Monday, September 30, 2019

Two new in vivo Drosophila RNAi screens reported

Zhou J, Xu L, Duan X, Liu W, Zhao X, Wang X, Shang W, Fang X, Yang H, Jia L, Bai J, Zhao J, Wang L, Tong C. Large-scale RNAi screen identified Dhpr as a regulator of mitochondrial morphology and tissue homeostasis. Sci Adv. 2019 Sep 18;5(9):eaax0365. PubMed PMID: 31555733; PubMed Central PMCID: PMC6750926.

 Umer Z, Akhtar J, Khan MHF, Shaheen N, Haseeb MA, Mazhar K, Mithani A, Anwar S, Tariq M. Genome-wide RNAi screen in Drosophila reveals Enok as a novel trithorax group regulator. Epigenetics Chromatin. 2019 Sep 23;12(1):55. PubMed PMID: 31547845.

Monday, August 19, 2019

New S2 cell assay reported -- Cell density

Romine ML, Li M, Liu KJ, Patel SK, Nelson JG, Shen P, Cai HN. A Cell Density-Dependent Reporter in the Drosophila S2 Cells. Sci Rep. 2019 Aug 14;9(1):11868. doi: 10.1038/s41598-019-47652-0. PubMed PMID: 31413273.

Abstract: "Cell density regulates many aspects of cell properties and behaviors including metabolism, growth, cytoskeletal structure and locomotion. Importantly, the responses by cultured cells to density signals also uncover key mechanisms that govern animal development and diseases in vivo. Here we characterized a density-responsive reporter system in transgenic Drosophila S2 cells. We show that the reporter genes are strongly induced in a cell density-dependent and reporter-independent fashion. The rapid and reversible induction occurs at the level of mRNA accumulation. We show that multiple DNA elements within the transgene sequences, including a metal response element from the metallothionein gene, contribute to the reporter induction. The reporter induction correlates with changes in multiple cell density and growth regulatory pathways including hypoxia, apoptosis, cell cycle and cytoskeletal organization. Potential applications of such a density-responsive reporter will be discussed."

Monday, August 12, 2019

in vivo RNAi screen in the wing

Rotelli MD, Bolling AM, Killion AW, Weinberg AJ, Dixon MJ, Calvi BR. An RNAi Screen for Genes Required for Growth of Drosophila Wing Tissue. G3 (Bethesda). 2019 Aug 6. pii: g3.400581.2019. doi: 10.1534/g3.119.400581. PubMed PMID: 31387856.

Abstract: "Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development."

Tuesday, April 30, 2019

in vivo RNAi screen for metabolic enzymes required for eye development

Rose C. Pletcher, Sara L. Hardman, Sydney F. Intagliata, Rachel L. Lawson, Aumunique Page and Jason M. Tennessen

A Genetic Screen Using the Drosophila melanogaster TRiP RNAi Collection To Identify Metabolic Enzymes Required for Eye Development

G3: GENES, GENOMES, GENETICS Early online April 29, 2019; https://doi.org/10.1534/g3.119.400193

http://www.g3journal.org/content/early/2019/04/29/g3.119.400193

Abstract: "The metabolic enzymes that compose glycolysis, the citric acid cycle, and other pathways within central carbon metabolism have emerged as key regulators of animal development. These enzymes not only generate the energy and biosynthetic precursors required to support cell proliferation and differentiation, but also moonlight as regulators of transcription, translation, and signal transduction. Many of the genes associated with animal metabolism, however, have never been analyzed in a developmental context, thus highlighting how little is known about the intersection of metabolism and development. Here we address this deficiency by using the Drosophila TRiP RNAi collection to disrupt the expression of over 1,100 metabolism-associated genes within cells of the eye imaginal disc. Our screen not only confirmed previous observations that oxidative phosphorylation serves a critical role in the developing eye, but also implicated a host of other metabolic enzymes in the growth and differentiation of this organ. Notably, our analysis revealed a requirement for glutamine and glutamate metabolic processes in eye development, thereby revealing a role of these amino acids in promoting Drosophila tissue growth. Overall, our analysis highlights how the Drosophila eye can serve as a powerful tool for dissecting the relationship between development and metabolism."

RNAi screen for genes in escort cells related to germ cell maintenance and differentiation

Gao Y, Mao Y, Xu RG, Zhu R, Zhang M, Sun J, Shen D, Peng P, Xie T, Ni JQ. Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen. J Genet Genomics. 2019 Jan 20;46(1):19-30. PMID: 30745214.

Abstract: "In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled."

Optogenetic tools for Drosophila S2 cells

Osswald M, Santos AF, Morais-de-Sá E. Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells. Biomolecules. 2019 Feb 12;9(2). pii: E61. doi: 10.3390/biom9020061. PubMed PMID: 30759894; PubMed Central PMCID: PMC6406598.

Abstract: "Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells."