Planar cell polarity and tissue design: Shaping the Drosophila wing membrane

Planar cell polarity (PCP) describes the orientation of a cell within the plane of an epithelial cell layer. During tissue development, epithelial cells normally align their PCP so that they face in the same direction. This alignment allows cells to move in a common direction, or to generate structures with a common orientation. A classic system for studying the coordination of epithelial PCP is the developing Drosophila wing. The alignment of epithelial PCP during pupal wing development allows the production of an array of cell hairs that point towards the wing tip. Multiple studies have established that the Frizzled (Fz) PCP signaling pathway coordinates wing PCP. Recently, we have found that the same pathway also controls the formation of ridges on the Drosophila wing membrane. However, in contrast to hair polarity, ridge orientation differs between the anterior and posterior wing. How can the Fz PCP pathway generate a different relationship between hair and ridge orientation in different parts of the wing? In this Extra View article, we discuss membrane ridge development drawing upon our recent PLoS Genetics paper and other, published and unpublished, data. We also speculate upon how our findings impact the ongoing debate concerning the interaction of the Fz PCP and Fat/Dachsous pathways in the control of PCP.     

The Frizzled Planar Cell Polarity signaling pathway controls Drosophila wing topography

The Drosophila wing is a primary model system for studying the genetic control of epithelial Planar Cell Polarity (PCP). Each wing epithelial cell produces a distally pointing hair under the control of the Frizzled (Fz) PCP signaling pathway. Here, we show that Fz PCP signaling also controls the formation and orientation of ridges on the adult wing membrane. Ridge formation requires hexagonal cell packing, consistent with published data showing that Fz PCP signaling promotes hexagonal packing in developing wing epithelia. In contrast to hair polarity, ridge orientation differs across the wing and is primarily anteroposterior (A-P) in the anterior and proximodistal (P-D) in the posterior. We present evidence that A-P ridge specification is genetically distinct from P-D ridge organization and occurs later in wing development. We propose a two-phase model for PCP specification in the wing. P-D ridges are specified in an Early PCP Phase and both A-P ridges and distally pointing hairs in a Late PCP Phase. Our data suggest that isoforms of the Fz PCP pathway protein Prickle are differentially required for the two PCP Phases, with the Spiny-legs isoform primarily active in the Early PCP Phase and the Prickle isoform in the Late PCP Phase.     

Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway

The regular array of distally pointing hairs on the mature Drosophila wing is evidence for the fine control of Planar Cell Polarity (PCP) during wing development. Normal wing PCP requires both the Frizzled (Fz) PCP pathway and the Fat/Dachsous (Ft/Ds) pathway, although the functional relationship between these pathways remains under debate. There is strong evidence that the Fz PCP pathway signals twice during wing development, and we have previously presented a Bidirectional-Biphasic Fz PCP signaling model which proposes that the Early and Late Fz PCP signals are in different directions and employ different isoforms of the Prickle protein. The goal of this study was to investigate the role of the Ft/Ds pathway in the context of our Fz PCP signaling model. Our results allow us to draw the following conclusions: (1) The Early Fz PCP signals are in opposing directions in the anterior and posterior wing and converge precisely at the site of the L3 wing vein. (2) Increased or decreased expression of Ft/Ds pathway genes can alter the direction of the Early Fz PCP signal without affecting the Late Fz PCP signal. (3) Lowfat, a Ft/Ds pathway regulator, is required for the normal orientation of the Early Fz PCP signal but not the Late Fz PCP signal. (4) At the time of the Early Fz PCP signal there are symmetric gradients of dachsous (ds) expression centered on the L3 wing vein, suggesting Ds activity gradients may orient the Fz signal. (5) Localized knockdown or over-expression of Ft/Ds pathway genes shows that boundaries/gradients of Ft/Ds pathway gene expression can redirect the Early Fz PCP signal specifically. (6) Altering the timing of ds knockdown during wing development can separate the role of the Ft/Ds pathway in wing morphogenesis from its role in Early Fz PCP signaling.     

Revisiting planar cell polarity in the inner ear

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

Twinstar, the Drosophila homolog of cofilin/ADF, is required for planar cell polarity patterning

Planar cell polarity (PCP) is a level of tissue organization in which cells adopt a uniform orientation within the plane of an epithelium. The process of tissue polarization is likely to be initiated by an extracellular gradient. Thus, determining how cells decode and convert this graded information into subcellular asymmetries is key to determining how cells direct the reorganization of the cytoskeleton to produce uniformly oriented structures. Twinstar (Tsr), the Drosophila homolog of Cofilin/ADF (actin depolymerization factor), is a component of the cytoskeleton that regulates actin dynamics. We show here that various alleles of tsr produce PCP defects in the wing, eye and several other epithelia. In wings mutant for tsr, Frizzled (Fz) and Flamingo (Fmi) proteins do not properly localize to the proximodistal boundaries of cells. The correct asymmetric localization of these proteins instructs the actin cytoskeleton to produce one actin-rich wing hair at the distal-most vertex of each cell. These results argue that actin remodeling is not only required in the manufacture of wing hairs, but also in the PCP read-out that directs where a wing hair will be secreted.     

Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility

Signalling through Frizzled (Fz)/planar cell polarity (PCP) is a conserved mechanism that polarizes cells along specific axes in a tissue. Genetic screens in Drosophila melanogaster pioneered the discovery of core PCP factors, which regulate the orientation of hairs on wings and facets in eyes. Recent genetic evidence shows that the Fz/PCP pathway is conserved in vertebrates and is crucial for disparate processes as gastrulation and sensory cell orientation. Fz/PCP signalling depends on complex interactions between core components, leading to their asymmetric distribution and ultimately polarized activity in a cell. Whereas several mechanistic aspects of PCP have been uncovered, the global coordination of this polarization remains debated.     

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

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

Planar cell polarization requires Widerborst,a B' regulatory subunit of protein phosphatase 2A

We have identified widerborst (wdb), a B' regulatory subunit of PP2A, as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

Multiple Roles of a Trimeric G Protein in Drosophila Cell Polarization

Polarization of the cellular cytoskeleton underlies many cellular processes including axon growth cone guidance, chemotaxis and yeast mating. Planar cell polarity (PCP) is a similar phenomenon in which cells in an epithelium become uniformly polarized to generate a field of aligned structures such as the hair cells of the cochlea. In Drosophila PCP is under the hierarchical control of Frizzled (Fz) - a serpentine receptor (that also functions in the Wnt signaling pathway). Serpentine receptors are routinely transduced by trimeric G-proteins, but until recently the general consensus was that Fzs were not G-protein linked. In Drosphila a G-protein (Gαo ) has now been identified that functions in both the Wnt and PCP pathways. Here we review the cell polarity phenotypes of Gαo mutants and discuss the evidence that it plays multifarious roles in PCP and the organization of the cytoskeleton.     

Cell flow and tissue polarity patterns

Planar tissue polarity is a fundamental feature of many epithelia. Large-scale cell polarity patterns govern the orientation of external structures such as hairs and cilia. Tissue polarity patterns arise from the collective organization of cells, which are polarized individually. Such cell and tissue polarities are reflected in anisotropic distributions of proteins of the planar cell polarity (PCP) pathway. Here we give an overview on recent progress in understanding how large-scale patterns of tissue polarity are controlled. We highlight the role of active mechanical events in the organization of polarity patterns during the development of the pupal fly wing. Patterns of cell flow are generated by mechanical stresses exerted on the tissue as well as by oriented cell divisions and neighbor exchanges. We discuss how the resulting tissue shear controls polarity orientation. We argue that the often-observed alignment of PCP either parallel or perpendicular to the long axis of developing tissues is a characteristic consequence of shear-induced polarity alignment. This principle allows for the versatile and robust generation of polarity patterns in tissues.     

Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair [5]. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.     

Novel regulators of planar cell polarity: a genetic analysis in Drosophila

Planar cell polarity (PCP) is a common feature of many epithelia and epithelial organs. Although progress has been made in the dissection of molecular mechanisms regulating PCP, many questions remain. Here we describe a screen to identify novel PCP regulators in Drosophila. We employed mild gain-of-function (GOF) phenotypes of two cytoplasmic Frizzled (Fz)/PCP core components, Diego (Dgo) and Prickle (Pk), and screened these against the DrosDel genome-wide deficiency collection for dominant modifiers. Positive genomic regions were rescreened and narrowed down with smaller overlapping deficiencies from the Exelixis collection and RNAi-mediated knockdown applied to individual genes. This approach isolated new regulators of PCP, which were confirmed with loss-of-function analyses displaying PCP defects in the eye and/or wing. Furthermore, knockdown of a subset was also sensitive to dgo dosage or dominantly modified a dishevelled (dsh) GOF phenotype, supporting a role in Fz/PCP-mediated polarity establishment. Among the new "PCP" genes we identified several kinases, enzymes required for lipid modification, scaffolding proteins, and genes involved in substrate modification and/or degradation. Interestingly, one of them is a member of the Meckel-Gruber syndrome factors, associated with human ciliopathies, suggesting an important role for cell polarity in nonciliated cells.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.     

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. Here, we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. We propose that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems.     

Planar cell polarity signaling in craniofacial development

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.     

Planar cell polarity signaling in craniofacial development

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.     

The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.     

FijiWingsPolarity: An open source toolkit for semi-automated detection of cell polarity

Epithelial cells are defined by apical-basal and planar cell polarity (PCP) signaling, the latter of which establishes an orthogonal plane of polarity in the epithelial sheet. PCP signaling is required for normal cell migration, differentiation, stem cell generation and tissue repair, and defects in PCP have been associated with developmental abnormalities, neuropathologies and cancers. While the molecular mechanism of PCP is incompletely understood, the deepest insights have come from Drosophila, where PCP is manifest in hairs and bristles across the adult cuticle and organization of the ommatidia in the eye. Fly wing cells are marked by actin-rich trichome structures produced at the distal edge of each cell in the developing wing epithelium and in a mature wing the trichomes orient collectively in the distal direction. Genetic screens have identified key PCP signaling pathway components that disrupt trichome orientation, which has been measured manually in a tedious and error prone process. Here we describe a set of image processing and pattern-recognition macros that can quantify trichome arrangements in micrographs and mark these directly by color, arrow or colored arrow to indicate trichome location, length and orientation. Nearest neighbor calculations are made to exploit local differences in orientation to better and more reliably detect and highlight local defects in trichome polarity. We demonstrate the use of these tools on trichomes in adult wing preps and on actin-rich developing trichomes in pupal wing epithelia stained with phalloidin. FijiWingsPolarity is freely available and will be of interest to a broad community of fly geneticists studying the effect of gene function on PCP.