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mRNAs、蛋白质和细胞器引导的，由 母系种系。当胚胎从母体控制切换到合子控制时，机制被部署以清除 母体信使RNA(MRNAs)在胚胎完成其功能后从胚胎中分离出来。分子 母体信使核糖核酸清除的机制尚不完全清楚，但引起了该领域的极大兴趣。 我们最近发现一个高度保守的rna结合蛋白是母体清除mrna所必需的。 在线虫秀丽线虫中。我们定义了与该蛋白相关的mRNAs，保守的 RBFox同源物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，我们发现许多与SPN-4相关的蛋白在受精后迅速不稳定并被清除。我们 正在测试一种假设，即SPN-4通过其在体细胞分裂球中清除许多与其相关的mRNA 与CCR4-非死烯基酶复合体相关，该复合体在mRNA失稳中发挥作用。因为 CCR4-非死烯基酶复合体是生存和生殖系发育所必需的，我们开发了新的工具来 利用生长素诱导的降解系统研究其在早期胚胎发育中的作用。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)。 家长奖的这一行政补充将使我们能够升级我们现有的荧光 增加电动化平台和去卷积的高通量SMFISH定量研究显微镜 软件。由于mRNAs天生不稳定，我们的实验方案需要显微成像 在发现后一天内立即完成。机动化阶段将实现快速和自动化 评估和记录RNA清除所需的不同阶段的许多胚胎的图像采集 机制。为了使图像达到最优分辨率并对信号进行量化，需要采用反卷积算法 信使核糖核酸转录。我们希望对现有显微镜的这些升级将使我们能够定义 保守的RNA结合蛋白招募CCR4-而不是死烯基酶的分子机制 在受精后不久清除母体mRNAs以重塑早期胚胎转录组的复合体。 这些在线虫实验生物体中的实验将指导我们对早期 如果不是同源的话，受精后的发育是通过类比来进行的。