Scabrous controls ommatidial rotation in the Drosophila compound eye

Establishment of planar polarity in the Drosophila compound eye requires precise 90 degrees rotation of the ommatidial clusters during development. We found that the morphogenetic furrow controls the stop of ommatidial rotation at 90 degrees by emitting signals to posterior ommatidial clusters. One such signal, Scabrous, is synthesized in the furrow cells and transported in vesicles to ommatidial row 6-8. Scabrous vesicles are transported through actin-based cellular extensions but not transcytosis. Scabrous functions nonautonomously to control the stop of ommatidial rotation by suppressing nemo activity in the second 45 degrees rotation. We propose that the morphogenetic furrow regulates precise ommatidial rotation by transporting Scabrous and perhaps other factors through actin-based cellular extensions.     

The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.     

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

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.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

Gliotactin,a novel marker of tricellular junctions,is necessary for septate junction development in Drosophila

Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. Gliotactin (Gli) is a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, and may, therefore, contribute to SJ development or function. To address this hypothesis, we analyzed Gli expression and the Gli mutant phenotype in Drosophila epithelia. In Gli mutants, localization of SJ markers neurexin-IV, discs large, and coracle are disrupted. Furthermore, SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. We propose a model, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development.     

Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90 degrees, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Here, we demonstrate a requirement for epidermal growth factor receptor (Egfr) signalling in rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. We propose that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, we have evidence that there is an error-correction mechanism operating during pupal stages to reposition inappropriately orientated ommatidia. Our results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling.     

Drosophila Gp150 is required for early ommatidial development through modulation of Notch signaling

Cellular signaling activities must be tightly regulated for proper cell fate control and tissue morphogenesis. Here we report that the Drosophila leucine-rich repeat transmembrane glycoprotein Gp150 is required for viability, fertility and development of the eye, wing and sensory organs. In the eye, Gp150 plays a critical role in regulating early ommatidial formation. Gp150 is highly expressed in cells of the morphogenetic furrow (MF) region, where it accumulates exclusively in intracellular vesicles in an endocytosis-independent manner. Loss of gp150 function causes defects in the refinement of photoreceptor R8 cells and recruitment of other cells, which leads to the formation of aberrant ommatidia. Genetic analyses suggest that Gp150 functions to modulate Notch signaling. Consistent with this notion, Gp150 is co-localized with Delta in intracellular vesicles in cells within the MF region and loss of gp150 function causes accumulation of intracellular Delta protein. Therefore, Gp150 might function in intracellular vesicles to modulate Delta-Notch signaling for cell fate control and tissue morphogenesis.     

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.     

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.     

A G beta protein in the Drosophila compound eye is different from that in the brain

A G protein beta subunit gene (Gbe) is expressed only in the eyes of adult D. melanogaster. This gene was identified by probing a Drosophila head cDNA expression library with monoclonal antibodies to a previously characterized Drosophila G protein beta subunit (Gbb). Immunoblot and Northern analyses demonstrate that Gbe protein and mRNA is not present in Drosophila mutants that lack eyes. Immunocytochemical and in situ hybridization analyses further demonstrate that Gbe is expressed in the eyes but not in the brain, whereas Gbb is abundantly expressed in the brain. The Gbe product is approximately 45% identical to previously identified G beta subunits and defines a new G beta class. Its localization suggests a possible role in phototransduction.     

Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila beta-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dalpha-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.     

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.     

The development of ommatidial patterning in metamorphosed eye imaginal discs implants ofDrosophila melanogaster

Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of in vitro³H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.     

Contribution of annexin 2 to the architecture of mature endothelial adherens junctions

The vascular endothelial cadherin (VE-cad)-based complex is involved in the maintenance of vascular endothelium integrity. Using immunoprecipitation experiments, we have demonstrated that, in confluent human umbilical vein endothelial cells, the VE-cad-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the cell-cell contact sites as cell confluence is established. Annexin 2, located in cholesterol rafts, binds to both the actin cytoskeleton and the VE-cad-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cad-based complex, thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the down-regulation of annexin 2 by small interfering RNA induces a delocalization of VE-cad from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data indicate that the decoupling of the annexin 2/p11 complex from the VE-cad-based junction, triggered by vascular endothelial growth factor treatment, facilitates the switch from a quiescent to an immature state.     

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.     

The extracellular architecture of adherens junctions revealed by crystal structures of type I cadherins

Adherens junctions, which play a central role in intercellular adhesion, comprise clusters of type I classical cadherins that bind via extracellular domains extended from opposing cell surfaces. We show that a molecular layer seen in crystal structures of E- and N-cadherin ectodomains reported here and in a previous C-cadherin structure corresponds to the extracellular architecture of adherens junctions. In all three ectodomain crystals, cadherins dimerize through a trans adhesive interface and are connected by a second, cis, interface. Assemblies formed by E-cadherin ectodomains coated on liposomes also appear to adopt this structure. Fluorescent imaging of junctions formed from wild-type and mutant E-cadherins in cultured cells confirm conclusions derived from structural evidence. Mutations that interfere with the trans interface ablate adhesion, whereas cis interface mutations disrupt stable junction formation. Our observations are consistent with a model for junction assembly involving strong trans and weak cis interactions localized in the ectodomain.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.