The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family.

Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-ß signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, we analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.

Cell competition removes segmental aneuploid cells from Drosophila imaginal disc-derived tissues based on ribosomal protein gene dose.

Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a potential marker for aneuploidy. We found that elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. Segmentally aneuploid cells derived from targeted chromosome excisions were eliminated by the RpS12-Xrp1 cell competition pathway if they differed from neighboring cells in Rp gene dose, whereas cells with normal doses of the Rp and eIF2γ genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer.

Aneuploid cells emerge when cellular division goes awry and a cell ends up with the wrong number of chromosomes, the tiny genetic structures carrying the instructions that control life's processes. Aneuploidy can lead to fatal conditions during development, and to cancer in an adult organism. A safety mechanism may exist that helps the body to detect and remove these cells. Yet, exactly this happens is still poorly understood: in particular, it is unclear how cells manage to 'count' their chromosomes. One way they could do so is through the ribosomes, the molecular 'factories' that create the building blocks required for life. In a cell, every chromosome carries genes that code for the proteins (known as Rps) forming ribosomes. Aneuploidy will alter the number of Rp genes, and in turn the amount and type of Rps the cell produces, so that ribosomes and the genes for Rps could act as a 'readout' of aneuploidy. Ji et al set out to test this theory in fruit flies. The first experiment used a genetic manipulation technique called site-specific recombination to remove parts of chromosomes from cells in the developing eye and wing. Cells which retained all their Rp genes survived, while those that were missing some usually died ­ but only when the surrounding cells were normal. In this situation, healthy cells eliminated their damaged neighbours through a process known as cell competition. A second experiment, using radiation as an alternative method of damaging chromosomes, also gave similar results. The work by Ji et al. reveals how the body can detect and eliminate aneuploid cells, potentially before they can cause harm. If the same mechanism applies in humans, boosting cell competition may, one day, helps to combat diseases like cancer.

Self-organized patterning of cell morphology via mechanosensitive feedback.

Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. Here, we investigate the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, we reveal a pattern of radially oriented cell rearrangements that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, we map tissue stresses and extract key parameters of tissue mechanics. We present a continuum theory showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. Our work reveals a mechanism for the emergence of cellular patterns in morphogenesis.

During development, carefully choreographed cell movements ensure the creation of a healthy organism. To determine their identity and place across a tissue, cells can read gradients of far-reaching signaling molecules called morphogens; in addition, physical forces can play a part in helping cells acquire the right size and shape. Indeed, cells are tightly attached to their neighbors through connections linked to internal components. Structures or proteins inside the cells can pull on these junctions to generate forces that change the physical features of a cell. However, it is poorly understood how these forces create patterns of cell size and shape across a tissue. Here, Dye, Popovic et al. combined experiments with physical models to examine how cells acquire these physical characteristics across the developing wing of fruit fly larvae. This revealed that cells pushing and pulling on one another create forces that trigger internal biochemical reorganization ­ for instance, force-generating structures become asymmetrical. In turn, the cells exert additional forces on their neighbors, setting up a positive feedback loop which results in cells adopting the right size and shape across the organ. As such, cells in the fly wing can spontaneously self-organize through the interplay of mechanical and biochemical signals, without the need for pre-existing morphogen gradients. A refined understanding of how physical forces shape cells and organs would help to grasp how defects can emerge during development. This knowledge would also allow scientists to better grow tissues and organs in the laboratory, both for theoretical research and regenerative medicine.

Role of D-GADD45 in JNK-Dependent Apoptosis and Regeneration in Drosophila.

The GADD45 proteins are induced in response to stress and have been implicated in the regulation of several cellular functions, including DNA repair, cell cycle control, senescence, and apoptosis. In this study, we investigate the role of D-GADD45 during Drosophila development and regeneration of the wing imaginal discs. We find that higher expression of D-GADD45 results in JNK-dependent apoptosis, while its temporary expression does not have harmful effects. Moreover, D-GADD45 is required for proper regeneration of wing imaginal discs. Our findings demonstrate that a tight regulation of D-GADD45 levels is required for its correct function both, in development and during the stress response after cell death.

The formation of the Thickveins (Tkv) gradient in Drosophila wing discs: A theoretical study.

The development of the wing imaginal disc (wing disc) is commonly adopted for the studies of patterning and growth which are two fundamental problems in developmental biology. Decapentaplegic (Dpp) signaling regulates several aspects of wing development, such as the anterior (A)-posterior (P) patterning, cellular growth rate, and cell adhesion. The distribution and activity of Dpp signaling are controlled in part by the expression level of its major type I receptor, Thickveins (Tkv). In this paper, we focus on theoretically investigating mechanisms by which the highly asymmetric pattern of Tkv is established in Drosophila wing discs. To the end, a mathematical model of Hh signaling and Dpp signaling is proposed and validated by comparisons with experimental observations. Our model provides a comprehensive view of the formation of Tkv gradients in wing discs. We found that engrailed (En), Hedgehog (Hh) signaling, and Dpp signaling cooperate to establish the asymmetric gradients of Tkv and pMad in the wing disc. Moreover, our model suggests a Brinker-mediated mechanism of Dpp-dependent repression of Tkv. Based on this mechanism, a couple of predicted experimental observations have been provided for further lab confirmation.

Glutamate signaling at cytoneme synapses.

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.

Fate mapping during regeneration: Cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types.

Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, we utilized a paradigm for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. Here we found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. We conclude that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation.

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.

Regenerative response of different regions of Drosophila imaginal discs.

Thanks to the introduction of new methods to induce massive damage under controlled conditions, much information about regeneration in Drosophila imaginal discs has accumulated in recent years. In this review, we discuss results concerning primarily the wing disc, putting emphasis on the different regenerative responses of the wing appendage, which exhibits a robust regenerative potential, and the trunk region, the notum, which regenerates very poorly. The wing disc may be a paradigm of a tissue in which a common original lineage generates cells with distinct regenerative potential. We argue that a key factor in those differences is the activity of the Jun N-terminal Kinase (JNK) pathway, which functions differently in the appendage and the body trunk.

JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster.

The regenerative process after tissue damage relies on a variety of cellular responses that includes compensatory cell proliferation and cell fate re-specification. The identification of the signalling networks regulating these cellular events is a central question in regenerative biology. Tissue regeneration models in Drosophila have shown that two of the signals that play a fundamental role during the early stages of regeneration are the c-Jun N-terminal kinase (JNK) and JAK/STAT signalling pathways. These pathways have been shown to be required for controlling regenerative proliferation, however their contribution to the processes of cellular reprogramming and cell fate re-specification that take place during regeneration are largely unknown. Here, we present evidence for a previously unrecognised function of the cooperative activities of JNK and JAK/STAT signalling pathways in inducing loss of cell fate specification in imaginal discs. We show that co-activation of these signalling pathways induces both the cell fate changes in injured areas, as well as in adjacent cells. We have also found that this function relies on the activity of the Caspase initiator encoded by the gene dronc.

Coordination of Septate Junctions Assembly and Completion of Cytokinesis in Proliferative Epithelial Tissues.

How permeability barrier function is maintained when epithelial cells divide is largely unknown. Here, we have investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. We report that, following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. We propose that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia.

Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture.

Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. Here, we establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.

Hormonal control of growth in the wing imaginal disks of Junonia coenia: the relative contributions of insulin and ecdysone.

The wing imaginal disks of Lepidoptera can be grown in tissue culture, but require both insulin and ecdysone to grow normally. Here, we investigate the contributions the two hormones make to growth. Ecdysone is required to maintain mitoses, whereas in the presence of insulin alone mitoses stop. Both ecdysone and insulin stimulate protein synthesis, but only ecdysone stimulates DNA synthesis. Insulin stimulates primarily cytoplasmic growth and an increase in cell size, whereas ecdysone, by virtue of its stimulation of DNA synthesis and mitosis, stimulates growth by an increase in cell number. Although both hormones stimulate protein synthesis, they do so in different spatial patterns. Both hormones stimulate protein synthesis in the inter-vein regions, but ecdysone stimulates synthesis more strongly in the veins and in the margin of the wing disk. We propose that the balance of insulin and ecdysone signaling must be regulated to maintain normal growth, and when growth appears to be due primarily to an increase in cell number, or an increase in cell size, this may indicate growth occurred under conditions that favored a stronger role for ecdysone, or insulin, respectively.

Cap-n-Collar Promotes Tissue Regeneration by Regulating ROS and JNK Signaling in the Drosophila melanogaster Wing Imaginal Disc.

Regeneration is a complex process that requires an organism to recognize and repair tissue damage, as well as grow and pattern new tissue. Here, we describe a genetic screen to identify novel regulators of regeneration. We ablated the Drosophila melanogaster larval wing primordium by inducing apoptosis in a spatially and temporally controlled manner and allowed the tissue to regenerate and repattern. To identify genes that regulate regeneration, we carried out a dominant-modifier screen by assessing the amount and quality of regeneration in adult wings heterozygous for isogenic deficiencies. We have identified 31 regions on the right arm of the third chromosome that modify the regenerative response. Interestingly, we observed several distinct phenotypes: mutants that regenerated poorly, mutants that regenerated faster or better than wild-type, and mutants that regenerated imperfectly and had patterning defects. We mapped one deficiency region to cap-n-collar (cnc), the Drosophila Nrf2 ortholog, which is required for regeneration. Cnc regulates reactive oxygen species levels in the regenerating epithelium, and affects c-Jun N-terminal protein kinase (JNK) signaling, growth, debris localization, and pupariation timing. Here, we present the results of our screen and propose a model wherein Cnc regulates regeneration by maintaining an optimal level of reactive oxygen species to promote JNK signaling.

Imaginal disc regeneration takes flight.

Drosophila imaginal discs, the larval precursors of adult structures such as the wing and leg, are capable of regenerating after damage. During the course of regeneration, discs can sometimes generate structures that are appropriate for a different type of disc, a phenomenon termed transdetermination. Until recently, these phenomena were studied by physically fragmenting discs and then transplanting them into the abdomens of adult female flies. This field has experienced a renaissance following the development of genetic ablation systems that can damage precisely defined regions of the disc without the need for surgery. Together with more traditional approaches, these newer methods have generated many novel insights into wound healing, the mechanisms that drive regenerative growth, plasticity during regeneration and systemic effects of tissue damage and regeneration.

The SWI/SNF Complex Protein Snr1 Is a Tumor Suppressor in Drosophila Imaginal Tissues.

Components of the SWI/SNF chromatin-remodeling complex are among the most frequently mutated genes in various human cancers, yet only SMARCB1/hSNF5, a core member of the SWI/SNF complex, is mutated in malignant rhabdoid tumors (MRT). How SMARCB1/hSNF5 functions differently from other members of the SWI/SNF complex remains unclear. Here, we use Drosophila imaginal epithelial tissues to demonstrate that Snr1, the conserved homolog of human SMARCB1/hSNF5, prevents tumorigenesis by maintaining normal endosomal trafficking-mediated signaling cascades. Removal of Snr1 resulted in neoplastic tumorigenic overgrowth in imaginal epithelial tissues, whereas depletion of any other members of the SWI/SNF complex did not induce similar phenotypes. Unlike other components of the SWI/SNF complex that were detected only in the nucleus, Snr1 was observed in both the nucleus and the cytoplasm. Aberrant regulation of multiple signaling pathways, including Notch, JNK, and JAK/STAT, was responsible for tumor progression upon snr1-depletion. Our results suggest that the cytoplasmic Snr1 may play a tumor suppressive role in Drosophila imaginal tissues, offering a foundation for understanding the pivotal role of SMARCB1/hSNF5 in suppressing MRT during early childhood. Cancer Res; 77(4); 862-73. ©2017 AACR.

How tissue damage MET metabolism: Regulation of the systemic damage response.

Living organisms experience tissue damage from both, the surrounding environment and from inside their bodies. Tissue repair/regeneration is triggered by local tissue injury to restore an injured, or lost, part of the body. Tissue damage results in a series of responses, not only locally but also systemically in distant tissues. In our recent publication, we established a "dual system" that induces spatiotemporal tissue damage simultaneously with gene manipulation in surrounding tissues. With this system, we demonstrated that appropriate regulation of methionine metabolism in the fat body is required for tissue repair in Drosophila wing discs, thus highlighting the importance of systemic damage response (SDR) in tissue repair. This "Extra View" aims to discuss our recent reports that propose methionine metabolism to be an essential part of SDR, together with related topics in several model organisms.

Analysis of the Function of Apoptosis during Imaginal Wing Disc Regeneration in Drosophila melanogaster.

Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.

Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster.

The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations.

Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

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.