A dual role for the βPS integrin myospheroid in mediating Drosophila embryonic macrophage migration

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main βPS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards wounds.This work was supported by the Spanish Ministerio de Ciencia y Tecnología [grant numbers BFU2010-16669 and CSD-2007-00008 to M.D.M.-B.] and by a Wellcome Trust Senior Research Fellowship [grant number 090899/Z/09/Z to W.W.]. R.R. was supported by the DFG as project B11 of the SFB 446 ‘Zellverhalten der Eukaryoten’]; the EMBO Young Investigator Programme [a Formación de. Personal Investigador studentship from the Spanish Ministerio de Ciencia y Tecnología [to B.J.S.-S.]. and an MRC Doctoral Training Grant to K.C.Peer reviewe

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

The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in Drosophila

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.Muscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though themolecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibrils at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, which are localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominalmuscles as amodel to investigate in vivo the mechanisms behind myofibrillogenesis.Research was funded by the Spanish Ministry of Science and Innovation [grant number BFU2011-26745]. B.E. was funded in part by the Ramon y Cajal program by the Universidad Pablo de Olavide; J.J.P.-M. was funded by the Proyecto de Excelencia of Junta de Andalucía; M.B. was funded by a Wellcome Trust Senior Investigator Award to Peter Lawrence [grant number WT096645MA]. M.D.M.-B. is funded by the Consejo Superior de Investigaciones Científicas.Peer Reviewe

Integrins regulate epithelial cell shape by controlling the architecture and mechanical properties of basal actomyosin networks.

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces

Las integrinas regulan positivamente la supervivencia celular en el disco imaginal de ala en Drosophila melanogaster

Motivación: Las integrinas son una amplia familia de receptores transmembrana que se unen preferentemente a componentes de la matriz extracelular. Además de su importancia como conectores mecánicos, las integrinas también participan en la activación de diferentes cascadas de señalización y en el control de diferentes procesos celulares como adhesión, migración, proliferación, diferenciación y supervivencia celular en la cual se centra este trabajo. La disrupción de la función de interacción de las integrinas resulta en un tipo de apoptosis denominada anoikis. La anoikis es esencial no solo durante el desarrollo y el mantenimiento de la homeostasis durante la vida adulta, sino también como un importante mecanismo de supervivencia asegurando que toda célula que pierde su posición apropiada en un tejido es señalizada para sufrir apoptosis. Aunque existe abundante información sobre el papel de las integrinas como promotores de supervivencia celular, se conoce poco acerca de la significancia biológica de esta función de las integrinas y el mecanismo molecular que regula la anoikis durante el desarrollo. Por ello, nuestro objetivo principal en este proyecto es comprender los mecanismos moleculares a través de los cuales las integrinas regulan la supervivencia celular durante el desarrollo utilizando el disco imaginal de ala de Drosophila como sistema modelo.Métodos: Para alcanzar nuestro objetivo hemos reducido los niveles de integrinas en el disco de ala y en este contexto hemos analizado el posible papel de diferentes proteínas proapoptóticas en la muerte celular mediada por la falta de función de integrinas. Por otra parte, hemos estudiado el papel de la tensión de miosina en la muerte celular mediada por la falta de función de integrinas. Los fenotipos resultantes los hemos analizado a través de la tinción inmunohistoquímica de los discos imaginales previamente fijados.Resultados: Mostramos que la falta de función de integrinas en el disco de ala resulta en anoikis dependiente de caspasa debida a la activación de la ruta JNK la cual a su vez activa a la proteína proapoptótica Hid.Mostramos por otra parte que la falta de función de integrinas en el disco de ala resulta en un aumento de tensión por miosina.Conclusiones: Tras nuestros estudios hemos concluido que la falta de función de integrinas promueve la activación de la ruta JNK la cual media la muerte celular a través de la expresión de la proteína proapoptótica Hid

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

ojoplano-mediated basal constriction is essential for optic cup morphogenesis

11 páginas, 7 figuras. To the memory of Dr José-Santiago Martínez-Vinjoy. Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/13/2165/DC1Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.This work was supported by grants from the Deutsche Forschungsgemeinschaft, Collaborative Research Centre 488, the EU and HFSPO to J.W.; and MEC:BFU2008-04362/BMC to J.R.M.-M.Peer reviewe

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

PS Integrins and Laminins: Key Regulators of Cell Migration during Drosophila Embryogenesis

During embryonic development, there are numerous cases where organ or tissue formation depends upon the migration of primordial cells. In the Drosophila embryo, the visceral mesoderm (vm) acts as a substrate for the migration of several cell populations of epithelial origin, including the endoderm, the trachea and the salivary glands. These migratory processes require both integrins and laminins. The current model is that αPS1βPS (PS1) and/or αPS3βPS (PS3) integrins are required in migrating cells, whereas αPS2βPS (PS2) integrin is required in the vm, where it performs an as yet unidentified function. Here, we show that PS1 integrins are also required for the migration over the vm of cells of mesodermal origin, the caudal visceral mesoderm (CVM). These results support a model in which PS1 might have evolved to acquire the migratory function of integrins, irrespective of the origin of the tissue. This integrin function is highly specific and its specificity resides mainly in the extracellular domain. In addition, we have identified the Laminin α1,2 trimer, as the key extracellular matrix (ECM) component regulating CVM migration. Furthermore, we show that, as it is the case in vertebrates, integrins, and specifically PS2, contributes to CVM movement by participating in the correct assembly of the ECM that serves as tracks for migration