Automated single cell expression analysis in C. elegans

描述（由申请人提供）：生物体基因组中储存的信息指导其发育和行为，但控制该信息表达的调控网络在后生动物中才刚刚开始被理解。精确确定基因，特别是转录因子基因，何时何地表达，是全面了解这些网络的核心。线虫C.秀丽线虫在确定基因表达模式以及控制它们的调控网络方面具有独特的优势。这些优势包括固定的细胞谱系，少于1,000个的体细胞总数，紧凑的，完全测序的基因组和整个生命周期的透明体。利用过去资助期间的这些优势，我们开发了4D成像技术，可以在胚胎发生的前半部分以高时间分辨率自动确定每个细胞中单个基因的表达模式。我们已经应用这种技术来确定胚胎的表达模式的约200个转录因子基因，揭示了各种各样的表达模式，这些基因在指定细胞身份的作用。最近，我们已经设计出收集流式细胞仪分选的胚胎细胞的时间序列数据的方法，以提供基因组中所有基因的胚胎表达的更粗略的时空估计。我们建议在未来的资助期内，使用4D技术扩展和完成转录因子表达模式的目录。我们将建立一个最近开发的diSPIM显微镜，并使用它来分析通过胚胎发生的表达。我们将完成带有GFP标记的转录因子的菌株的构建，并使用这些来确定基因组中大量转录因子的详细表达模式。为了补充这一点，我们将进行RNA-seq。在从大致同步的胚胎中分选的组织和细胞群上，测量基因组中所有基因的基因表达动态。我们还将探索提供胚胎单个细胞表达数据的技术。这些数据集与ChIP-seq相结合。而其他数据集将被用于构建在整个胚胎发生过程中在每个细胞中活跃的调节网络。所有的数据和菌株将通过我们的网站，蠕虫基地和Cairrhaditis遗传学中心提供给社区。这种简单的后生动物的调控网络的知识将有直接的影响，了解人类在健康和疾病的调控网络。

DESCRIPTION (provided by applicant): The information stored in an organism's genome directs its development and behavior, but the regulatory networks that control the expression of that information are only beginning to be understood in metazoans. Determining precisely when and where genes, particularly transcription factor genes, are expressed is central to gaining a comprehensive understanding of these networks. The nematode C. elegans offers unique advantages for determining gene expression patterns and in turn the regulatory networks that control them. These advantages include a fixed cell lineage, a total somatic cell number of less than 1,000, a compact, fully sequenced genome and a transparent body throughout the life cycle. Exploiting these advantages in the past grant periods, we have developed 4D imaging technology that automatically determines expression patterns of individual genes in each cell with high temporal resolution over the first half of embryogenesis. We have applied this technology to determine the embryonic expression patterns of some 200 transcription factor genes, revealing a wide variety of expression patterns that suggest roles for these genes in specifying cell identity. More recently we have devised methods to collect timed series data for FACS sorted embryonic cells to provide coarser spatiotemporal estimates of embryonic expression of all genes in the genome. We propose in the coming grant period to extend and complete the catalog of expression patterns for transcription factors using the 4D technology. We will build a recently developed diSPIM microscope and use that to assay expression through embryogenesis. We will complete the construction of strains with GFP tagged transcription factors and use these to determine the detailed expression patterns for the bulk of transcription factors in the genome. To complement this we will perform RNA-seq. on FACS sorted tissues and cell populations from roughly synchronized embryos to measure the dynamics of gene expression for all genes in the genome. We will also explore technology to provide expression data on individual cells of the embryo. These data sets, combined with ChIP-seq. and other data sets will be used to build regulatory networks active in each cell throughout embryogenesis. All the data and strains will be made available to the community through our website, Worm Base and the Caenorhaditis Genetics Center. Knowledge of the regulatory networks of this simple metazoan will have direct implications for understanding regulatory networks in humans both in health and in disease.