Mendenhall Alexander R, Tedesco Patricia M, Sands Bryan, Johnson Thomas E, Brent Roger
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado, United States of America.
PLoS One. 2015 May 6;10(5):e0124289. doi: 10.1371/journal.pone.0124289. eCollection 2015.
In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.
在多细胞生物如秀丽隐杆线虫中，诸如寿命等复杂表型的差异与特定工程报告基因的表达水平相关。在单细胞生物中，对细胞外信号反应以及反应中细胞间差异的定量理解依赖于报告基因表达的精确测量。在此，我们开发了基于显微镜的方法来定量测量秀丽隐杆线虫细胞中报告基因的表达，且测量误差较低。然后，我们对携带不同构型的热休克蛋白16.2-荧光蛋白报告基因的菌株、整个动物以及负责大部分荧光信号的肠道组织的所有20个细胞中的表达进行了定量。一些动物携带最近开发的整合在特定染色体位点的单拷贝热休克蛋白16.2报告基因，另一些则是整合在随机位点的“经典”多拷贝报告基因阵列。在整个动物水平上，基因表达的差异相似：单拷贝报告基因的菌株与多拷贝报告基因的菌株表现出相同程度的动物个体间差异。在细胞水平上，对于单拷贝报告基因的动物，组织内细胞的表达模式高度定型。对于多拷贝报告基因的动物，细胞特异性表达模式也是定型的，但有所不同，且变异性稍大。我们的方法快速且温和，可以在成年期的不同时间对同一只动物的相同细胞中的表达进行定量。它们应能让研究人员将组织中单个细胞报告基因表达的变化用作定量表型，并将这些变化与分子差异联系起来。此外，通过减少测量误差，应该能够剖析剩余的真实表达差异的原因。理解这种差异应有助于揭示其对多细胞生物复杂表型结果差异的贡献。
