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，但控制该信息表达的调节网络在后生动物中才刚刚开始被理解。精确地确定基因，特别是转录因子基因在何时何地表达，对于全面了解这些网络至关重要。秀丽隐杆线虫在确定基因表达模式和控制它们的调控网络方面具有独特的优势。这些优势包括一个固定的细胞谱系，体细胞总数少于1000，一个紧凑的，完全测序的基因组和整个生命周期的透明体。利用这些优势，我们开发了4D成像技术，可以在胚胎发生的前半段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。我们已经应用这项技术来确定大约200个转录因子基因的胚胎表达模式，揭示了各种各样的表达模式，表明这些基因在指定细胞身份中的作用。最近，我们设计了一些方法来收集FACS分类胚胎细胞的时间序列数据，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。我们建议在即将到来的资助期内，利用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，并使用它来检测胚胎发生过程中的表达。我们将完成构建带有GFP标记的转录因子的菌株，并使用这些来确定基因组中大部分转录因子的详细表达模式。为了补充这一点，我们将执行RNA-seq。在FACS上对大致同步胚胎的组织和细胞群进行分类，以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，结合ChIP-seq。其他数据集将用于构建在胚胎发生过程中每个细胞中活跃的调控网络。所有的数据和菌株将通过我们的网站、蠕虫基地和隐杆线虫遗传中心提供给社区。了解这种简单后生动物的调控网络将对理解人类健康和疾病中的调控网络具有直接意义。

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.