Automated single-cell gene expression and phenotype analysis in C. elegans

摘要： 人们对生物体的基因组如何指导其发育和行为的了解才刚刚开始。 了解每个细胞中的基因功能将是朝着全面的 理解。我们已经开发了一种利用线虫线虫不变谱系的系统 在单细胞水平上自动定义发育过程中的基因表达和表型 高时间分辨率。该系统使用双色、3D、延时共聚焦显微镜来记录 胚胎发生，随后通过自动图像分析识别和跟踪每个细胞核 运动、分裂和死亡。到目前为止，它可以通过十轮中的前九轮追溯血统 胚胎细胞以高精度分裂，并将表达分配给单个细胞。在这里，我们建议 系统的进一步开发。我们将通过最后一轮细胞分裂来扩展系统。我们会 还通过测量各种时间和空间特征使系统适用于自动表型分析 细胞和细胞群的变化，以及基因表达的变化。此外，我们将使用这些方法来 系统地破译转录因子和其他已知RNAi表型的基因的功能 是胚胎致死的。 相关性： 完成的系统将用于广泛的功能基因组学应用，并将 为全面阐明线虫的基因功能和调控提供了途径。是这样的 对线虫的知识不仅将提供对动物发育中的基因和网络的洞察，而且 表明同源基因在人类健康和疾病中的作用。

Summary: How an organism's genome directs its development and behavior is only beginning to be understood. Knowledge of what genes function in each cell would be a significant step toward a comprehensive understanding. We have developed a system that exploits the invariant lineage of the nematode C. elegans to automatically define gene expression and phenotype throughout development at the single cell level with high temporal resolution. The system uses 2-color, 3D, time-lapse confocal microscopy to record embryogenesis, followed by automated image analysis that identifies and tracks each nucleus through movement, division and death. To date, it can trace the lineage through the first nine out of ten rounds of embryonic cell divisions with high accuracy and assign expression to individual cells. Here, we propose further development of the system. We will extend the system through the last round of cell division. We will also adapt the system for automated phenotypic analysis by measuring various temporal and spatial features of cells and cell groups, as well as changes of gene expression. Furthermore, we will use the methods to systematically decode the function of transcription factors and other genes whose RNAi phenotype is known to be embryonic lethal. Relevance: The completed system will be useful for a wide variety of functional genomics applications and would provide an avenue to comprehensive elucidation of gene function and regulation in C. elegans. Such knowledge in C. elegans would not only provide insights into genes and networks in animal development, but would suggest roles of homologous genes in human health and disease.