Integrin Signaling Regulates Spindle Orientation in Drosophila to Preserve the Follicular-Epithelium Monolayer

SummaryEpithelia act as important physiological barriers and as structural components of tissues and organs. In the Drosophila ovary, follicle cells envelop the germline cysts to form a monolayer epithelium. During division, the orientation of the mitotic spindle in follicle cells is such that both daughter cells remain within the same plane, and the simple structure of the follicular epithelium is thus preserved. Here we show that integrins, heterodimeric transmembrane receptors that connect the extracellular matrix to the cell's cytoskeleton [1, 2], are required for maintaining the ovarian monolayer epithelium in Drosophila. Mosaic egg chambers containing integrin mutant follicle cells develop stratified epithelia at both poles. This stratification is due neither to abnormal cell proliferation nor to defects in the apical-basal polarity of the mutant cells. Instead, integrin function is required for the correct orientation of the mitotic apparatus both in mutant cells and in their immediately adjacent wild-type neighbors. We further demonstrate that integrin-mediated signaling, rather than adhesion, is sufficient for maintaining the integrity of the follicular epithelium. The above data show that integrins are necessary for preserving the simple organization of a specialized epithelium and link integrin-mediated signaling to the correct orientation of the mitotic spindle in this epithelial cell type

Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assembliesResearch in our laboratories is funded by the Spanish Ministerio de Economía y Competitividad and the FEDER programme (BFU2013-48988-C2-1-P to M.D.M.-B, BFU2012-35446 to A.G.-R., BFU2011-30303 and BFU2010-18959 to D.G.M.) and by the Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058). I.G. was supported by a JAE-DOC (CSIC) and D.G.M. by a Ramon y Cajal Fellowship (Ministerio Español de Economía y Competitividad Ref. RYC-2010-07450) and a Marie Curie International Reintegration Grant (EU, Ref. 248346-NMSSBLS

Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary.

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.We thank J.C.-G. Hombría and A. Page-McCaw for fly stocks and the Developmental Studies Hybridoma Bank from the University of Iowa (USA) for antibodies. The Proteomics Facility at the CNB (CSIC; Madrid, Spain) provided technical support with the iTRAQ analysis. The TEM analysis was performed at the CIC, University of Granada. The help of J. Garrido with S5 Fig is acknowledged. This work was funded by the Spanish MINECO (Grants BFU2009-08013, BFU2012-35446 to AGR, BFU2010-16669 to MDMB and Consolider CSD-2007-00008 to MDMB and AGR), by the Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058 to MDMB and AGR) and by the European Regional Development Fund (FEDER). JRP was supported by a JAE-Doc contract from the Spanish National Research Council (CSIC) and KF by a Career Development Award from the UK Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pgen.100576

Laminin Levels Regulate Tissue Migration and Anterior-Posterior Polarity during Egg Morphogenesis in Drosophila.

Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. We study the role of laminin and its integrin receptor in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. We show that laminin content in the BM increases with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation

Mitochondria are subcellular organelles critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, as well as their coordinated translation, import and respiratory complex assembly. Here we describe the characterization of exonuclease domain like 2 (EXD2), a nuclear encoded gene that we show is targeted to the mitochondria and prevents the aberrant association of mRNAs with the mitochondrial ribosome. The loss of EXD2 resulted in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of EXD2/CG6744 in D.melanogaster caused developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that could be reversed with an anti-oxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and aging

Cytoneme-Mediated Delivery of Hedgehog Regulates the Expression of Bone Morphogenetic Proteins to Maintain Germline Stem Cells in Drosophila

Genetic manipulation of the germline stem cell niche in Drosophila ovaries reveals that support cells ensure the maintenance of stem cells by modulating the spread of Hedgehog within the niche

Stem cells, niches and cadherins: a view from Drosophila

Stem cells are essential for the correct development and homeostasis of adult organisms, as well as having obvious potential therapeutic importance. Analysis of the biology of stem cells and their regulatory microenvironment in adult organs has, however, been hindered by the rarity of these cells in mature tissues and by the lack of positive markers for them. The ovary of the Drosophila melanogaster female is a stem cell niche in which such analyses can be performed. The stromal cells of the microenvironment act as a regulatory centre to control the proliferation and differentiation of the germline stem cells, using several signalling molecules, among them the protein DPP - a Drosophila homologue of the human bone morphogenetic proteins BMP2 and BMP4. Recent work shows that DE-cadherin-mediated adhesion is used for the initial recruitment and posterior anchoring of the germline-derived stem cells in their niche.Financial support from the EMBO-Young Investigator Programme, the Spanish Plan Nacional de Biotecnología (PM99-0107) and Junta de Andalucía (CVI-280) is acknowledged.Peer reviewe

Organization of the Drosophila head as revealed by the ectopic expression of the Ultrabithorax product

By using a hsp70-Ubx fusion gene, we have ectopically expressed a Ubx product in the embryonic head primordia and studied the developmental effects on the larval head. We find that after high and persistent levels of Ubx product, the head is replaced by three (Cl, C2 and C3) abdominal-like denticle belts. The C2 and C3 belts are the homeotic transformations of parasegments 1 and 2, respectively, while the Cl belt probably derives from the transformation and subsequent fusion of the most anterior procephalic primordia. On the basis of their response to the Ubx product and other arguments, we propose that the larval head is made of two genetically distinct components; one is the procephalon and the anterior region of the mandihular lobe, and the other is part of the parasegmental trunk and includes parasegments 1 and 2. Our results also indicate that most or all the larval head structures derive from precursor cells of ventral originDireccion General de Investigation Cientifica y tecnica and by a institutional grant of the Fundacion Ramon ArecesPeer Reviewe