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.     

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.     

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.     

The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.     

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.     

Analysis of the role of the Rac/Cdc42 GTPases during planar cell polarity generation in Drosophila

Initial genetic studies in Drosophila suggested that several members of the Rho subfamily (RhoA, Rac1 and Cdc42) are involved in planar cell polarity (PCP) establishment. However, analyses of Rac1, Rac2 and Mtl loss-of-function (LOF) mutants have argued against their role in this process. Here, we investigate in detail the role of the Rho GTPases Mtl, Cdc42, Rac1 and Rac2 in PCP generation. These functional analyses were performed by overexpressing Mtl in eyes and wings, by performing genetic interaction assays and by using a combination of triple and quadruple mutant LOF clones. We found that Mtl overexpression caused PCP phenotypes and that it interacted genetically with other Rho GTPases, such as Rac1 and Cdc42 as well as with several PCP genes, such as stbm, pk and aos. However, Mtl was not found to interact with Rac2, RhoA and other members of the Fz/PCP pathway. Triple mutant clones of Rac1, Rac2 and Mtl were found to exhibit mild PCP defects which were enhanced by reduction of Cdc42 function with a hypomorphic Cdc42 allele. Taken together, these and previous results suggest that Rho GTPases may have partially overlapping functions during PCP generation. Alternatively, it is also possible that the mild PCP phenotypes observed could indicate that they are required at low levels in that process. However, since not all of them function upstream of a JNK cassette, we propose that they may act in at least two parallel pathways.     

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.     

Control of planar cell polarity by interaction of DWnt4 and four-jointed

The Drosophila eye and the wing display specific planar cell polarity. Although Frizzled (Fz) signaling has been implicated in the establishment of ommatidial and wing hair polarity, evidence for the Wnt gene function has been limited. Here we examined the function of a Drosophila homolog of Wnt4 (DWnt4) in the control of planar polarity. We show that DWnt4 mRNA and protein are preferentially expressed in the ventral region of eye disc. DWnt4 mutant eyes show polarity reversals mostly in the ventral domain, consistent with the ventral expression of DWnt4. Ectopic expression of DWnt4 in the dorsoventral (DV) polar margins is insufficient to induce ommatidial polarity but becomes inductive when coexpressed with Four-jointed (Fj). Similarly, DWnt4 and Fj result in synergistic induction of hair polarity toward the source of expression in the wing. Consistent with genetic interaction, we provide evidence for direct interaction of DWnt4 and Fj transmembrane protein. The extracellular domain of Fj is required for direct binding to DWnt4 and for the induction of hair polarity. In contrast to the synergy between DWnt4 and Fj, DWnt4 antagonizes the polarizing effect of Fz. Our results suggest that DWnt4 is involved in ommatidial polarity signaling in the ventral region of the eye and its function is mediated by interacting with Fj.     

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.     

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.     

CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.     

Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6

Epidermal Growth Factor-receptor (Egfr) signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Here we provide evidence for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90 degrees rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90 degrees. Here we show that rlt is a rotation-specific allele of the inhibitory Egfr ligand Argos and that modulation of Egfr activity shows defects in ommatidial rotation. Our data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. We provide further evidence for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway.     

Drosophila eye disc margin is a center for organizing long-range planar polarity

Planar polarity patterning involves long-range signaling and signal transduction. In Drosophila eye, Dishevelled (Dsh) is not only crucial for cell-autonomous transduction of a polarity signal(s) but is also involved in nonautonomous signaling function. To identify the sites for long-range polarity signaling in eye disc, we examined spatial and temporal conditions for nonautonomous Dsh function. Here we show that Dsh and its downstream factor Armadillo (Arm) are required in the border region of eye disc between the peripodial membrane (PM) and the disc proper (DP) for nonautonomous signaling. Conditional misexpression of Dsh or Arm at the posterior margin of the disc was sufficient to induce nonautonomous polarity reversals. A critical time window for the induction of such changes was approximately coincident with the timing of morphogenetic furrow initiation. Our data suggest that the disc margin is an essential site for organizing planar polarity during the initial stage of retinal morphogenesis.     

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.     

A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.     

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.     

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.