Cytoskeletal turnover and Myosin contractility drive cell autonomous oscillations in a model of Drosophila Dorsal Closure

Oscillatory behaviour in force-generating systems is a pervasive phenomenon in cell biology. In this work, we investigate how oscillations in the actomyosin cytoskeleton drive cell shape changes during the process of Dorsal Closure, a morphogenetic event in Drosophila embryo development whereby epidermal continuity is generated through the pulsatile apical area reduction of cells constituting the amnioserosa (AS) tissue. We present a theoretical model of AS cell dynamics by which the oscillatory behaviour arises due to a coupling between active Myosin-driven forces, actin turnover and cell deformation. Oscillations in our model are cell-autonomous and are modulated by neighbour coupling, and our model accurately reproduces the oscillatory dynamics of AS cells and their amplitude and frequency evolution. A key prediction arising from our model is that the rate of actin turnover and Myosin contractile force must increase during DC in order to reproduce the decrease in amplitude and period of cell area oscillations observed in vivo. This prediction opens up new ways to think about the molecular underpinnings of AS cell oscillations and their link to net tissue contraction and suggests the form of future experimental measurements.Comment: 17 pages, 6 figures; added references, modified and corrected Figs. 1 and 3, corrected typos, expanded discussio

From pulsatile apicomedial contractility to effective epithelial mechanics.

We review recent developments in the understanding of the biomechanics of apicomedial actomyosin and how its contractility can tense and deform tissue. Myosin pulses are driven by a biochemical oscillator but how they are modulated by the mechanical context remains unclear. On the other hand, the emergence of tissue behaviour is highly dependent on the material properties of actin, on how strongly components are connected and on the influence of neighbouring tissues. We further review the use of constitutive equations in exploring the mechanics of epithelial apices dominated by apicomedial Myosin contractility

Emergent material properties of developing epithelial tissues.

BACKGROUND: Force generation and the material properties of cells and tissues are central to morphogenesis but remain difficult to measure in vivo. Insight is often limited to the ratios of mechanical properties obtained through disruptive manipulation, and the appropriate models relating stress and strain are unknown. The Drosophila amnioserosa epithelium progressively contracts over 3 hours of dorsal closure, during which cell apices exhibit area fluctuations driven by medial myosin pulses with periods of 1.5-6 min. Linking these two timescales and understanding how pulsatile contractions drive morphogenetic movements is an urgent challenge. RESULTS: We present a novel framework to measure in a continuous manner the mechanical properties of epithelial cells in the natural context of a tissue undergoing morphogenesis. We show that the relationship between apicomedial myosin fluorescence intensity and strain during fluctuations is consistent with a linear behaviour, although with a lag. We thus used myosin fluorescence intensity as a proxy for active force generation and treated cells as natural experiments of mechanical response under cyclic loading, revealing unambiguous mechanical properties from the hysteresis loop relating stress to strain. Amnioserosa cells can be described as a contractile viscoelastic fluid. We show that their emergent mechanical behaviour can be described by a linear viscoelastic rheology at timescales relevant for tissue morphogenesis. For the first time, we establish relative changes in separate effective mechanical properties in vivo. Over the course of dorsal closure, the tissue solidifies and effective stiffness doubles as net contraction of the tissue commences. Combining our findings with those from previous laser ablation experiments, we show that both apicomedial and junctional stress also increase over time, with the relative increase in apicomedial stress approximately twice that of other obtained measures. CONCLUSIONS: Our results show that in an epithelial tissue undergoing net contraction, stiffness and stress are coupled. Dorsal closure cell apical contraction is driven by the medial region where the relative increase in stress is greater than that of stiffness. At junctions, by contrast, the relative increase in the mechanical properties is the same, so the junctional contribution to tissue deformation is constant over time. An increase in myosin activity is likely to underlie, at least in part, the change in medioapical properties and we suggest that its greater effect on stress relative to stiffness is fundamental to actomyosin systems and confers on tissues the ability to regulate contraction rates in response to changes in external mechanics.We thank the following funding bodies for their support: Herchel Smith Fund (PFM), Ministerio de Ciencia e Innovación (NG and JD, BFU2011-25828 and “Ramón y Cajal” fellowship award), Marie Curie Career Integration Grant (NG, PCIG09-GA-2011-293479), Biotechnology and Biological Sciences Research Council (GBB, grant No. BB/J010278/1) and Rhône-Alpes Complex System Institute (JE)

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a longrange gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Iglike and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.This work was supported by Grants BFU2005-04183 and BFU2008-03320/BMC and by Consolider Program CDS 2007-00008 from the Spanish MICINN, by Marie Curie RTN FP6 (RTN 035528-2) and FP7 (ITN 238186) projects, and by an institutional grant from the Fundación Areces to I.G. It was also financially supported by fellowships awarded by the Junta para la Ampliación de Estudios-Consejo Superior de Investigaciones Cientificas program (to N.G. and A.C.), a Juan de la Cierva fellowship from the Spanish MICINN (to A.B.), a Marie Curie RTN 035528-2 FP6 contract (to E.M.), a contract from the Spanish MICINN (to L.D.), and the senior researcher Programa Amarouto from Severo Ochoa Fondation program of the Comunidad Autónoma de Madrid (G.A.)Peer Reviewe

Endocytic and Recycling Endosomes Modulate Cell Shape Changes and Tissue Behaviour during Morphogenesis in Drosophila

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis

Laboratorio en abierto: aprendendiendo a copiar ADN.2

El objetivo principal del proyecto es la puesta a punto de recursos educativos en abierto (REA) dirigidos a los alumnos de secundaria. La propuesta pretende desarrollar habilidades, para la resolución de problemas científicos, a través de retos que despierten el interés y la imaginación de los alumnos de secundaria. En esta propuesta la resolución de los problemas planteados estaría basada en la aplicación de una herramienta que ha revolucionado la genética y biología, la reacción en Cadena de la Polimerasa, conocida como PCR

Integration of actomyosin contractility with cell-cell adhesion during dorsal closure

In this work, we combine genetic perturbation, time-lapse imaging and quantitative image analysis to investigate how pulsatile actomyosin contractility drives cell oscillations, apical cell contraction and tissue closure during morphogenesis of the amnioserosa, the main force-generating tissue during the dorsal closure in Drosophila. We show that Myosin activity determines the oscillatory and contractile behaviour of amnioserosa cells. Reducing Myosin activity prevents cell shape oscillations and reduces cell contractility. By contrast, increasing Myosin activity increases the amplitude of cell shape oscillations and the time cells spend in the contracted phase relative to the expanded phase during an oscillatory cycle, promoting cell contractility and tissue closure. Furthermore, we show that in AS cells, Rok controls Myosin foci formation and Mbs regulates not only Myosin phosphorylation but also adhesion dynamics through control of Moesin phosphorylation, showing that Mbs coordinates actomyosin contractility with cell-cell adhesion during amnioserosa morphogenesis.Ministerio de Ciencia e Innovación (BFU-2011-25828 and ‘Ramón y Cajal’ fellowship award) and a European Commission Marie Curie Career Integration Grant (PCIG09-GA-2011-293479). J.D. is a recipient of a PhD FPI-fellowship (BES-2012-051839) from the Ministerio de Ciencia e InnovaciónPeer Reviewe

Mechano-chemical coupling drives cell area oscillations during morphogenesis

Marie Curie Career Integration Grant (No. PCIG09-GA-2011-293479); Spanish Ministry of Science (No. BFU2011-25828)Peer Reviewe

Interactions between the amnioserosa and the epidermis revealed by the function of the u-shaped gene

Dorsal closure (DC) is an essential step during Drosophila development whereby a hole is sealed in the dorsal epidermis and serves as a model for cell sheet morphogenesis and wound healing. It involves the orchestrated interplay of transcriptional networks and dynamic regulation of cell machinery to bring about shape changes, mechanical forces, and emergent properties. Here we provide insight into the regulation of dorsal closure by describing novel autonomous and non-autonomous roles for U-shaped (Ush) in the amnioserosa, the epidermis, and in mediation of communication between the tissues. We identified Ush by gene expression microarray analysis of Dpp signaling targets and show that Ush mediates some DC functions of Dpp. By selectively restoring Ush function in either the AS or the epidermis in ush mutants, we show that the AS makes a greater (Ush-dependent) contribution to closure than the epidermis. A signal from the AS induces epidermal cell elongation and JNK activation in the DME, while cable formation requires Ush on both sides of the leading edge, i.e. in both the AS and epidermis. Our study demonstrates that the amnioserosa and epidermis communicate at several steps during the process: sometimes the epidermis instructs the amnioserosa, other times the AS instructs the epidermis, and still other times they appear to collaborate.Biotechnology and Biological Sciences Research CouncilPeer reviewe

El papel del gen 'Distal-less' en la determinación de la identidad de los apéndices ventrales de 'Drosophila melanogaster'

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-5-1998El trabajo presentado en esta tesis estudia el papel del gen Distal-less (D11) en el desarrollo de las patas, de las antenas, de las alas y dela terminalia de Drosophila melanogaster. La falta de DI1 en los apéndices ventrales (patas y antenas) produce una pérdida de la región dista1 de los mismos y un cambio de identidad en las células. La función de Dlt en la especificación de la identidad de las c6lulas de pata y antena es distinta. La expresión ectópica de DI1 en los apéndices ventrales produce una duplicación del eje P/D mediante la formación de una nueva zona de confluencia de Wingless (Wg) y Decapentaplegic (Dpp). La expresión ectópica en los apéndices dorsales produce una transformación de los mismos a apéndices ventrales. En el ala y en el halterio, se desamllan patas, y en la región de la cabeza y en el ojo, se desarrollan antenas. Este tipo de transformaciones está acompañada por una activación del DI1 endógeno y en el caso de ala, por una represión del gen determinante de ala vestigial y una activación de marcadores específicos de pata. Se propone que DI1 participa eh la especificación de los apéndices ventrales y discrimina apéndices ventrales versus dorsales en el adulto. DI1 también tiene una función más tardía en la formación del margen del ala y en el mantenimiento de la identidad de las cedas del margen posterior. ,. 7 En la terminalia, DI1 se requiere para la formación de la analia. Se expresa en el disco genital de machos y hembras y está regulado por Wg y Dpp como en los discos ventrales. Las interacciones entre los genes hedgehog, patched, wg, dpp y DI1 sugieren que el disco genital puede ser un disco de origenventi-al y apoyanla hipótesis de que las estmcturas de la terminalia se formaron por modificación de un apéndice ancestral