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      Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis

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          Abstract

          The orientation of cell division along the interphase cell long-axis, the century old Hertwig’s rule, has profound roles in tissue proliferation, morphogenesis, architecture and mechanics 1, 2. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways 312. At mitosis, epithelial cells usually round up to ensure faithful chromosome segregation and to promote morphogenesis 1. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. We found that in Drosophila epithelia, tricellular junctions (TCJ) localize microtubule force generators, orienting cell division via the Dynein associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJ emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.

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          Most cited references48

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          Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

          We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
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            Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

            We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.
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              P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.

              We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                20 March 2017
                17 February 2016
                25 February 2016
                31 May 2017
                : 530
                : 7591
                : 495-498
                Affiliations
                [1 ]Polarity, Division and Morphogenesis Team, Institut Curie, CNRS UMR 3215, INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France
                [2 ]Institut Jacques Monod, CNRS UMR7592 15 rue Hélène Brion, 75205 Paris cedex 13, France
                [3 ]Department of Physics, University of Michigan, Ann Arbor, MI 48109-1040, USA
                Author notes
                Correspondence and requests for material should be addressed to F.B. ( floris.bosveld@ 123456curie.fr ) and Y.B. ( yohanns.bellaiche@ 123456curie.fr )
                [#]

                Present address: Institut Curie, CNRS UMR 3348, Université Paris Sud, Bâtiment 110, 91405 Orsay, France.

                [*]

                Present address: INRA, Institut Jean-Pierre Bourgin, UMR 1318, ERL CNRS 3559, Saclay Plant Sciences, RD10, Versailles, France.

                [**]

                Present address: The Francis Crick Institute, Lincoln's Inn Fields Laboratory, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom

                Article
                EMS66649
                10.1038/nature16970
                5450930
                26886796
                caf7cc46-15da-4faa-b998-520a64849756

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