Acquisition of an Olympus SZX7 fluorescent stereo microscope for dissecting late-stage Drosophila embryos and selecting Drosophila embryos with GFP/RFP tagged genes

获取奥林巴斯 SZX7 荧光体视显微镜，用于解剖晚期果蝇胚胎并选择带有 GFP/RFP 标记基因的果蝇胚胎

• 批准号: 10795289
• 负责人: Lan Jiang
• 金额: $ 2.35万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795289
• 关键词: Air; Antibodies; Apical; Autophagosome; Award; Binding; Binding Proteins; Biological Assay; Biological Models; Blood Vessels; Cessation of life; Chromosomes; Co-Immunoprecipitations; Cytoplasm; Cytoplasmic Vesicles; Defect; Deposition; Disease; Dissection; Drosophila eye; Drosophila genus; Drug Metabolic Detoxication; Embryo; Endosomes; Epithelial Cells; Epithelium; Extracellular Matrix; Fluorescence; Gases; Gene Family; Genes; Genetic; Glycocalyx; Glycolysis; Goals; Golgi Apparatus; Human; Image; Immunohistochemistry; Individual; Insecta; Invertebrates; Kidney; Knowledge; Lactoylglutathione Lyase; Life; Light; Liquid substance; Lung; Lysosomes; Mediating; Metabolic; Microscope; Morphogenesis; Organ; Organism; Pathway interactions; Phenotype; Polycystic Kidney Diseases; Property; Proteins; Pyruvaldehyde; Recombinants; Research Project Grants; Role; Shapes; Structure; System; Testing; Time; Trachea; Tube; Tubular formation; United States National Institutes of Health; Vertebrates; Vesicle; Yeasts; body system; exosome; experimental study; fluorescence microscope; fly; gene function; in vivo; malformation; member; monolayer; mutant; protein biomarkers; protein transport; repaired; secretion process; spine bone structure; trafficking

Acquisition of an Olympus SZX7 fluorescent stereo microscope for dissecting late-stage Drosophila embryos and selecting Drosophila embryos with GFP/RFP tagged genes This proposal is for the acquisition of an Olympus SZX7 fluorescent stereo microscope to replace an old and broken fluorescence stereo microscope, which was used to select GFP- positive fly embryos and dissect fly embryos when ring light is provided. The awarded NIH R15 research project is to study the role of a poorly understood Osiris (Osi) gene family in regulating tube maturation in Drosophila trachea, which is a premier system to reveal fundamental mechanisms of tubular organ formation. The Drosophila trachea is a ramifying network of epithelial tubes with a monolayer of epithelial cells surrounding an apical lumen. During tube maturation, the apical secretion burst deposits large amounts of luminal matrix components to the apical lumen. Then, this lumen is cleared before air fills the lumen. The goal of the awarded project is to reveal the functions of Osi proteins as “broader coordinators” of protein trafficking during tube maturation. To test this hypothesis, we will complete the following three specific aims: Aim. 1 Determine the function of Osi genes in the trafficking of the apical luminal matrix during tube maturation. Aim. 2: Determine the function of Osi genes as coordinators to control endosome-mediated protein trafficking. Aim. 3: Identify proteins that directly bind to Osi proteins. To carry out the proposed experiments, we will analyze protein trafficking and structural changes in late-stage embryos by immunostaining. In addition, we will analyze the dynamic coordination of vesicular trafficking in vivo by observing GFP-tagged vesicle markers and RFP-tagged protein markers simultaneously in Osi mutant background. The tracheal phenotypes in Osi mutants appear in late-stage embryos (mid-stage 17). Due to cuticle formation, antibodies cannot enter embryos effectively without dissection. Therefore, we need to dissect the late-stage embryos before immunostaining. In addition, we will generate fly strains that carry GFP-tagged vesicle markers and RFP-tagged luminal protein markers in Osi mutant background. Since some marker genes are located on the same chromosome as Osi genes, we will recombine these marker genes to Osi mutant background genetically. Therefore the potential live recombinant fly embryos that carry GFP- or RFP- tagged marker genes can be selected using a stereo fluorescent microscope. Unfortunately, our old fluorescence stereo microscope that can be used to dissect late-stage fly embryos as well as to select live embryos that carry fluorescently tagged genes is broken beyond repair. Thus, this requested Olympus SZX7 fluorescent stereo microscope will allow us to carry out the proposed experiments (Aim. 1 and Aim. 2) on time. Without it, our progress on the project will be significantly delayed.

获取奥林巴斯SZX7荧光立体显微镜，用于解剖后期 果蝇胚胎并使用GFP/RFP标记的基因选择果蝇胚胎 该建议是为了获取Olympus SZX7荧光立体显微镜 更换荧光立体显微镜，用于选择GFP- 提供环光时，正蝇胚胎并解剖蝇胚。授予的NIH R15 研究项目是研究一个知识的Osiris（OSI）基因家族在调节中的作用 果蝇气管中的管成熟，这是一个揭示基本的首要系统 结节器官形成的机制。 果蝇气管是一个上皮管的刺激网络，具有上皮的单层 围绕根尖管腔的细胞。在管成熟期间，顶端分泌爆发沉积 顶端腔的大量腔基质组件。然后，该管腔被清除 在空气填充管腔之前。授予项目的目标是揭示OSI蛋白的功能 作为蛋白质运输过程中蛋白质运输的“更广泛的协调员”。为了检验这一假设，我们 将完成以下三个特定目标：目标。 1确定OSI基因在 管道成熟过程中顶端腔基质的运输。目的。 2：确定功能 OSI基因作为控制内体介导的蛋白质运输的协调员。目的。 3：识别 直接与OSI蛋白结合的蛋白质。为了进行提出的实验，我们将分析 通过免疫染色，晚期胚胎的蛋白质运输和结构变化。此外， 我们将通过观察GFP标签来分析体内囊泡贩运的动态协调 仅在OSI突变体背景下，囊泡标记和RFP标记的蛋白质标记物。 OSI突变体中的气管表型出现在晚期胚胎中（中期17）。由于 角质层形成，抗体不能在没有解剖的情况下有效地进入胚胎。因此，我们 需要在免疫染色之前剖析晚期胚胎。此外，我们将产生苍蝇 OSI中携带GFP标记的囊泡标记和RFP标记的腔蛋白标记的菌株 突变背景。由于某些标记基因位于与OSI同一染色体上 基因，我们将从遗传上将这些标记基因重组为OSI突变体背景。所以 携带GFP-或RFP标记的标记基因的潜在活蝇胚胎可能是 使用立体荧光显微镜选择。不幸的是，我们的旧荧光立体声 可用于剖析晚期蝇胚和选择活胚的显微镜 携带荧光标记的基因的基因无法修复。那要求奥林巴斯 SZX7荧光立体显微镜将使我们能够进行提出的实验（AIM。 1和目标。 2）准时。没有它，我们在项目上的进展将大大延迟。

项目成果

The novel gene apnoia regulates Drosophila tracheal tube size.

新基因 apnoia 调节果蝇气管的大小。

• DOI: 10.1002/dvdy.29
• 发表时间: 2019
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Scholl,Aaron;O'Brien,MichaelJ;Chandran,RachanaR;Jiang,Lan
• 通讯作者: Jiang,Lan

The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea.

• DOI: 10.1371/journal.pgen.1010571
• 发表时间: 2023-01
• 期刊: PLoS genetics
• 影响因子: 4.5