing hair phenotypes [29,52], suggesting that these genes function together in PCP and might interact with each other directly As we know very little at the molecular level about Fy and Frtz, and their subcellular localization has not been described yet, In recruitment remains an open issue We note, however, that the combination of the above alternatives is also a valid possibility In that scenario, Stbm and ⁄ or Pk would be the key to the initial proximal recruitment of In or Fy or Frtz, which in turn would promote the assembly of a functional In ⁄ Fy ⁄ Frtz complex Previous observations have suggested that the spatial restriction of cytoskeleton activation and prehair formation to the distal vertex of the wing cells largely or entirely depends on the local (distal) accumulation and activation of Fz and Dsh, whereas the proximal accumulation of other PCP proteins has been only suggested to play a role in the establishment of proximal and distal cortical domains It has now been found that local Fz ⁄ Dsh signaling alone is not sufficient to restrict prehair formation to the distal vertex: surprisingly, this signaling has to be coordinated with downstream reguFEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS ´ J Mihaly et al latory events that depend on the proximally localized factor, Inturned [28] How can this proximally recruited protein ensure that hairs form at the distal edge? Adler et al considered three alternatives: proximal In might stimulate hair formation at the distal edge of the neighboring cells; alternatively, proximal In might organize the polarized intracellular transport of cellular components that play a role in hair morphogenesis; and finally, the old idea that proximal In might function as an inhibitor of hair initiation The available data not permit a clear distinction between these alternatives, though the first appears very unlikely as it predicts a strong nonautonomous effect, which has not been seen in in mutants It is difficult to raise formal arguments against the second alternative, but the third seems to be the most appealing to us Following the finding that In is proximally localized in wing cells, the idea that In functions as an inhibitor, offers a refined view of the proximal and distal cortical domains According to this hypothesis, once the core PCP proteins have redistributed asymmetrically, the proximal and distal cortical domains become established Subsequently, Fz ⁄ Dsh promotes hair formation at the distalmost part of the cell, but only there, while at the same time Stbm ⁄ Pk inhibits hair formation in the proximal part, acting through an In complex Thus, this model predicts that the restriction of hair outgrowth exclusively to the distal vertex of the cell requires that the positively acting distal Fz ⁄ Dsh PCP signal is paralleled by an In inhibitory signal on the opposite side of the cell that might form an intracellular gradient with its high end at the proximal pole Interestingly, this hypothesis may help to explain a previously described set of results that were difficult to reconcile with a simple linear regulatory relationship between the core elements and the in-like genes Notably, Lee and Adler reported that a weak multiple wing hair phenotype induced by hypomorphic in or fy alleles is strongly enhanced both by the removal and by the overexpression of fz, fmi or dsh [52] If we consider that, in the absence of fz, the site of actin accumulation is already somewhat ‘delocalized’, leading to a weak multiple hair phenotype, the inhibitory model predicts that the introduction of a hypomorphic in allele into this background will enhance the multiple wing hair phenotype, because the proximally acting inhibitor of cytoskeleton activation is also impaired This prediction fits perfectly with the published data If we now consider the case of Fz overexpression, we find that the excess of Fz induces a failure in asymmetric PCP protein redistribution, leading to an imperfect restriction of actin accumulation even in the presence of In (which itself fails to undergo proper localization in this situation) If Fz overexpresFEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS Planar cell polarity in Drosophila sion is accompanied by a parallel impairment of the inhibitor of hair formation (In), the original multiple wing hair phenotype is again expected to be enhanced, as is indeed the case At present this model remains very speculative, but it is noteworthy that, if such opposite, but complementary In and Fz ⁄ Dsh effects exist, this would represent a very powerful mechanism whereby hair initiation is restricted exclusively to the distal vertex, and it could contribute substantially to the remarkable precision with which the site of actin accumulation and, ultimately, wing hair number and orientation are determined Nevertheless, further experiments will clearly be required to resolve the intriguing questions of how the In ⁄ Fy ⁄ Frtz module is linked to the core PCP complexes and what the in vivo function of In is Perspectives The earlier discovery that PCP proteins build up asymmetrically localized complexes was considered a major breakthrough in the field Much effort has subsequently been devoted to elucidating how such complexes are formed and to clarifying the protein–protein interactions between the components However, despite the great progress regarding certain details, the overall picture remains unclear The mechanistic details on protein localization are largely missing, the link between Fz signaling and asymmetric localization is not understood, and it remains a mystery how asymmetric localization is coupled to upstream elements, such as the atypical cadherins It appears likely that the extension of double mutant analysis will furnish additional valuable insights, however, it is equally clear that genetic and cell biological methods must be combined with biochemistry in order to reach the heart of these problems A detailed biochemical characterization of the core group may allow a functional dissection of the process Moreover, we need a better understanding of the protein–protein interactions between the core elements, we need to describe the complexes formed in vivo, and we need to learn about their spatial and temporal regulation during PCP establishment A second area of PCP research that has recently received considerable attention is the generation of long-range polarity cues While much has been learnt, the mysterious story of factor X continues, as the exact nature, source and mode of action of the polarity signal are still unknown Interestingly, however, important new results (not discussed in this minireview) have led to the identification of further upstream elements, one of which (widerborst) is asymmetrically localized 3249 Planar cell polarity in Drosophila long before the redistribution of the core elements [53], while the other (encoded by the grainy head transcription factor) is indirectly required for the apical localization of the core PCP proteins via the regulation of fmi transcription [54] These findings tend to lead us to question the view that asymmetric core PCP protein localization is the key to proximodistal polarization of the wing cells It rather appears that proximodistal polarity is established much before these players act Quite conceivably, Lawrence et al have proposed that cells are perhaps polarized throughout all or most of their development [55], and hence the core PCP proteins should already be regarded as effector elements that begin to execute the final steps of tissue differentiation, resulting in an overt manifestation of planar organization In the course of this process, they appear to organize downstream acting asymmetric complexes, as revealed by the analysis of In Research in this line is very likely to give rise to changes in the prevailing models on the downstream effectors Overall, the PCP field continues to provide ample opportunities for hard work to generate a new wave of exciting results, bright ideas and fun for developmental biologists Acknowledgements ´ We are grateful to Henrik Gyurkovics, Peter Vilmos, ´ ´ Laszlo Sipos, Gishnu Das, Marek Mlodzik, David Durham and one of the anonymous reviewers for their helpful comments and critical reading of the manuscript J M is an EMBO ⁄ HHMI Scientist and a Bol´ yai Janos Research Scholar, and 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polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein Development 124, 4029–4037 52 Lee H & Adler PN (2002) The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy Genetics 160, 1535–1547 3252 ´ J Mihaly et al 53 Hannus M, Feiguin F, Heisenberg CP & Eaton S (2002) Planar cell polarization requires Widerborst, a B’ regulatory subunit of protein phosphatase 2A Development 129, 3493–3503 54 Lee H & Adler PN (2004) The grainy head transcription factor is essential for the function of the frizzled pathway in the Drosophila wing Mech Dev 121, 37– 49 55 Lawrence PA, Casal J & Struhl G (2004) Cell interactions and planar polarity in the abdominal epidermis of Drosophila Development 131, 4651–4664 FEBS Journal 272 (2005) 3241–3252 ª 2005 FEBS ... cell boundary in the wing Consistent with the protein distribution in the eye, for fz and dsh there are genetic requirements in R3 [32], for stbm and pk in R4 [5,41], and for fmi in both R3 and. .. for Fj function, but instead it acts intracellularly in the Golgi apparatus [27]; and (c) the finding that the inturned (in) gene product is localized proximally in the wing cells [28], although... FEBS Planar cell polarity in Drosophila in the wing) is directly coupled to growth and patterning of the tissue (see also [23]) Direct evidence in support of this view is missing, however, indicating