Regulation of wingless signaling by the CKI family in Drosophila limb development

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. Here we examine the roles of seven CKI family members in Wg signaling during Drosophila limb development. We find that increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, we provide evidence that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway.     

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.     

Protection of Armadillo/β-Catenin by Armless,a Novel Positive Regulator of Wingless Signaling

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94''s positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated.     

A positive role for the PP2A catalytic subunit in Wnt signal transduction

Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. Using early Xenopus embryos, we studied how PP2A functions in Wnt signal transduction. The catalytic subunit of PP2A (PP2A-C) potentiated secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibited Dishevelled-mediated signal transduction, as did the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C did not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin was sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, we propose that PP2A regulates the Wnt pathway at multiple levels.     

CELLULAR LOCALIZATION AND EXPRESSION OF pygo DURING DROSOPHILA DEVELOPMENT

Abstract Wg/Wnt signaling is a key signaling pathway in Drosophila. Many genes involved in Wingless(wg) signal transduction pathway downstream of Wg, or it s vertebrate Wg homologue Wnt, have been identified. Transduction of the Wg signal downstream of Wg is mediated by nuclear TCF/LEF-1, through association with Armadillo (Arm)β-catenin. Pygopus (pygo) is a new identified component in this pathway. Cellular localization experiment showed that pygo was expressed specifically in the nucleus. The expression profile of pygo in embryos was examined using in situ hybridization. Although pygo expressed ubiquitously in the embryos, it expressed at relatively high level in pre-blastoderm embryos which indicate a high degree of maternally provided message, followed by a low level of ubiquitous zygotic expression. This continues into larval tissues (including wing disc, eye disc and leg disc), where pygo appears to be expressed at low level. Comparison of pygo expression levels, in the wing disc, eye disc and leg disc, showed pygo expression level in the wing disc pouch and leg disc were relative higher.     

Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway.

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087-1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.     

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.     

Wnt/Wingless signaling through β-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

Background

Wnt/Wingless (Wg) signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6) or Arrow. The aminotermini of LRP and Fz were reported to associate only in the presence of Wnt, implying that Wnt ligands form a trimeric complex with two different receptors. However, it was recently reported that LRPs activate the Wnt/β-catenin pathway by binding to Axin in a Dishevelled – independent manner, while Fzs transduce Wnt signals through Dishevelled to stabilize β-catenin. Thus, it is possible that Wnt proteins form separate complexes with Fzs and LRPs, transducing Wnt signals separately, but converging downstream in the Wnt/β-catenin pathway. The question then arises whether both receptors are absolutely required to transduce Wnt signals.

Results

We have established a sensitive luciferase reporter assay in Drosophila S2 cells to determine the level of Wg – stimulated signaling. We demonstrate here that Wg can synergize with DFz2 and function cooperatively with LRP to activate the β-catenin/Armadillo signaling pathway. Double-strand RNA interference that disrupts the synthesis of either receptor type dramatically impairs Wg signaling activity. Importantly, the pronounced synergistic effect of adding Wg and DFz2 is dependent on Arrow and Dishevelled. The synergy requires the cysteine-rich extracellular domain of DFz2, but not its carboxyterminus. Finally, mammalian LRP6 and its activated forms, which lack most of the extracellular domain of the protein, can activate the Wg signaling pathway and cooperate with Wg and DFz2 in S2 cells. We also show that the aminoterminus of LRP/Arr is required for the synergy between Wg and DFz2.

Conclusion

Our study indicates that Wg signal transduction in S2 cells depends on the function of both LRPs and DFz2, and the results are consistent with the proposal that Wnt/Wg signals through the aminoterminal domains of its dual receptors, activating target genes through Dishevelled.

     

Powerful Drosophila screens that paved the wingless pathway

The Wnt/Wingless (Wg) signaling cascade controls a number of biological processes in animal development and adult life; aberrant Wnt/Wg signaling can cause diseases. In the 1980s genes were discovered that encode core Wnt/Wg pathway components: their mutant phenotypes were similar and an outline of a signaling cascade emerged. Over the years our knowledge of this important signaling system increased and more components were uncovered that are instrumental for Wnt/Wg secretion and transduction. Here we provide an overview of these discoveries, the technologies involved, with a particular focus on the important role Drosophila screens played in this process.     

Powerful Drosophila screens that paved the wingless pathway

The Wnt/Wingless (Wg) signaling cascade controls a number of biological processes in animal development and adult life; aberrant Wnt/Wg signaling can cause diseases. In the 1980s genes were discovered that encode core Wnt/Wg pathway components: their mutant phenotypes were similar and an outline of a signaling cascade emerged. Over the years our knowledge of this important signaling system increased and more components were uncovered that are instrumental for Wnt/Wg secretion and transduction. Here we provide an overview of these discoveries, the technologies involved, with a particular focus on the important role Drosophila screens played in this process.