The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.     

RacGap50C negatively regulates wingless pathway activity during Drosophila embryonic development

The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these elements work together to generate cellular identities. In the ventral epidermis of Drosophila embryos, Wg specifies cells to secrete a characteristic pattern of denticles and naked cuticle that decorate the larval cuticle at the end of embryonic development. We have used the Drosophila ventral epidermis as our assay system in a series of genetic screens to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning represent the first loss-of-function mutations in the RacGap50C gene. These mutations on their own cause increased stabilization of Armadillo and cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which suggests that the mutant embryos suffer from ectopic Wg pathway activation. In addition, RacGap50C mutations interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.     

The canonical Wg and JNK signaling cascades collaborate to promote both dorsal closure and ventral patterning

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.     

Control of tracheal tubulogenesis by Wingless signaling

The tubular epithelium of the Drosophila tracheal system forms a network with a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching induced by the FGF homolog Branchless, differentiates cells with specialized functions such as fusion cells, which perform target recognition and adhesion during branch fusion, and extends branches toward specific targets. Specification of a unique identity for each primary branch is essential for directed migration, as a defect in either the EGFR or the Dpp pathway leads to a loss of branch identity and the misguidance of tracheal cell migration. Here, we investigate the role of Wingless signaling in the specification of cell and branch identity in the tracheal system. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, including specifying the dorsal trunk through activation of Spalt expression and inducing differentiation of fusion cells in all fusion branches. Moreover, we show that Wingless signaling regulates Notch signaling by stimulating delta expression at the tip of primary branches. These activities of Wingless signaling together specify the shape of the dorsal trunk and other fusion branches.     

A critical role of teashirt for patterning the ventral epidermis is masked by ectopic expression of tiptop, a paralog of teashirt in Drosophila

The teashirt gene encodes a protein with three widely spaced zinc finger motifs that is crucial for specifying trunk identity in Drosophila embryos. Here, we describe a gene called tiptop, which encodes a protein highly similar to Teashirt. We have analyzed the expression patterns and functions of these two genes in the trunk of the embryo. Initially, teashirt and tiptop expressions are detected in distinct domains; teashirt in the trunk and tiptop in parts of the head and tail. In different mutant situations, we show that, in the trunk and head, they repress each other's expression. Unlike teashirt, we found that deletion of tiptop is homozygous viable and fertile. However, embryos lacking both gene activities display a more severe trunk phenotype than teashirt mutant embryos alone. Ectopic expression of either gene produces an almost identical phenotype, indicating that Teashirt and Tiptop have, on the whole, common activities. We conclude that Teashirt and Tiptop repress each other's expression and that Teashirt has a crucial role for trunk patterning that is in part masked by ectopic expression of Tiptop.     

Transcription under the control of nuclear Arm/beta-catenin

The Wingless/Wnt pathway controls cell fates during animal development and regulates tissue homeostasis as well as stem cell number and differentiation in epithelia. Deregulation of Wnt signaling has been associated with cancer in humans. In the nucleus, the Wingless/Wnt signal is transmitted via the key effector protein Armadillo/beta-catenin. The recent identification and functional analysis of novel Armadillo/beta-catenin interaction partners provide new and exciting insights into the highly complex mechanism of Wingless/Wnt target gene activation.     

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.     

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. We show that the positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively.     

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.     

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic wingless signal transduction

The highly conserved Wnt family of growth factors is essential for generating embryonic pattern in many animal species [1]. In the fruit fly Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and DFrizzled2 (Dfz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately [2], suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Here, we report that overexpression of Dfz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz was able to substitute for Dfz2 only under conditions where the Wg ligand was present in excess. The wgPE2 mutant phenotype was also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. We conclude that wild-type Wg signaling requires efficient interaction between ligand and the Dfz2-proteoglycan receptor complex to promote the naked cuticle cell fate.