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Innexin 3, a New Gene Required for Dorsal Closure in Drosophila Embryo

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      Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described.

      Results and Discussion

      Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.

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      Most cited references 60

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      Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

       N Perrimon,  H. Brand (1993)
      We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
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        Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila.

        The crumbs protein of Drosophila is an integral membrane protein, with 30 EGF-like and 4 laminin A G domain-like repeats in its extracellular segment, which is expressed on the apical plasma membrane of all ectodermally derived epithelia. Here, we present evidence to show that the insertion of crumbs into the plasma membrane is necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of crumbs alone is sufficient to produce this dominant phenotype. Our results suggest that crumbs plays a key role in specifying the apical plasma membrane domain of ectodermal epithelial cells of Drosophila.
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          Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure.

          Dorsal closure is a tissue-modeling process in the developing Drosophila embryo during which an epidermal opening is closed. It begins with the appearance of a supracellular actin cable that surrounds the opening and provides a contractile force. Amnioserosa cells that fill the opening produce an additional critical force pulling on the surrounding epidermal tissue. We show that this force is not gradual but pulsed and occurs long before dorsal closure starts. Quantitative analysis, combined with laser cutting experiments and simulations, reveals that tension-based dynamics and cell coupling control the force pulses. These constitutively pull the surrounding epidermal tissue dorsally, but the displacement is initially transient. It is translated into dorsal-ward movement only with the help of the actin cable, which acts like a ratchet, counteracting ventral-ward epidermis relaxation after force pulses. Our work uncovers a sophisticated mechanism of cooperative force generation between two major forces driving morphogenesis.

            Author and article information

            [1 ]Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
            [2 ]UMC Utrecht, Utrecht, The Netherlands
            [3 ]LIMES-Institute, Program Unit Development, Genetics and Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany
            [4 ]Department of Cell Biology, UMC Utrecht, Utrecht, The Netherlands
            University of North Carolina at Chapel Hill, United States of America
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: FG GG CR. Performed the experiments: FG GG. Analyzed the data: FG GG CR. Contributed reagents/materials/analysis tools: RB. Wrote the paper: FG GG RB CR.

            Role: Editor
            PLoS One
            PLoS ONE
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            24 July 2013
            : 8
            : 7
            23894431 3722180 PONE-D-12-36772 10.1371/journal.pone.0069212

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Pages: 16
            Funding came from ZonMW TOP subsidie 912.080.24 from NWO (Netherlands wetenschappelijke Organisatie: Dutch Organisation for scientific Research) and SFB 645 from DFG (Deutsche Forschungsgemainschaft: German Research Foundation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Research Article
            Developmental Biology
            Molecular Development
            Adhesion Molecules
            Organism Development
            Molecular Genetics
            Gene Identification and Analysis
            Animal Genetics
            Gene Expression
            Gene Function
            Genetic Mutation
            Model Organisms
            Animal Models
            Drosophila Melanogaster
            Molecular Cell Biology
            Cellular Types
            Epithelial Cells
            Cell Adhesion
            Membranes and Sorting



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