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Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster

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      Abstract

      The Johnston’s Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. The shakB 2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. However, the identity of the innexin that forms the presynaptic hemichannels in the JONs remains unknown. We used electrophysiology, immunocytochemistry and dye injection, along with presynaptically-driven RNA interference, to investigate this question. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakB(L) or ShakB(N) isoforms alone did not reduce the synaptic strength, implying that it is ShakB(N+16) that is required in the presynaptic neurons. Overexpression of ShakB(N+16) in JONs caused the formation of ectopic dye coupling, whereas ShakB(N) prevented it altogether, supporting this conclusion and also suggesting that gap junction proteins may have an instructive role in synaptic target choice.

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      A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.

      Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
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        Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways.

        The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.
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          A genome-scale shRNA resource for transgenic RNAi in Drosophila.

          Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.
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            Author and article information

            Affiliations
            [1 ]Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico, United States of America
            [2 ]School of Life Sciences, University of Sussex, Brighton, United Kingdom
            Universitaet Regensburg, GERMANY
            Author notes

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

            Conceived and designed the experiments: APP SHJ JPB JMB. Performed the experiments: APP SHJ. Analyzed the data: APP SHJ JMB. Contributed reagents/materials/analysis tools: APP JPB JMB. Wrote the paper: APP SHJ JPB JMB.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, CA USA )
            1932-6203
            4 April 2016
            2016
            : 11
            : 4
            27043822 4833477 10.1371/journal.pone.0152211 PONE-D-15-43480
            © 2016 Pézier et al

            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.

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            Figures: 9, Tables: 0, Pages: 22
            Product
            Funding
            The authors declare no competing financial interests. This work was supported by an NINDS grant SC1NS081726 to JMB. The Institute Pascal confocal microscope was funded by NSF DBI 0115825, DoD 52680-RT-ISP and NIMHD G12 MD007600 (RCMI). The Nikon confocal microscope was funded by NSF DBI 1337284. The Institute Drosophila resource center was supported by NIMHD G12 MD007600 (RCMI). Some of the antibodies and transgenic fly lines used in this study were made with the support of a grant from the BBSRC (85/S15289) to JPB. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. The authors thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks used in this study. The authors also acknowledge the Vienna Drosophila RNAi Center for the supply of fly lines. This work was made possible in part by software funded by the NIH: Fluorender: An Imaging Tool for Visualization and Analysis of Confocal Data as Applied to Zebrafish Research, R01-GM098151-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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