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      Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing

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          Abstract

          RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked “shadow RNAi” clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc.

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          Most cited references40

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          Evidence that stem cells reside in the adult Drosophila midgut epithelium.

          Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. Here, we characterize a proliferating cellular compartment in the adult Drosophila midgut. Using genetic mosaic analysis we demonstrate that differentiated cells in the epithelium arise from a common lineage. Furthermore, we show that reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract.
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            Analysis of genetic mosaics in developing and adult Drosophila tissues.

            T Xu, G Rubin (1993)
            We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cell-autonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.
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              Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.

              Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.
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                Author and article information

                Journal
                Genetics
                Genetics
                genetics
                genetics
                genetics
                Genetics
                Genetics Society of America
                0016-6731
                1943-2631
                May 2016
                15 March 2016
                15 March 2016
                : 203
                : 1
                : 109-118
                Affiliations
                [1]Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
                Author notes
                [1 ]Corresponding author: University of California, 361 LSA, Berkeley, CA 94720. E-mail: ikh@ 123456berkeley.edu
                Article
                187062
                10.1534/genetics.116.187062
                4858766
                26984059
                b7a824e9-baea-48e6-80c9-ea072c2700c6
                Copyright © 2016 by the Genetics Society of America

                Available freely online through the author-supported open access option.

                History
                : 14 January 2016
                : 09 March 2016
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 54, Pages: 10
                Categories
                Investigations
                Methods, Technology, and Resources

                Genetics
                rnai,shrna,gal4/uas,lineage tracing,compartment boundary
                Genetics
                rnai, shrna, gal4/uas, lineage tracing, compartment boundary

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