Epithelial Cell Division: Keeping Aneuploidy Levels in Check

SummaryAneuploidy is deleterious at the cellular and organismal level and can promote tumorigenesis. Two new studies in Drosophila imaginal discs underscore the cellular and tissue-wide mechanisms that prevent the accumulation of aneuploid cells in symmetrically dividing epithelial tissues upon changes in centrosome number

Two new distinct mechanisms drive epithelial folding in Drosophila wing imaginal discs

Epithelial folding is an important morphogenetic process that is essential in transforming simple sheets of cells into complex three-dimensional tissues and organs during animal development (Davidson, 2012). Epithelial folding has been shown to rely on constriction forces generated by the apical actomyosin network (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). However, the contributions of mechanical forces acting along lateral and basal cell surfaces to epithelial folding remain poorly understood. Here we combine live imaging with force measurements of epithelial mechanics to analyze the formation of two epithelial folds in the Drosophila larval wing imaginal disc. We show that these two neighboring folds form via two distinct mechanisms. These two folds are driven either by decrease of basal tension or increase of lateral tension, none of them depends on apical constriction. In the first fold, a local decrease in extracellular matrix (ECM) density in prefold cells results in a reduction of mechanical tension on the basal cell surface, leading to basal expansion and fold formation. Consistent with that, a local reduction of ECM by overexpression of Matrix metalloproteinase II is sufficient to induce ectopic folding. In the second fold a different mechanism is at place. Here basal tension is not different with neighboring cells, but pulsed dynamic F-actin accumulations along the lateral interface of prefold cells lead to increased lateral tension, which drives cell shortening along the apical-basal axis and fold formation. In this thesis I described two distinct mechanisms driving epithelial folding, both basal decrease and lateral increase in tension can generate similar morphological changes and promote epithelial folding in the Drosophila wing discs.Die Faltung von Epithelien ist ein wichtiger morphogenetischer Prozess, der die Entstehung komplexer, dreidimensionaler Gewebe und Organe aus einfachen Zellschichten ermöglicht (Davidson, 2012). Es ist bekannt, dass Kräfte erzeugt durch das apikale Aktomyosin-Netzwerk wichtig sind für die erfolgreiche Faltung von Epithelien (Martin et al., 2009; Roh-Johnson et al., 2012; Sawyer et al., 2010). Die Rolle von mechanischen Kräften, die entlang der lateralen und basalen Seite wirken, ist jedoch kaum verstanden. Wir verbinden Lebendmikroskopie mit der Messung von mechanischen Eigenschaften, um die Entstehung von 2 Epithelfalten in den Imaginalscheiben von Drosophila zu verstehen. Wir können dadurch zeigen, dass die beiden Falten durch unterschiedliche Mechanismen entstehen. Sie entstehen entweder durch eine Verringerung der Spannung auf der basalen Seite oder durch eine Erhöhung der Spannung auf der lateralen Seite, aber keine von beiden entsteht durch zusammenziehende Kräfte auf der apikalen Seite. Die erste Falte entsteht durch eine lokale Verringerung der extrazellulären Matrix in den Vorläuferzellen, was zu einer Reduktion der Spannung auf der basalen Seite und zur Ausbildung der Falte führt. Die zweite Falte wird durch einen anderen Mechanismus ausgebildet. Hier ist nicht die Spannung auf der basalen Seite reduziert sondern dynamische Anreicherungen von F-Aktin auf der lateralen Seite resultieren in einer erhöhten lateralen Spannung, die zu einer Verkürzung der Zellen und damit zur Ausbildung einer Falte führt. In meiner Arbeit zeige ich 2 neue Mechanismen zur Entstehung von Epithelfalten auf, durch Absenken der Spannung auf der basalen oder Erhöhen auf der lateralen Seite

Spatial discontinuity of Optomotor-blind expression in the Drosophila wing imaginal disc disrupts epithelial architecture and promotes cell sorting

<p>Abstract</p> <p>Background</p> <p>Decapentaplegic (Dpp) is one of the best characterized morphogens, required for dorso-ventral patterning of the <it>Drosophila </it>embryo and for anterior-posterior (A/P) patterning of the wing imaginal disc. In the larval wing pouch, the Dpp target gene <it>optomotor-blind </it>(<it>omb</it>) is generally assumed to be expressed in a step function above a certain threshold of Dpp signaling activity.</p> <p>Results</p> <p>We show that the transcription factor Omb forms, in fact, a symmetrical gradient on both sides of the A/P compartment boundary. Disruptions of the Omb gradient lead to a re-organization of the epithelial cytoskeleton and to a retraction of cells toward the basal membrane suggesting that the Omb gradient is required for correct epithelial morphology. Moreover, by analysing the shape of <it>omb </it>gain- and loss-of-function clones, we find that Omb promotes cell sorting along the A/P axis in a concentration-dependent manner.</p> <p>Conclusions</p> <p>Our findings show that Omb distribution in the wing imaginal disc is described by a gradient rather than a step function. Graded Omb expression is necessary for normal cell morphogenesis and cell affinity and sharp spatial discontinuities must be avoided to allow normal wing development.</p

Apico-basal forces exerted by apoptotic cells drive epithelium folding

© 2015 Macmillan Publishers Limited. All rights reserved. Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.Agence Nationale de la Recherche (ANR), Fondation de la Recherche et de l’Innovation The´rapeutique en Cance´rologie (RITC) and the University of Toulouse.Peer Reviewe

ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila

Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects

Systematic characterization of Rab GTPase cell type expression and subcellular localization in Drosophila melanogaster

The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases. In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab. For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues. I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific. I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans. The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning. My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease

Cell-Sorting at the A/P Boundary in the Drosophila Wing Primordium: A Computational Model to Consolidate Observed Non-Local Effects of Hh Signaling

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach

Genetic analysis of Drosophila adult muscle type specification

Muscles of all higher animals comprise different muscle types adapted to perform distinct functions in the body. These express different sets of genes controlled by distinct combinations of transcriptional programs and extracellular signals, and thus differ in their myofibrillar organization and contractile properties. Despite major progress in our understanding of myogenesis, the genetic pathways controlling the formation and function of different muscle types are still largely uncharacterized. Flying insects possess specialized flight muscles enabling wing oscillations with frequencies of up to 1000 Hz together with high power outputs of 80 W per kg muscle. To achieve these parameters, flight muscles contain stretch-activated myofibrils with a unique fibrillar organization, whereas all other, more slowly contracting muscles, such as leg muscles, display a tubular morphology. To delineate the genetic regulation of muscle development and function, and, in particular, muscle type specification, we performed a genome-wide RNA interference (RNAi) screen in Drosophila, in which we systematically inactivate genes exclusively in muscle tissue. We uncovered more than 2000 genes with putative roles in muscles, many of which we were able to assign to specific functions in muscle, myofibril or sarcomere organization by phenotypic characterization. Muscle-specific knockdown of 315 genes resulted in viable, but completely flightless animals, indicating a specific function of those genes in fibrillar flight muscles. Detailed morphological analysis of these 315 genes revealed a striking phenotype upon knockdown of the zinc finger transcription factor spalt major (salm): the fibrillar flight muscles are switched to tubular muscles, whereas tubular leg muscles are wild type, demonstrating that salm is a key determinant of fibrillar muscle fate. We could show that the transcription factor vestigial (vg) acts upstream of salm to induce its expression specifically in fibrillar flight muscles. Importantly, salm is not only required but also sufficient to induce the fibrillar muscle fate upon ectopic expression in other muscle types. Microarray analysis, comparing mRNA expression from adult wild-type flight and leg muscles to salm knockdown flight muscles, indicates that salm instructs most features of fibrillar muscles by regulating both gene expression as well as alternative splicing. Remarkably, we could show that spalt’s function in programming stretch-activated fibrillar muscles is conserved in insect species separated by 280 million years of evolution. Interestingly, in mouse two of the four spalt-like (sall) genes are expressed in heart, a stretch-activated muscle, sharing some features with insect fibrillar flight muscles. Since heart abnormalities observed in patients suffering from the Towns-Brocks syndrome are caused by a mutation in SALL1, it is possible that Spalt’s function to determine a fibrillar, stretch-modulated muscle type is conserved to vertebrates

Studying fibroblast growth factor (FGF) mediated cell migration in "Drosophila" larval air sacs

Invertebrates and vertebrates use FGF signaling in many developmental processes. Mesoderm formation, limb outgrowth but also the development of the vascular system and the lung rely on FGF ligands. We have chosen to study the Drosophila FGF signaling pathway that has been shown to be required for mesodermal- as well as tracheal cell migration. We aimed at a better understanding of FGF signaling to elucidate how the extracellular information, provided by the FGF/Bnl ligand is interpreted in tracheal cells. Using Downstream of FGFR (Dof), an adaptor protein of the FGF signaling pathway, as an entry point, we have previously identified interacting proteins and focused on one prime candidate as a potential linker of FGFR to the cytoskeleton. This candidate protein Receptor of protein kinase C (Rack1) is conserved throughout evolution. rack1 is expressed in the early embryonic tracheal system and has been proposed to play important roles in cell migration as well as in the regulation of the actin cytoskeleton. We have identified and characterized rack1 mutants; these mutants are zygotic lethal but neither show a detectable embryonic- nor any other larval phenotype, due to a very high maternal contribution. Removing the maternal store by generating germline clones results in eggs that fail to develop. This developmental arrest is due to an incomplete transfer of maternal product into the oocyte (nurse cell dumping). In order to characterize the function of rack1 in the context of FGF signaling, we started to characterize the development of third instar larval air sacs. It has been reported that this structure develops via cell migration as well as cell division in response to FGF/Bnl signaling. First we confirm the occurrence of cell division and found that in early air sacs, division is ubiquitous and becomes restricted later to the central part of the air sac. We also documented cell behavior during cell migration using live imaging. To initiate a genetic analysis of rack1 and other candidate target genes in tracheal cell migration, strains and methods were established, allowing the generation of mosaic air sacs consisting of marked wild-type or mutant cells in an otherwise heterozygous background based on the MARCM system. This system was also applied to characterize cellular shape and dynamics of individual or small groups of air sac tracheoblasts in different parts of the air sac. We found that air sac tip cells extend long and dynamic actin based protrusions and further demonstrated that cells not directly located at the tip do form similar protrusions. Finally, we took advantage of the our knowledge of air sac architecture and development to study the cell-autonomous requirement of candidate genes in genetic mosaics. We showed that marked wild-type clones have a preference to be positioned at the tip. Mutants lacking btl or dof, two genes required for embryonic tracheal cell migration, never populate regions at the migratory front. We inferred that air sac tracheoblast cells lacking btl or dof are deficient in migration and take this as a readout for measuring cell migration. Having established criteria for measuring cell migration in air sacs, we tested rack1 mutants for their involvement in air sac tracheoblast migration and find that this gene is not required for this process. We also analyzed other candidate genes as well as components of the FGF signaling pathway and found evidence that Ras plays a dual role during third instar air sac formation. It appears to integrate signaling input from the EGFR pathway to trigger cell division as well as input from the FGF pathway to activate a cell migratory response. In contrast to border cells, mutants affecting the transcription factor Slow border cells (Slbo), the VEGFR (PVR) or DE-Cadherin (Shg) do not impede air sac tracheoblast migration. Components shown to regulate the actin cytoskeleton in response to PVR signaling such as Myoblast city (Mbc) the Drosophila Dock180 homologue or the small Rho family GTPases Rac1, Rac2 and Mig-2-like (Mtl) as well as the effector Chickadee, the Drosophila homologue of Profilin, are essential for air sac tracheoblast migration. Thus, recruitment of these actin cytoskeleton regulators and effectors is mediated via different ligands/receptors in trachea and border cells. Our studies demonstrate that the development of the air sac during late larval stages is a good system to study guided cell migration and allows the genetic dissection of the FGF signaling pathway. The tools we developed allow to assay any candidate gene for which a mutant is available and also laid the foundation for the isolation and characterization of genes in a genome wide EMS screen