Automated single cell expression analysis in C. elegans

描述（由申请人提供）：存储在生物体基因组中的信息指导其发育和行为，但如何发生才刚刚开始被理解。了解每个细胞在发育的每个阶段都使用什么基因，将是迈向全面理解的重要一步。在过去的资助期内，我们在秀丽隐杆线虫中开发了一种技术，可以在胚胎细胞分裂的最后一轮以高时间分辨率自动将基因表达分配给单个细胞。该技术通过无所不在的核荧光标记来追踪胚胎共聚焦3D电影中的已知谱系，并利用谱系将第二种荧光报告基因的表达分配给特定细胞。我们已经开始将这项技术应用于系统地阐明胚胎表达的转录因子的表达模式。在即将到来的拨款期内，我们将提出技术的进步，以提高谱系测定的准确性，并将其扩展到最后一轮细胞分裂。反过来，我们建议应用这种改进的技术来完成胚胎转录因子的表达模式目录，并在时间允许的情况下将这项研究扩展到其他关键的发育基因，如受体介导的蛋白激酶。我们将继续开发工具来查看和分析数据，使我们能够推断调节基因之间的关系。结合新兴的数据集，如通过ChIP-seq研究的转录因子结合位点和这些基因的缺失突变的表型，我们期望从这些分析中开始出现强有力的预测。我们将扩展我们的技术，以方便使用表达式模式来验证这种推断关系。最后，我们将开发基于网络的展示和工具，允许访问我们的数据，并使我们的分析工具可供科学界使用。这些对秀丽隐杆线虫基因表达模式的研究不仅将提供对这些基因在发育中的作用的见解，而且将提示同源基因在人类健康和疾病中的作用。

DESCRIPTION (provided by applicant): The information stored in an organism's genome directs its development and behavior, but how that occurs is only beginning to be understood. Knowledge of what genes are used in each cell at every stage of development would be a significant step toward a comprehensive understanding. In the past grant period, we have developed technology in C. elegans that automatically assigns gene expression to individual cells at high temporal resolution up to the last round of embryonic cell division. The technology tracks the known lineage in confocal 3D movies of embryos with a ubiquitous, nuclear fluorescent label and exploits the lineage to assign expression of a second fluorescent reporter to particular cells. We have begun to apply this technology to the systematic elucidation of the expression patterns of the embryonically expressed transcription factors. In the coming grant period, we propose advances to the technology that will increase the accuracy of the lineage determination and extend it through the last round of cell division. In turn, we propose to apply this improved technology to complete the catalog of expression patterns for the embryonic transcription factors and as time permits extend this study to other key developmental genes such as receptor mediated protein kinases. We will continue to develop tools to view and analyze the data, allowing us to infer relationships between regulatory genes. Combined with emerging data sets, such as the sites of transcription factor binding through ChIP-seq studies and the phenotypes of deletion mutants for these genes, we expect that strong predictions will begin to emerge from these analyses. We will extend our technology to facilitate the use of expression patterns to validate such inferred relationships. Finally, we will develop web-based displays and tools that will permit access to our data as well as make our analytical tools available to the scientific community. These studies of gene expression patterns in C. elegans will not only provide insights into the role of these genes in development, but will suggest roles of homologous genes in human health and disease. PUBLIC HEALTH RELEVANCE: This grant proposes to create an atlas of embryonic transcription factor gene expression for the nematode C. elegans through the development and application of novel methods. Studies on C. elegans have provided insight into the function of animal genes, which in turn can lead to a greater understanding of related genes involved in human diseases. We expect the atlas we create as well as the methods we develop will speed those insights.