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

 描述(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-线虫秀丽线虫的晚期胚胎发生-并极大地扩大必要基因的条件突变的可用性，以研究这种模式动物的生物过程。我们将使用基于Illumina的全基因组测序来快速确定250个温度敏感的、胚胎致命的线虫突变体中的因果突变，这些突变体通常在胚胎发生的早期阶段进行，但在胚胎发生的后期停止。这些突变体中约有30个因形态发生严重缺陷而停止生长--即主要由有丝分裂后细胞组成的椭圆形团块延伸成又长又细的虫子。其余的在广泛的形态发生后在发育后期停滞。虽然线虫的胚胎形态发生多年来一直是一个研究的主题，但我们对它的了解仍然不完整。我们的突变体库有望为调节和调节这一复杂过程的遗传网络提供重要的新见解。除了鉴定致病基因外，我们还将在细胞分辨率上系统地鉴定晚期胚胎的发育缺陷，由于技术上的困难，这些缺陷几乎完全被忽视了。为了高分辨率地描述这两类突变体的发育缺陷，并将我们的数据整合到后期胚胎发生的系统视图中，我们将(I)应用最近开发的自动胚胎细胞谱系分析以及定量图像分析的新方法来确定整个胚胎发育过程中的异常，(Ii)使用CRISPR/Cas9技术来产生携带GFP与我们鉴定的基因的翻译融合的转基因菌株，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从基因到器官形态的晚期胚胎发育的多尺度视图。我们还将利用我们分离的条件突变来确定受影响的基因在发育过程中何时履行其基本的胚胎功能，并在线虫的整个生命周期中确定其他必要的功能。全基因组RNA干扰筛选已在线虫中鉴定出约2500个必需基因，其中大部分在其他动物中保守。温度敏感突变为研究必需基因的需求提供了一个独特而强大的工具，因为它们中的许多，如果不是大多数，在动物的整个生命过程中都具有多种功能。此外，线虫作为一种动物模型是独一无二的，在这种模型中，人们可以可行地分离出大量相对罕见的基本基因条件突变，但在2500个基本线虫基因中，只有大约100个被确定为条件突变。除了大大提高我们对形态发生和晚期胚胎发生的理解外，我们还将极大地扩大这些强大的遗传工具的可获得性，供世界各地使用线虫作为动物模型的研究人员使用。

 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.