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

基本信息

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
项目类别：
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

项目摘要

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、蛋白质和细胞器提供的指导的。母系种系。当胚胎从母体控制切换到合子控制时，就会部署一些机制来清除完成其功能后，来自胚胎的母体信使 RNA (mRNA)。分子母体 mRNA 清除机制尚不完全清楚，但引起了该领域的广泛关注。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质相关的 mRNA，即保守的 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 SPN-4 相关蛋白在受精后迅速不稳定并被清除。我们正在验证 SPN-4 通过其清除体细胞卵裂球中许多相关 mRNA 的假设与 CCR4-NOT 去腺苷酶复合体相关，该复合体在 mRNA 不稳定中发挥作用。因为 CCR4-NOT 去腺苷酶复合体是活力和种系发育所必需的，我们开发了新工具使用生长素诱导降解系统研究其在早期胚胎发生过程中的作用。一项关键技术完成这些研究需要定量单分子荧光原位杂交（smFISH）。这项对家长奖的行政补充将使我们能够升级现有的荧光通过添加电动载物台和反卷积进行高通量定量 smFISH 研究的显微镜软件。由于 mRNA 本质上不稳定，我们的实验方案需要显微成像检测后一天内立即完成。电动平台将实现快速和自动化采集评估和记录 RNA 清除所需的不同阶段的许多胚胎的图像机制。需要反卷积算法来实现图像的最佳分辨率并量化单个 mRNA 转录本。我们期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-NOT去腺苷酸酶的分子机制受精后不久清除母体 mRNA 的复合体，以重塑早期胚胎的转录组。这些在秀丽隐杆线虫实验生物体中的实验将指导我们了解早期受精后发育即使不是同源，也可以类比。