Drosophila twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of ∼36 kDa catalytic and ∼65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of PP2A role in Wg/Wnt pathway in different organisms are conflicting. We show that twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is a positive regulator of Wg/Wnt signaling. In tws-wing discs both short-and long-range targets of Wingless morphogen are downregulated. Analyses of tws-mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. Our results suggest that Tws is required for the stabilization of Armadillo/β-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits

A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems

A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems

Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System.

International audienceCell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo.

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ( undead ) model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer

Genome-wide RNAi Pvr modifier screen.

<p>(A) Screen scheme of the Primary and Secondary Screens, and subsequent single gene analysis. (B) Cluster analysis of primary screen hits, highlighting a fraction of <i>Pvr</i> Enhancers including InR (arrow) and <i>Pvr</i> Suppressors including <i>EcR</i> and <i>usp</i> (arrows). (C) Protein complexes enriched in the list of <i>Pvr</i> Suppressors from the RNAi screen, identified using COMPLEAT [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005056#pgen.1005056.ref154" target="_blank">154</a>]. Node color correlates with phenotypic strength where red is strongest and purple is weakest, while gray nodes were not identified in the screen. Note p-values are approximate given that the random proteins used to generate the p-value are unique for each of 1000 random sets.</p

Role of EcR and InR in embryonic hemocytes <i>in vivo</i>.

<p>(A) In vivo rescue experiment, combining Pvr1 mutant or hemocyte specific expression of UAS-PvrΔC with various UAS-transgenes. Inhibition of EcR signaling by dominant-negative EcR (EcRA dn or EcRB dn) rescues hemocyte numbers in <i>Pvr</i>^<i>1</i> mutant embryos, while co-expression of dominant-negative InR enhances the <i>Pvr</i>^<i>1</i> phenotype. Embryonic hemocyte numbers in embryos of the indicated genetic combinations. Hemocytes were marked by nuclear β-Gal driven by srpHemoGAL4; total hemocytes of individual stage 16 embryos were counted. For full genotype, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005056#sec013" target="_blank">Methods</a>. (B-F) Confocal images of representative embryos. (G) Summary model of Pvr Suppressors and Pvr Enhancers as shown in B-F. In Pvr1 mutants, apoptotic hemocytes are phagocytosed by remaining, viable hemocytes, which increase in size [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005056#pgen.1005056.ref012" target="_blank">12</a>]. Additional lack of a Pvr Enhancer aggravates the phenotype, while lack of a Pvr Suppressor rescues hemocyte death.</p