Automated single cell expression analysis in C. elegans

描述(申请人提供)：存储在生物体基因组中的信息指导着它的发育和行为，但它是如何发生的才刚刚开始被理解。了解每个细胞在发育的每个阶段都使用了哪些基因，将是通向全面理解的重要一步。我们建议开发一种方法，利用线虫不变的谱系，以高时间分辨率在单细胞水平上定义整个发育过程中的基因表达模式。这将通过两个平行的报告系统来实现。我们将通过计算跟踪每个细胞核在每个细胞中表达组蛋白-GFP融合的发育胚胎的3D电影中的移动、分裂和死亡，从而自动确定每个细胞的谱系，从而确定其身份。同时，我们将检测到在候选转录调控区域控制下表达的第二颜色记者。通过将产生的时间和空间表达模式映射到胚胎谱系，我们将识别表达该报告的细胞。这些方法将需要创造新的技术和修改现有的技术，以便能够产生新的蠕虫菌株、活细胞荧光成像和图像模式识别。我们将通过多项质量控制测试来评估该方法的准确性，并开始开发一条管道，以便将该方法系统地应用于大量基因。完成的系统将用于广泛的功能基因组学应用，并进一步适应可能提供一条途径，全面分析线虫的基因表达。了解线虫的基因表达模式不仅可以深入了解这些基因在发育中的作用，还可以表明同源基因在包括人类在内的其他动物的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. We propose to develop methods that will exploit the invariant lineage of the nematode C. elegans to define gene expression patterns throughout development at the single cell level with high temporal resolution. This will be achieved with two parallel reporter systems. We will computationally track each nucleus through movement, division and death in 3D movies of developing embryos expressing histone-GFP fusions in each cell, and thus automatically determine the lineage for each cell and hence its identity. Simultaneously, we will detect a second-color reporter expressed under the control of a candidate transcriptional regulatory region. By mapping the resulting temporal and spatial expression patterns onto the embryonic lineage, we will identify the cells expressing the reporter. These methods will require the creation of new techniques and the modification of existing one to enable the generation of new worm strains, live-cell fluorescence imaging and pattern recognition in images. We will assess the accuracy of the method through multiple quality control tests and begin to develop a pipeline to allow systematic application of the methods to large numbers of genes. The completed system will be useful for a wide variety of functional genomics applications and with further adaptation could provide an avenue to comprehensive analysis of gene expression in C. elegans. Knowledge of gene expression patterns in C. elegans would not only provide insights into the role of these genes in development, but would suggest roles of homologous genes in health and disease in other animals, including humans.