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.     

Planar polarity in the Drosophila eye: a multifaceted view of signaling specificity and cross-talk

Functional tissues not only polarize their epithelia in the apical-basolateral axis, but also often display a polarity within the plane of the epithelium. In Drosophila, all adult structures are derived from epithelia called imaginal discs and display planar polarization; the eye and the wing are particularly well suited for analysis. Studies of their polarization have identified several conserved genes that regulate both nuclear signaling and cytoskeletal architecture. In particular, the Frizzled (Fz) receptor has been identified as a key component of polarity establishment in all tissues. The Fz signaling pathway and associated events are beginning to be unraveled, shedding light on a novel Wnt/Fz signaling cascade.     

Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless

The Frizzled (Fz) protein in Drosophila is a bifunctional receptor that acts through a GTPase pathway in planar polarity signaling and as a receptor for Wingless (Wg) using the canonical Wnt pathway. We found that the ligand-binding domain (CRD) of Fz has an approximately 10-fold lower affinity for Wg than the CRD of DFz2, a Wg receptor without polarity activity. When the Fz CRD is replaced by the high-affinity CRD of DFz2, the resulting chimeric protein gains Wg signaling activity, yet also retains polarity signaling activity. In contrast, the reciprocal exchange of the Fz CRD onto DFz2 is not sufficient to confer polarity activity to DFz2. This suggests that Fz has an intrinsic capacity for polarity signaling and that high-affinity interaction with Wg couples it to the Wnt pathway.     

The atypical cadherin Flamingo links Frizzled and Notch signaling in planar polarity establishment in the Drosophila eye

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. We show that the atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. Our results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing.     

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.     

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.     

DWnt4 and wingless elicit similar cellular responses during imaginal development

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. We previously reported that DWnt4 and wingless (wg), two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Here we provide evidence that both genes can elicit similar cellular responses during imaginal development. Ectopic expression of DWnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, DWnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, we demonstrate that DWnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. We also show that both genes are transcribed in overlapping domains in imaginal discs, suggesting that DWnt4 may cooperate with wg during limb patterning.     

EGF signaling and ommatidial rotation in the Drosophila eye

The ommatidia of the Drosophila eye initiate development by stepwise recruitment of photoreceptors into symmetric ommatidial clusters. As they mature, the clusters become asymmetric, adopting opposite chirality on either side of the dorsoventral midline and rotating exactly 90 degrees (Figures 1A and 1B, ). The choice of chirality is governed by higher activity of the frizzled (fz) gene in one cell of the R3/R4 photoreceptor pair and by Notch-Delta (N-Dl) signaling. The 90 degrees rotation also requires activity of planar polarity genes such as fz as well as the roulette (rlt) locus. We now show that two regulators of EGF signaling, argos and sprouty (sty), and a gain-of-function Ras85D allele, interact genetically with fz in ommatidial polarity. Furthermore, we find that argos is required for ommatidial rotation, but not chirality, and that rlt is a novel allele of argos. We present evidence that there are two pathways by which EGF signaling affects ommatidial rotation. In the first, typified by the rlt phenotype, there is partial transformation of the "mystery cells" toward a neuronal fate. Although most of these mystery cells subsequently fail to develop as neurons, their partial transformation results in inappropriate subcellular localization of the Fz receptor, a likely cue for regulating ommatidial rotation. Secondly, reducing EGF signaling can specifically affect ommatidial rotation without showing transformation of the mystery cells or defects in polarity protein localization.     

Cleavage and secretion is not required for Four-jointed function in Drosophila patterning

Four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. We show that Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localised to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. We find that cleavage and secretion of Fj is not necessary for activity, and that Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). We show that fj interacts genetically with ft and ds in planar polarity and proximodistal patterning. We propose that Fj may act in the Golgi to regulate the activity of Ft and Ds.     

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.     

Planar cell polarity and tissue design

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.     

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.     

Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/β-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/β-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.     

Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins

Planar polarity development in the Drosophila wing is under the control of the frizzled (fz) pathway. Recent work has established that the planar polarity (PP) proteins become localized to either the distal, proximal, or both sides of wing cells. Fz and Dsh distal accumulation is thought to locally activate the cytoskeleton to form a hair . Planar polarity effector (PPE) genes such as inturned (in) are not required for the asymmetric accumulation of PP proteins, but they are required for this to influence hair polarity. in mutations result in abnormal hair polarity and are epistatic to mutations in the PP genes. We report that In localizes to the proximal side of wing cells in a PP-dependent and PP-instructive manner. We further show that the function of two other PPE genes (fuzzy and fritz) is essential for In protein localization, a finding consistent with previous genetic data that suggested these three genes function in a common process. These data indicate that accumulation of proteins at the proximal side of wing cells is a key event for the distal activation of the cytoskeleton to form a hair.     

Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels

Background: The frizzled (fz) gene of Drosophila encodes the founding member of the large family of receptors for the Wnt family of signaling molecules. It was originally studied in the adult epidermis, where it plays a key role in the generation of tissue polarity. Mutations in components of the fz signal transduction pathway disrupt tissue polarity; on the wing, hairs normally point distally but their polarity is altered by these mutations.Results: We devised a method to induce a gradient of fz expression with the highest levels near the distal wing tip. The result was a large area of proximally pointing hairs in this region. This reversal of polarity was seen when fz expression was induced just before the start of hair morphogenesis when polarity is established, suggesting that the gradient of Fz protein acted fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, resulted in a distinct tissue polarity phenotype, but no reversal of polarity; this argues that fz signaling was required for polarity reversal. Furthermore, the finding that functional dsh was required for the reversal of polarity argues that the reversal requires normal fz signal transduction.Conclusions: The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. Our observations enable us to propose a general mechanism to explain how Wnts polarize target cells.     

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development.     

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.