Fly最新文献

{"title":"A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in <i>Drosophila</i>.","authors":"Kevin G Nyberg, Joseph Q Nguyen, Yong-Jae Kwon, Shelby Blythe, Greg J Beitel, Richard Carthew","doi":"10.1080/19336934.2020.1832416","DOIUrl":"10.1080/19336934.2020.1832416","url":null,"abstract":"<p><p>Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of <i>Drosophila melanogaster</i> as well as to other <i>Drosophila</i> species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against <i>eyes absent</i>. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":"14 1-4","pages":"34-48"},"PeriodicalIF":1.2,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2020.1832416","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9278192","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Short is still sweet.","authors":"Howy Jacobs","doi":"10.1080/19336934.2020.1831879","DOIUrl":"https://doi.org/10.1080/19336934.2020.1831879","url":null,"abstract":"Shortly after taking over as Chief Editor of my former journal, EMBO Reports, I penned an editorial outlining the kind of research articles we were seeking as submissions [1]. The message was straightforward: we were looking for manuscripts reporting single key findings of note, which were backed up by multiple lines of evidence. Although EMBO Reports embodied the idea of short-format papers in its very name, I’m trying to bring the same philosophy to Fly. A common fault of many submitted ‘in-brief’ papers is that they present the results of just one experiment, often accompanied by exorbitant claims as to their significance. Reviewers, unconvinced that what was presented is a substantial advance, or even doubting its veracity entirely, then propose other experiments that they themselves dreamed up, which go off on a tangent, even if it’s an interesting tangent. The end result is that the paper becomes an unwieldy composite of two, three, or four disparate experiments, none of them backed up by independent evidence. When authors carry out an experiment demanded by a reviewer, which produces the anticipated result (‘the authors must demonstrate that ....’), the satisfied reviewer is often inclined to disregard the fact that the finding is of small magnitude, statistically questionable, or lacks independent repeats which the journal’s revision timetable made impossible. The published paper therefore ends up full of holes, is fundamentally unreliable, meanders all over the place and is rarely cited because nobody knows what it is really trying to say, even if the data are believable. At the opposite extreme, junior investigators often try to bend the parameters of a manuscript so that it includes every possibly relevant experiment they have ever done. The submitted draft thus resembles a ramble built from scraps demanded by reviewers, but from a much earlier stage. If the reviewers are awake, they will often seek removal of extraneous materials until, finally, little of substance is left, and it risks actually becoming the one-experiment paper that lacks evidentiary rigour; unless the editor is also awake. To me, the perfect short-format paper should have a laser focus on one simply formulated scientific question, that is then tested by different experimental approaches. Beyond that I would not seek to be prescriptive. The various streams of verification could be, for example, a bioinformatic prediction of an interaction, plus a biophysical test in vitro, plus a set of pulldown assays from a cell-line grown under different conditions and, finally, a phenotypic analysis of an in vivo model. Or any of hundreds of other possible combinations. The experiments should not simply be linked by a keyword, but should constitute parallel or sequential experimental tests of the specific hypothesis, where the outcome could be either ‘positive’ or ‘negative’, but should be coherent and statistically sound. Note that negative findings can provide at least as much ","PeriodicalId":12128,"journal":{"name":"Fly","volume":" ","pages":"1-2"},"PeriodicalIF":1.2,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2020.1831879","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38506343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SILAC-based quantitative proteomic analysis of <i>Drosophila</i> gastrula stage embryos mutant for fibroblast growth factor signalling.","authors":"Hamze Beati, Alistair Langlands, Sara Ten Have, H-Arno J Müller","doi":"10.1080/19336934.2019.1705118","DOIUrl":"10.1080/19336934.2019.1705118","url":null,"abstract":"<p><p>Quantitative proteomic analyses in combination with genetics provide powerful tools in developmental cell signalling research. <i>Drosophila melanogaster</i> is one of the most widely used genetic models for studying development and disease. Here we combined quantitative proteomics with genetic selection to determine changes in the proteome upon depletion of Heartless (Htl) Fibroblast-Growth Factor (FGF) receptor signalling in <i>Drosophila</i> embryos at the gastrula stage. We present a robust, single generation SILAC (stable isotope labelling with amino acids in cell culture) protocol for labelling proteins in early embryos. For the selection of homozygously mutant embryos at the pre-gastrula stage, we developed an independent genetic marker. Our analyses detected quantitative changes in the global proteome of <i>htl</i> mutant embryos during gastrulation. We identified distinct classes of downregulated and upregulated proteins, and network analyses indicate functionally related groups of proteins in each class. In addition, we identified changes in the abundance of phosphopeptides. In summary, our quantitative proteomic analysis reveals global changes in metabolic, nucleoplasmic, cytoskeletal and transport proteins in <i>htl</i> mutant embryos.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":" ","pages":"10-28"},"PeriodicalIF":2.4,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746239/pdf/KFLY_14_1705118.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37487946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A method for the permeabilization of live <i>Drosophila melanogaster</i> larvae to small molecules and cryoprotectants.","authors":"Alex Murray,&nbsp;Daniel Palmer,&nbsp;Daimark Bennett,&nbsp;Venkata Dwarampudi,&nbsp;João Pedro de Magalhães","doi":"10.1080/19336934.2020.1724051","DOIUrl":"https://doi.org/10.1080/19336934.2020.1724051","url":null,"abstract":"<p><p>The larvae of <i>Drosophila melanogaster</i> is a model organism widely used to study the muscular and nervous systems. <i>Drosophila</i> larvae are surrounded by a waxy cuticle that prevents permeation by most substances. Here we develop a method to remove this layer, rendering the larvae permeable to small molecules without causing death, allowing the larvae to develop to adulthood and reproduce. Permeability was assessed using fluorescein diacetate dye uptake, and mortality upon exposure to toxic levels of ethylene glycol (EG) and Dimethyl sulfoxide (DMSO). Potential uses for this method include drug delivery, toxicity assays, cryopreservation, staining, and fixation.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":" ","pages":"29-33"},"PeriodicalIF":1.2,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2020.1724051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37626666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mitochondrial dysfunction generates a growth-restraining signal linked to pyruvate in Drosophila larvae","authors":"Jack George, Tea Tuomela, E. Kemppainen, A. Nurminen, Samuel T. Braun, Cagri Yalgin, H. Jacobs","doi":"10.1080/19336934.2019.1662266","DOIUrl":"https://doi.org/10.1080/19336934.2019.1662266","url":null,"abstract":"ABSTRACT The Drosophila bang-sensitive mutant tko25t, manifesting a global deficiency in oxidative phosphorylation due to a mitochondrial protein synthesis defect, exhibits a pronounced delay in larval development. We previously identified a number of metabolic abnormalities in tko25t larvae, including elevated pyruvate and lactate, and found the larval gut to be a crucial tissue for the regulation of larval growth in the mutant. Here we established that expression of wild-type tko in any of several other tissues of tko25t also partially alleviates developmental delay. The effects appeared to be additive, whilst knockdown of tko in a variety of specific tissues phenocopied tko25t, producing developmental delay and bang-sensitivity. These findings imply the existence of a systemic signal regulating growth in response to mitochondrial dysfunction. Drugs and RNAi-targeted on pyruvate metabolism interacted with tko25t in ways that implicated pyruvate or one of its metabolic derivatives in playing a central role in generating such a signal. RNA-seq revealed that dietary pyruvate-induced changes in transcript representation were mostly non-coherent with those produced by tko25t or high-sugar, consistent with the idea that growth regulation operates primarily at the translational and/or metabolic level.","PeriodicalId":12128,"journal":{"name":"Fly","volume":"13 1","pages":"12 - 28"},"PeriodicalIF":1.2,"publicationDate":"2019-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2019.1662266","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48725842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A standardized nomenclature and atlas of the male terminalia of Drosophila melanogaster","authors":"Gavin R. Rice, J. David, Y. Kamimura, John P. Masly, A. McGregor, Olga Nagy, S. Noselli, M. D. Nunes, P. O'Grady, E. Sánchez-Herrero, M. Siegal, M. Toda, Mark Rebeiz, V. Courtier-Orgogozo, A. Yassin","doi":"10.1080/19336934.2019.1653733","DOIUrl":"https://doi.org/10.1080/19336934.2019.1653733","url":null,"abstract":"ABSTRACT Animal terminalia represent some of the most diverse and rapidly evolving structures in the animal kingdom, and for this reason have been a mainstay in the taxonomic description of species. The terminalia of Drosophila melanogaster, with its wide range of experimental tools, have recently become the focus of increased interest in the fields of development, evolution, and behavior. However, studies from different disciplines have often used discrepant terminologies for the same anatomical structures. Consequently, the terminology of genital parts has become a barrier to integrating results from different fields, rendering it difficult to determine what parts are being referenced. We formed a consortium of researchers studying the genitalia of D. melanogaster to help establish a set of naming conventions. Here, we present a detailed visual anatomy of male genital parts, including a list of synonymous terms, and suggest practices to avoid confusion when referring to anatomical parts in future studies. The goal of this effort is to facilitate interdisciplinary communication and help newcomers orient themselves within the exciting field of Drosophila genitalia.","PeriodicalId":12128,"journal":{"name":"Fly","volume":"13 1","pages":"51 - 64"},"PeriodicalIF":1.2,"publicationDate":"2019-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2019.1653733","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48298400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A modular toolset of phiC31-based fluorescent protein tagging vectors for <i>Drosophila</i>.","authors":"Jun Luo,&nbsp;Pingping Shen,&nbsp;Jiong Chen","doi":"10.1080/19336934.2019.1595999","DOIUrl":"https://doi.org/10.1080/19336934.2019.1595999","url":null,"abstract":"<p><p>The <i>Drosophila</i> transgenic technology and fluorescent protein fusions are powerful tools to analyze protein expression patterns, subcellular localization and protein dynamics. Recently, the <i>Drosophila</i> transgenic technology has been improved by the highly efficient phiC31 site-specific integration system. Many new and improved fluorescent proteins with desirable advantages have been developed. However, the phiC31 system and the newly developed fluorescent proteins have not been systematically applied in <i>Drosophila</i> transgenic vectors. Here, we have constructed a modular toolset of C-terminal fluorescent protein fusion vectors based on phiC31 site-specific integration system for the generation of transgenic <i>Drosophila</i> lines. These cloning vectors contain a variety of fluorescent tags, including blue, cyan, green or red fluorescent proteins, photoactivatable or photoswitchable fluorescent proteins, fluorescent timers, photosensitizers and bimolecular fluorescence complementation tags. These vectors provide a range of transcriptional regulation options including UAST, UASP, UASC, LexAop, QUAS, Ubi, αTub67C and αTub84B promoters, and two screening marker options including <i>white</i> and <i>vermilion</i> gene. The vectors have been tested <i>in vivo</i> and can produce fluorescent chimeric proteins that are functional.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":"13 1-4","pages":"29-41"},"PeriodicalIF":1.2,"publicationDate":"2019-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2019.1595999","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37067893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bivalency in <i>Drosophila</i> embryos is associated with strong inducibility of Polycomb target genes.","authors":"Arslan Akmammedov,&nbsp;Marco Geigges,&nbsp;Renato Paro","doi":"10.1080/19336934.2019.1619438","DOIUrl":"https://doi.org/10.1080/19336934.2019.1619438","url":null,"abstract":"<p><p>Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation is bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. Here, we provide evidence for the existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, we observed a strong reporter inducibility in the embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in the embryo. Using re-chromatin immunoprecipitation experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":"13 1-4","pages":"42-50"},"PeriodicalIF":1.2,"publicationDate":"2019-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2019.1619438","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37245835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extending julius seizure, a bang-sensitive gene, as a model for studying epileptogenesis: Cold shock, and a new insertional mutation.","authors":"Derek Dean,&nbsp;Hannah Weinstein,&nbsp;Seema Amin,&nbsp;Breelyn Karno,&nbsp;Emma McAvoy,&nbsp;Ronald Hoy,&nbsp;Andrew Recknagel,&nbsp;Casey Jarvis,&nbsp;David Deitcher","doi":"10.1080/19336934.2017.1402993","DOIUrl":"https://doi.org/10.1080/19336934.2017.1402993","url":null,"abstract":"<p><p>The bang-sensitive (BS) mutants of Drosophila are an important model for studying epilepsy. We recently identified a novel BS locus, julius seizure (jus), encoding a protein containing two transmembrane domains and an extracellular cysteine-rich loop. We also determined that jus^{sda iso7.8}, a previously identified BS mutation, is an allele of jus by recombination, deficiency mapping, complementation testing, and genetic rescue. RNAi knockdown revealed that jus expression is important in cholinergic neurons and that the critical stage of jus expression is the mid-pupa. Finally, we found that a functional, GFP-tagged genomic construct of jus is expressed mostly in axons of the neck connectives and of the thoracic abdominal ganglia. In this Extra View article, we show that a MiMiC GFP-tagged Jus is localized to the same nervous system regions as the GFP-tagged genomic construct, but its expression is mostly confined to cell bodies and it causes bang-sensitivity. The MiMiC GFP-tag lies in the extracellular loop while the genomic construct is tagged at the C-terminus. This suggests that the alternate position of the GFP tag may disrupt Jus protein function by altering its subcellular localization and/or stability. We also show that a small subset of jus-expressing neurons are responsible for the BS phenotype. Finally, extending the utility of the BS seizure model, we show that jus mutants exhibit cold-sensitive paralysis and are partially sensitive to strobe-induced seizures.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":"12 1","pages":"55-61"},"PeriodicalIF":1.2,"publicationDate":"2018-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/19336934.2017.1402993","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35243197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Keeping it tight: The relationship between bacterial dysbiosis, septate junctions, and the intestinal barrier in Drosophila.","authors":"Martin Resnik-Docampo, Vivien Sauer, Joseph M Schinaman, Rebecca I Clark, David W Walker, D Leanne Jones","doi":"10.1080/19336934.2018.1441651","DOIUrl":"10.1080/19336934.2018.1441651","url":null,"abstract":"<p><p>Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability^1,2. However, the causal relationships between these phenotypes are only beginning to be unravelled. We recently characterized the age-related changes that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ)³. Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed us to begin to explore the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.</p>","PeriodicalId":12128,"journal":{"name":"Fly","volume":"12 1","pages":"34-40"},"PeriodicalIF":1.2,"publicationDate":"2018-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927685/pdf/kfly-12-01-1441651.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35841932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}