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Drosophila embryo

Antoine Ducuing, Stéphane Vincent
The actin cable is a supracellular structure that embryonic epithelia produce to close gaps. However, the action of the cable remains debated. Here, we address the function of the cable using Drosophila dorsal closure, a paradigm to understand wound healing. First, we show that the actin cytoskeleton protein Zasp52 is specifically required for actin cable formation. Next, we used Zasp52 loss of function to dissect the mechanism of action of the cable. Surprisingly, closure dynamics are perfect in Zasp52 mutants: the cable is therefore dispensable for closure, even in the absence of the amnioserosa...
October 17, 2016: Nature Cell Biology
Yad Ghavi-Helm, Bingqing Zhao, Eileen E M Furlong
Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is an invaluable technique to assess transcription factor binding and histone modifications in a genome-wide manner, an essential step towards understanding the mechanisms that govern embryonic development. Here, we provide a detailed protocol for all steps involved in generating a ChIP-seq library, starting from embryo collection, fixation, chromatin preparation, immunoprecipitation, and finally library preparation. The protocol is optimized for Drosophila embryos, but can be easily adapted for any model organism...
2016: Methods in Molecular Biology
Pruthvi C Shivakumar, Pierre-François Lenne
Laser ablation is nowadays a widespread technique to probe tissue mechanics during development. Here we describe the setup of one such ablation system and ablation experiments performed on the embryo and pupa of Drosophila. We describe in detail the process of sample preparation, how to disrupt single-cell junctions and perform linear or circular cuts at the tissue scale, and how to analyze the data to determine relevant mechanical parameters.
2016: Methods in Molecular Biology
Christopher Schmied, Pavel Tomancak
Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva...
2016: Methods in Molecular Biology
Emmanuel Caussinus, Markus Affolter
Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]...
2016: Methods in Molecular Biology
Anne Pélissier-Monier, Bénédicte Sanson, Bruno Monier
Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis...
2016: Methods in Molecular Biology
Felix Muerdter, Alexander Stark
A recent study visualizes nascent RNAs in live Drosophila embryos to establish a connection between enhancer strength and the frequency of transcriptional bursts. Interestingly, one enhancer can simultaneously activate two core promoters, challenging models of enhancer-core-promoter communication via direct protein-protein contacts.
October 10, 2016: Current Biology: CB
Boyin Liu, Torsten Bossing
We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching...
October 11, 2016: Scientific Reports
Amanda Field, Jie Xiang, W Ray Anderson, Patricia Graham, Leslie Pick
The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success...
2016: PloS One
Barbara Horváth, Andrea J Betancourt, Alex T Kalinka
BACKGROUND: Embryogenesis is a highly conserved, canalized process, and variation in the duration of embryogenesis (DOE), i.e., time from egg lay to hatching, has a potentially profound effect on the outcome of within- and between-species competition. There is both intra- and inter-specific variation in this trait, which may provide important fuel for evolutionary processes, particularly adaptation. However, while genetic variation underlying simpler morphological traits, or with large phenotypic effects is well described in the literature, less is known about the underlying genetics of traits, such as DOE, partly due to a lack of tools with which to study them...
October 7, 2016: BMC Evolutionary Biology
Delia Ricolo, Myrto Deligiannaki, Jordi Casanova, Sofia J Araújo
Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen...
September 17, 2016: Current Biology: CB
Fernanda Hernandes Figueira, Lais Mattos de Aguiar, Carlos Eduardo da Rosa
The herbicide atrazine has been used worldwide with subsequent residual contamination of water and food, which may cause adverse effects on non-target organisms. Animal exposure to this herbicide may affect development, reproduction and energy metabolism. Here, the effects of atrazine regarding survival and redox metabolism were assessed in the fruit fly D. melanogaster exposed during embryonic and larval development. The embryos (newly fertilized eggs) were exposed to different atrazine concentrations (10μM and 100μM) in the diet until the adult fly emerged...
September 27, 2016: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
Na Xu, Xingwu Lu, Harsh Kavi, Alexander V Emelyanov, Travis J Bernardo, Elena Vershilova, Arthur I Skoultchi, Dmitry V Fyodorov
Metazoan linker histones are essential for development and play crucial roles in organization of chromatin, modification of epigenetic states and regulation of genetic activity. Vertebrates express multiple linker histone H1 isoforms, which may function redundantly. In contrast, H1 isoforms are not present in Dipterans, including D. melanogaster, except for an embryo-specific, distantly related dBigH1. Here we show that Drosophila BEN domain protein Elba2, which is expressed in early embryos and was hypothesized to have insulator-specific functions, can compensate for the loss of H1 in vivo...
September 30, 2016: Scientific Reports
Peiwen Liu, Yunqiao Dong, Jinbao Gu, Santhosh Puthiyakunnon, Yang Wu, Xiao-Guang Chen
BACKGROUND: In eukaryotic organisms, Piwi-interacting RNAs (piRNAs) control the activities of mobile genetic elements and ensure genome maintenance. Recent evidence indicates that piRNAs are involved in multiple biological pathways, including transcriptional regulation of protein-coding genes, sex determination and even interactions between host and pathogens. Aedes albopictus is a major invasive species that transmits a number of viral diseases in humans. Ae. albopictus has the largest genome and the highest abundance of repetitive sequences when compared with members that belong to Culicidae with a published genome...
September 29, 2016: Parasites & Vectors
Thierry Cheutin, Giacomo Cavalli
During the last two decades, observation of cell nuclei in live microscopy evidences motion of nuclear compartments. Drosophila embryos constitute a good model to study nuclear dynamic during cell differentiation because they can easily be observed in live microscopy. Inside the cell nucleus, Polycomb group proteins accumulate in foci named Pc bodies. Here, we describe a method to visualize and analyze the motion of these nuclear compartments inside cell nuclei of Drosophila embryos.
2016: Methods in Molecular Biology
Eva Löser, Daniel Latreille, Nicola Iovino
This protocol provides specific details on how to perform Chromatin immunoprecipitation (ChIP) from Drosophila embryos. ChIP allows the matching of proteins or histone modifications to specific genomic regions. Formaldehyde-cross-linked chromatin is isolated and antibodies against the target of interest are used to determine whether the target is associated with a specific DNA sequence. This can be performed in spatial and temporal manner and it can provide information about the genome-wide localization of a given protein or histone modification if coupled with deep sequencing technology (ChIP-Seq)...
2016: Methods in Molecular Biology
Toshiyuki Harumoto, Hisashi Anbutsu, Bruno Lemaitre, Takema Fukatsu
Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. Here, we uncover the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos...
September 21, 2016: Nature Communications
Vincent Loubiere, Anna Delest, Aubin Thomas, Boyan Bonev, Bernd Schuettengruber, Satish Sati, Anne-Marie Martinez, Giacomo Cavalli
Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, we identify a large set of genes that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity...
September 19, 2016: Nature Genetics
Monn Monn Myat, Unisha Patel
Lumen formation and maintenance are important for the development and function of essential organs such as the lung, kidney and vasculature. In the Drosophila embryonic trachea, lumena form de novo to connect the different tracheal branches into an interconnected network of tubes. Here, we identify a novel role for the receptor type guanylyl cyclase at 76C (Gyc76C) in de novo lumen formation in the Drosophila trachea. We show that in embryos mutant for gyc76C or its downsteam effector protein kinase G (PKG) 1, tracheal lumena are disconnected...
2016: PloS One
Chun-Chieh Lin, Christopher J Potter
The CRISPR/Cas9 system has revolutionized genomic editing. The Cas9 endonuclease targets genomic DNA via an experimentally determined guide RNA (gRNA). This results in a double-strand break at the target site. We generated transgenic Drosophila melanogaster in which the CRISPR/Cas9 system was used to target a GAL4 transgene in vivo To our surprise, progeny whose genomes did not contain CRISPR/Cas9 components were still capable of mutating GAL4 sequences. We demonstrate this effect was caused by maternal deposition of Cas9 and gRNAs into the embryo, leading to extensive GAL4 mutations in both somatic and germline tissues...
September 16, 2016: G3: Genes—Genomes—Genetics
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