Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.
ABSTRACT: Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.
Project description:The proper number of cells in developing tissues is achieved by coordinating cell division with apoptosis. In Drosophila, the adult wing is derived from wing imaginal discs, which undergo a period of growth and proliferation during larval stages without much programmed cell death. In this report, we demonstrate that the Drosophila casein kinase Iepsilon/delta, known as Discs overgrown (Dco), is required for maintaining this low level of apoptosis. Expression of dco can suppress the apoptotic activity of Head involution defective (Hid) in the developing eye. Loss of dco in the wing disc results in a dramatic reduction in expression of the caspase inhibitor DIAP1 and a concomitant activation of caspases. The regulation of DIAP1 by Dco occurs by a post-transcriptional mechanism that is independent of hid. Mutant clones of dco are considerably smaller than controls even when apoptosis is inhibited, suggesting that Dco promotes cell division/growth in addition to its role in cell survival. The dco phenotype cannot be explained by defects Wingless (Wg) signaling. We propose that Dco coordinates tissue size by stimulating cell division/growth and blocking apoptosis via activation of DIAP1 expression.
Project description:We report a phenomenon wherein induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which we identified in a genetic screen for modifiers of ban. tie mutants were hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. We propose that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect we report differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.
Project description:Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion.
Project description:BACKGROUND:Archipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (?-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers. RESULTS:We found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of ?-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER. CONCLUSIONS:We concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.
Project description:Cancer treatments including ionizing radiation (IR) can induce cancer stem cell-like properties in non-stem cancer cells, an outcome that can interfere with therapeutic success. Yet, we understand little about what consequences of IR induces stem cell like properties and why some cancer cells show this response but not others. In previous studies, we identified a pool of epithelial cells in Drosophila larval wing discs that display IR-induced stem cell-like properties. These cells are resistant to killing by IR and, after radiation damage, change fate and translocate to regenerate parts of the disc that suffered more cell death. Here, we report the identification of two new pools of cells with IR-induced regenerative capability. We addressed how IR exposure results in the induction of stem cell-like behavior, and found a requirement for IR-induced caspase activity and for Zfh2, a transcription factor and an effector in the JAK/STAT pathway. Unexpectedly, the requirement for caspase activity was cell-autonomous within cell populations that display regenerative behavior. We propose a model in which the requirement for caspase activity and Zfh2 can be explained by apoptotic and non-apoptotic functions of caspases in the induction of stem cell-like behavior.
Project description:Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. Here, we sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. We compared the activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster and found strong functional conservation among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, we found three novel wg enhancer activities in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. Our results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. We suggest that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.
Project description:We report here a study of regeneration in Drosophila larval wing imaginal discs after damage by ionizing radiation. We detected faithful regeneration that restored a wing disc and abnormal regeneration that produced an extra wing disc. We describe a sequence of changes in cell number, location and fate that occur to produce an ectopic disc. We identified a group of cells that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.
Project description:Wnts are secreted proteins that regulate cell fate during development of all metazoans. Wnt proteins were proposed to spread over several cells to activate signaling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of the Wnt1 homolog Wingless (Wg) was observed extending over several cells away from producing cells. Surprisingly, however, it was also shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can largely replace endogenous Wg, leading to proper patterning of the wing. Therefore, the functional range of Wg and whether Wg spreading is required for correct tissue patterning remains controversial. Here, by capturing secreted Wg on cells away from the source, we show that Wg acts over a distance of up to 11 cell diameters to induce signaling. Furthermore, cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain signaling. Frizzled2 expression is increased in the absence of Wg secretion and is required to maintain signaling and cell survival in NRT-wg wing discs. Together, these results provide insight into the mechanisms by which robust Wnt signaling is achieved in proliferating tissues.
Project description:In holometabolous insects, a species-specific size, known as critical weight, needs to be reached for metamorphosis to be initiated in the absence of further nutritional input. Previously, we found that reaching critical weight depends on the insulin-dependent growth of the prothoracic glands (PGs) in Drosophila larvae. Because the PGs produce the molting hormone ecdysone, we hypothesized that ecdysone signaling switches the larva to a nutrition-independent mode of development post-critical weight. Wing discs from pre-critical weight larvae [5 hours after third instar ecdysis (AL3E)] fed on sucrose alone showed suppressed Wingless (WG), Cut (CT) and Senseless (SENS) expression. Post-critical weight, a sucrose-only diet no longer suppressed the expression of these proteins. Feeding larvae that exhibit enhanced insulin signaling in their PGs at 5 hours AL3E on sucrose alone produced wing discs with precocious WG, CT and SENS expression. In addition, knocking down the Ecdysone receptor (EcR) selectively in the discs also promoted premature WG, CUT and SENS expression in the wing discs of sucrose-fed pre-critical weight larvae. EcR is involved in gene activation when ecdysone is present, and gene repression in its absence. Thus, knocking down EcR derepresses genes that are normally repressed by unliganded EcR, thereby allowing wing patterning to progress. In addition, knocking down EcR in the wing discs caused precocious expression of the ecdysone-responsive gene broad. These results suggest that post-critical weight, EcR signaling switches wing discs to a nutrition-independent mode of development via derepression.
Project description:Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. We identified pdm3m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by Pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that Pdm3 acts upstream to Wg. Furthermore, transient expression of Pdm3 induced ectopic expression of wg-LacZ reporter and Wg effector proteins in wing discs. We propose that Pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles.