Systems Analysis of Conditional C. elegans Mutants with Late Embryonic Defects

具有晚期胚胎缺陷的条件性秀丽隐杆线虫突变体的系统分析

• 批准号: 9230401
• 负责人: BRUCE A BOWERMAN
• 金额: $ 32.4万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9230401
• 关键词: Affect; Animal Model; Animals; Area; Biochemical; Biological Models; Biological Process; CRISPR/Cas technology; Caenorhabditis elegans; Cell Lineage; Cell division; Cells; Chimeric Proteins; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collection; Communities; Complex; Data; Defect; Development; Developmental Biology; Embryo; Embryonic Development; Essential Genes; Exhibits; Gene Expression; Gene Expression Profile; Gene Transfer Techniques; Genes; Genetic; Image Analysis; Investigation; Laboratories; Life; Life Cycle Stages; Light; Mediating; Methods; Microscope; Mitotic; Modeling; Molecular; Molecular Genetics; Morphogenesis; Morphology; Mutation; Nematoda; Pattern; Phenotype; Positioning Attribute; Process; Proteins; RNA interference screen; Research; Research Personnel; Resolution; Resources; Shapes; Somatic Cell; Specific qualifier value; System; Systems Analysis; Systems Biology; Technology; Temperature; Thinness; Transgenic Organisms; base; blastomere structure; cell type; conditional mutant; data integration; experience; gene discovery; gene function; genome sequencing; genome-wide; improved; insight; multi-scale modeling; mutant; neglect; new technology; novel; phenotypic data; positional cloning; public health relevance; quantitative imaging; tool; whole genome

 DESCRIPTION (provided by applicant): We propose to combine powerful new technologies to investigate at a systems level an almost entirely neglected area of developmental biology research-late embryogenesis in the nematode Caenorhabditis elegans-and to greatly expand the availability of conditional mutations in essential genes for the investigation of biological processes in this model animal. We will use Illumina-based whole genome sequencing to rapidly identify the causal mutations in a collection of 250 temperature-sensitive, embryonic-lethal C. elegans mutants that progress normally through the early stages of embryogenesis but arrest late in embryogenesis. About 30 of these mutants arrest with severe defects in morphogenesis - the elongation of an oval mass of largely post-mitotic cells into a long thin worm. The remainder arrest later in development after extensive morphogenesis. While embryonic morphogenesis in C. elegans has been a subject of investigation for many years, our understanding of it remains incomplete. Our mutant collection promises important new insights into the genetic networks that regulate and mediate this complex process. In addition to identifying the causal genes, we will systematically identify the developmental defects in late embryos at cellular resolution, which has been almost entirely neglected due to technical difficulties. To characterize the developmental defects at high resolution in both classes of mutants, and assemble our data into a systems view of late embryogenesis, we will (i) apply recently developed automated embryonic cell lineage analysis as well as new methods for quantitative image analysis to identify abnormalities throughout embryogenesis, (ii) use CRISPR/Cas9 technology to generate transgenic strains bearing translational fusions of GFP to the genes we identify so as to determine when and where in embryogenesis they are expressed, and to guide our phenotype analysis, and (iii) assemble the phenotype and expression data into a multi-scale view of late embryonic development from genes to organismal morphology. We also will take advantage of the conditional mutations we have isolated to determine when in development the affected genes perform their essential embryonic functions, and to identify additional essential functions throughout the nematode life cycle. Genome-wide RNA interference screens have identified about 2500 essential genes in C. elegans, most of which are conserved in other animals. Temperature-sensitive mutations provide a uniquely powerful tool for investigating the requirements for essential genes, as many if not most of them have multiple functions throughout the life of the animal. Moreover, C. elegans is unique as an animal model in which one can feasibly isolate large numbers of relatively rare conditional mutations in essential genes, and yet conditional mutations have been identified in only about one hundred of the 2500 essential C. elegans genes. In addition to substantially advancing our understanding of morphogenesis and late embryogenesis, we also will greatly expand the availability of these powerful genetic tools to investigators throughout th world who use C. elegans as an animal model.

 描述（由申请人提供）：我们建议结合强大的新技术，在系统水平上研究发育生物学研究中几乎完全被忽视的领域——线虫秀丽隐杆线虫的晚期胚胎发生——并大大扩展条件突变的可用性我们将使用基于 Illumina 的全基因组测序来快速识别 250 个对温度敏感、胚胎致死的 C.线虫突变体在胚胎发生的早期阶段进展正常，但在胚胎发生后期停滞，其中约 30 种突变体因形态发生的严重缺陷而停滞——主要有丝分裂后细胞的椭圆形团块伸长为细长的蠕虫。虽然线虫的胚胎形态发生多年来一直是研究的主题，但我们对它的理解仍然不完整，我们的突变体收集为调控遗传网络提供了重要的新见解。除了识别因果基因之外，我们还将以某种方式以细胞分辨率识别晚期胚胎的发育缺陷，由于技术困难，这几乎完全被忽视了。突变体，并将我们的数据组装成晚期胚胎发生的系统视图，我们将（i）应用最近开发的自动化胚胎细胞谱系分析以及定量图像分析的新方法来识别整个胚胎发生过程中的异常，（ii）使用CRISPR/Cas9产生转基因菌株的技术携带 GFP 与我们识别的基因的翻译融合，以确定它们在胚胎发生中何时何地表达，并指导我们的表型分析，以及 (iii) 将表型和表达数据组装成晚期胚胎发育的多尺度视图我们还将利用我们分离出的条件突变来确定受影响的基因何时发挥其基本的胚胎功能，并确定整个线虫生命周期中的其他基本功能。已确定关于秀丽隐杆线虫中有 2500 个必需基因，其中大多数在其他动物中是保守的，为研究必需基因的需求提供了独特的强大工具，因为其中许多（如果不是大多数）在动物的整个生命周期中具有多种功能。此外，线虫作为一种独特的动物模型，可以在必需基因中分离出大量相对罕见的条件突变，但在 2500 个必需基因中，仅在大约 100 个中发现了条件突变。除了大幅增进我们对形态发生和晚期胚胎发生的理解之外，我们还将极大地扩大这些强大的遗传工具对世界各地使用线虫作为动物模型的研究人员的可用性。