Automated single cell expression analysis in C. elegans

储存在生物体基因组中的信息指导着它的发育和行为，但它是如何发生的呢？ 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哪些基因。 发展将是迈向全面理解的重要一步。我们建议发展 方法，将利用不变的谱系线虫C。来定义基因表达模式 在单细胞水平上以高时间分辨率贯穿整个发育过程。这将通过两个 平行报告系统。我们将通过计算追踪每个细胞核的运动、分裂和死亡 在每个细胞中表达组蛋白-GFP融合的发育胚胎的3D电影中， 确定每个细胞的谱系，从而确定其身份。同时，我们将检测到第二种颜色 在候选转录调控区的控制下表达的报告基因。通过映射 由此产生的时间和空间表达模式到胚胎谱系，我们将确定细胞 记者表示。这些方法将需要创造新的技术和修改 现有的一个，使新的蠕虫菌株，活细胞荧光成像和模式的产生 图像识别。我们将通过多次质量控制测试评估该方法的准确性， 开始开发一个管道，以允许系统地将这些方法应用于大量基因。的 完成的系统将可用于各种各样的功能基因组学应用， 适应可以为全面分析C.优雅 对C.秀丽线虫不仅能让我们深入了解 基因在发育中的作用，但也暗示了同源基因在其他动物的健康和疾病中的作用， 包括人类

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.