The frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/beta-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz(-) patches reorient toward the fz(-) cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Our in vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, we demonstrate biochemical and physical interactions between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). We show that this function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. Our data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field.     

Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.     

Kermit Interacts with Gαo,Vang, and Motor Proteins in Drosophila Planar Cell Polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue – the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.     

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.     

Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V‐ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co‐localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E‐Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V‐ATPase subunits. By contrast, the V‐ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.     

Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila

The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.     

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.     

Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.     

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs.     

Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis

Abstract

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40?years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.     

The Drosophila GIPC Homologue Can Modulate Myosin Based Processes and Planar Cell Polarity but Is Not Essential for Development

Epithelia often show, in addition to the ubiquitous apico-basal (A/B) axis, a polarization within the plane of the epithelium, perpendicular to the A/B axis. Such planar cell polarity (PCP) is for example evident in the regular arrangement of the stereocilia in the cochlea of the mammalian inner ear or in (almost) all Drosophila adult external structures. GIPCs (GAIP interacting protein, C terminus) were first identified in mammals and bind to the Gαi GTPase activating protein RGS-GAIP. They have been proposed to act in a G-protein coupled complex controlling vesicular trafficking. Although GIPCs have been found to bind to numerous proteins including Frizzled receptors, which participate in PCP establishment, there is little in vivo evidence for the functional role(s) of GIPCs. We show here that overexpressed Drosophila dGIPC alters PCP generation in the wing. We were however unable to find any binding between dGIPC and the Drosophila receptors Fz1 and Fz2. The effect of overexpressed dGIPC is likely due to an effect on the actin cytoskeleton via myosins, since it is almost entirely suppressed by removing a genomic copy of the Myosin VI/jaguar gene. Surprisingly, although dGIPC can interfere with PCP generation and myosin based processes, the complete loss-of-function of dGIPC gives viable adults with no PCP or other detectable defects arguing for a non-essential role of dGIPC in viability and normal Drosophila development.     

How do the Fat–Dachsous and core planar polarity pathways act together and independently to coordinate polarized cell behaviours?

Planar polarity describes the coordinated polarization of cells within the plane of a tissue. This is controlled by two main pathways in Drosophila: the Frizzled-dependent core planar polarity pathway and the Fat–Dachsous pathway. Components of both of these pathways become asymmetrically localized within cells in response to long-range upstream cues, and form intercellular complexes that link polarity between neighbouring cells. This review examines if and when the two pathways are coupled, focusing on the Drosophila wing, eye and abdomen. There is strong evidence that the pathways are molecularly coupled in tissues that express a specific isoform of the core protein Prickle, namely Spiny-legs. However, in other contexts, the linkages between the pathways are indirect. We discuss how the two pathways act together and independently to mediate a diverse range of effects on polarization of cell structures and behaviours.     

C. elegans Brat homologs regulate PAR protein-dependent polarity and asymmetric cell division

The evolutionary conserved PAR proteins control polarization and asymmetric division in many organisms. Recent work in Caenorhabditis elegans demonstrated that nos-3 and fbf-1/2 can suppress par-2(it5ts) lethality, suggesting that they participate in cell polarity by regulating the function of the anterior PAR-3/PAR-6/PKC-3 proteins. In Drosophila embryos, Nanos and Pumilio are homologous to NOS-3 and FBF-1/2 respectively and control cell polarity by forming a complex with the tumor suppressor Brat to inhibit Hunchback mRNA translation. In this study, we investigated the possibility that Brat could control cell polarity and asymmetric cell division in C. elegans. We found that disrupting four of the five C. elegans Brat homologs (Cebrats) individually results in suppression of par-2(it5ts) lethality, indicating that these genes are involved in embryonic polarity. Two of the Cebrats, ncl-1 and nhl-2, partially restore the localization of PAR proteins at the cortex. While mutations in the four Cebrat genes do not severely impair polarity, they display polarity-associated defects. Surprisingly, these defects are absent from nos-3 mutants. Similarly, while nos-3 controls PAR-6 protein levels, this is not the case for any of the Cebrats. Our results, together with results from Drosophila, indicate that Brat family members function in generating cellular asymmetries and suggest that, in contrast to Drosophila embryos, the C. elegans homologs of Brat and Nanos could participate in embryonic polarity via distinct mechanisms.     

Establishing cell polarity by the Lgl family proteins

The lethal giant larvae (lgl) gene was first identified more than 30 years ago in Drosophila and characterized as a tumor suppressor gene. Studies in budding yeast, flies and mammals all indicate that the evolutionarily conserved Lgl family proteins play an important role in cell polarity. Sro7/77, the yeast Lgl homologues, are important for the establishment and reinforcement of cell polarity through their localized interaction and kinetic activation of the post-Golgi secretion machinery. As for higher eukaryotes, both in epithelial polarity and asymmetric cell division, the role of Lgl protein is deployed by localizing proteins to the membrane in a polarized fashion. In addition, Lgl is transiently required during the establishment phase of polarity, implicating that Lgl functions at strategic time points for proliferation control. Studies in cancer biology provide direct connections between malfunction of Lgl and formation, progression and metastasis of various cancers. Here, we review recent advances in the field, focusing on the function of the Lgl family in cellular polarization.     

Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. We provide evidence that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. We show that the initial membrane association of Dgo depends on Fz, and that Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi.     

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.     

Planar polarization of Drosophila and vertebrate epithelia

Our understanding of the actin and microtubule rearrangements that generate planar polarity in Drosophila and in vertebrate epithelia has been extended by recent discoveries. Three different Rho family proteins have been shown to mediate polarization in the wing and the eye of Drosophila. In vertebrates, the importance of myosin Vlla has been uncovered by mutations that cause defects in planar polarization in the ear. Advances in our understanding of the Frizzled pathway, which coordinates planar polarization in Drosophila, are moving the field closer to understanding the links between signal transduction and polarized cytoskeletal reorganization.