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.

总结/摘要 胚胎发育的最早阶段是由基因组提供的mRNA、蛋白质和细胞器指导的。 母系生殖系当胚胎从母体控制切换到合子控制时， 母体信使RNA（mRNA）在完成其功能后从胚胎中释放出来。分子 母体mRNA清除的机制还不完全清楚，但对本领域具有强烈的兴趣。 我们最近发现一个高度保守的RNA结合蛋白是母体mRNA清除所必需的 在秀丽隐杆线虫中。我们定义了与该蛋白相关的mRNA，保守的 Rbfox同系物SPN-4，在卵母细胞中使用RNA结合蛋白免疫纯化和RNA测序。 值得注意的是，我们发现许多SPN-4相关的在受精后迅速不稳定和清除。我们 他们正在测试这样一种假设，即SPN-4通过其自身的作用清除体细胞卵裂球中许多与其相关的mRNA。 与CCR 4-NOT去腺苷酶复合物相关，其在mRNA不稳定中起作用。因为 CCR 4-NOT去腺苷酶复合物是生存力和种系发育所必需的，我们开发了新的工具， 利用生长素诱导降解系统研究其在早期胚胎发生中的作用。一项关键技术 完成这些研究所需的是定量单分子荧光原位杂交（smFISH）。 这一行政补充的父奖将使我们能够升级我们现有的荧光 通过添加电动载物台和去卷积，用于高通量定量smFISH研究的显微镜 软件因为mRNA本身是不稳定的，我们的实验方案需要显微成像， 在发现后一天内立即完成。电动载物台将实现快速和自动化 对不同阶段的许多胚胎进行图像采集，以评估和记录RNA清除率 机制等需要反卷积算法来实现对图像的最佳分辨率并量化单个 mRNA转录物。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 保守的RNA结合蛋白募集CCR 4-NOT去腺苷酶的分子机制 受精后不久清除母体mRNA以重塑早期胚胎的转录组是复杂的。 这些实验在C. elegans实验生物将指导我们对早期 如果不是同源的话，受精后发育也是类似的。