The C. elegans Germline: A Test Tube for Cell and Developmental Biology

线虫种系：细胞和发育生物学的试管

• 批准号: 10893272
• 负责人: David Irwin Greenstein
• 金额: $ 6.22万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10893272
• 关键词: Administrative Supplement; Algorithms; Auxins; Award; Caenorhabditis elegans; Cellular biology; Complex; Computer software; Congenital Abnormality; Data; Defect; Detection; Development; Developmental Biology; Embryo; Embryonic Development; Ethics; Experimental Models; Fertilization; Fluorescent in Situ Hybridization; Gametogenesis; Homologous Gene; Human; Image; Infertility; Laboratory Organism; Maternal Messenger RNA; Messenger RNA; Microscope; Molecular; Motor; Nematoda; Oocytes; Organelles; Parents; Proteins; Protocols documentation; RNA; RNA-Binding Proteins; Reproduction; Resolution; Role; Signal Transduction; Spontaneous abortion; System; Technology; Testing; Transcript; Translational Regulation; Tube; embryo stage 2; experimental study; fluorescence microscope; interest; microscopic imaging; programs; recruit; single molecule; tool; transcriptome; transcriptome sequencing

SUMMARY/ABSTRACT The earliest stages of embryonic development are guided by mRNAs, proteins, and organelles provided by the maternal germline. As the embryo switches from maternal to zygotic control, mechanisms are deployed to clear maternal messenger RNAs (mRNAs) from the embryo after they have completed their functions. The molecular mechanisms of maternal mRNA clearance are incompletely understood but are of intensive interest for the field. We recently discovered that a highly conserved RNA-binding protein is required for maternal mRNA clearance in the nematode Caenorhabditis elegans. We defined the mRNAs associated with this protein, the conserved Rbfox homolog SPN-4, in oocytes using RNA-binding protein immunopurification and RNA sequencing. Remarkably, we found that many SPN-4-associated are rapidly destabilized and cleared after fertilization. We are testing the hypothesis that SPN-4 clears many of its associated mRNAs in somatic blastomeres through its association with the CCR4-NOT deadenylase complex, which functions in mRNA destabilization. Because the CCR4-NOT deadenylase complex is required for viability and germline development, we developed new tools to study its role during early embryogenesis using the auxin-inducible degradation system. A key technology needed to complete these studies is quantitative single-molecule fluorescence in situ hybridization (smFISH). This administrative supplement to the parent award would enable us to upgrade our existing fluorescence microscope for high-throughput quantitative smFISH studies by adding a motorized stage and deconvolution software. Because mRNAs are inherently unstable, our experimental protocols require the microscopic imaging to be completed immediately within a day of detection. The motorized stage will enable rapid and automated image acquisition of the many embryos of different stages required to assess and document the RNA clearance mechanisms. Deconvolution algorithms are needed to achieve optimal resolution to image and quantify single mRNA transcripts. We expect that these upgrades to our existing microscope will enable us to define the molecular mechanisms by which a conserved RNA-binding protein recruits the CCR4-NOT deadenylase complex to clear maternal mRNAs shortly after fertilization to remodel the transcriptome of the early embryo. These experiments in the C. elegans experimental organism will instruct our understanding of early postfertilization development by analogy if not homology.

摘要/摘要 胚胎发育的最早阶段是由mRNA，蛋白质和细胞器指导的 产妇种系。随着胚胎从母体切换到合子控制，部署了机制以清除 产妇Messenger RNA（mRNA）从胚胎完成功能后。分子 母体mRNA清除的机制尚未完全理解，但对该领域具有密集的兴趣。 我们最近发现，母体mRNA清除需要高度保守的RNA结合蛋白 在线虫秀丽隐杆线虫中。我们定义了与该蛋白相关的mRNA，保守的 RBFOX同源性SPN-4，使用RNA结合蛋白免疫疗法和RNA测序在卵母细胞中。 值得注意的是，我们发现受精后许多与SPN-4相关的相关性迅速不稳定和清除。我们 正在测试以下假设：SPN-4通过其通过其清除了体细胞囊体中的许多相关mRNA 与CCR4-not dearenylase复合物的关联，该复合物在mRNA不稳定中起作用。因为 CCR4-不denenylase复合物是可行性和种系开发所必需的，我们开发了新的工具 使用生长素诱导的降解系统研究其在早期胚胎发生过程中的作用。关键技术 完成这些研究所需的是定量的单分子荧光原位杂交（Smfish）。 父母奖的这种行政补充将使我们能够升级现有的荧光 通过添加电动阶段和反卷积的显微镜用于高通量定量Smfish研究 软件。由于mRNA本质上是不稳定的，因此我们的实验协议需要微观成像 在发现一天之内立即完成。电动阶段将使快速自动化 评估和记录RNA清除所需的不同阶段的许多胚胎的图像获取 机制。需要反卷积算法来实现最佳分辨率以形象和量化单个 mRNA转录本。我们希望这些升级到我们现有的显微镜将使我们能够定义 分子机制，保守的RNA结合蛋白募集CCR4-not dearenylase ynosegult 施肥后不久以清除母体mRNA，以重塑早期胚胎的转录组。 秀丽隐杆线虫实验生物中的这些实验将指导我们对早期的理解 剥夺后通过类比开发，即使不是同源性。