Department of Biology, Duke University, Durham, NC, USA.
Department of Mathematics, University of British Columbia, Vancouver, BC, Canada.
Methods Mol Biol. 2021;2179:303-314. doi: 10.1007/978-1-0716-0779-4_23.
An epithelial-mesenchymal transition (EMT) occurs in almost every metazoan embryo at the time mesoderm begins to differentiate. Several embryos have a long record as models for studying an EMT given that a known population of cells enters the EMT at a known time thereby enabling a detailed study of the process. Often, however, it is difficult to learn the molecular details of these model EMT systems because the transitioning cells are a minority of the population of cells in the embryo and in most cases there is an inability to isolate that population. Here we provide a method that enables an examination of genes expressed before, during, and after the EMT with a focus on just the cells that undergo the transition. Single cell RNA-seq (scRNA-seq) has advanced as a technology making it feasible to study the trajectory of gene expression specifically in the cells of interest, in vivo, and without the background noise of other cell populations. The sea urchin skeletogenic cells constitute only 5% of the total number of cells in the embryo yet with scRNA-seq it is possible to study the genes expressed by these cells without background noise. This approach, though not perfect, adds a new tool for uncovering the mechanism of EMT in this cell type.
上皮-间充质转化（EMT）几乎在每个后生动物胚胎中都会在中胚层开始分化时发生。由于已知的细胞群体在已知的时间进入 EMT，因此有几个胚胎作为研究 EMT 的模型具有悠久的历史，从而可以对该过程进行详细研究。然而，通常情况下，很难了解这些 EMT 模型系统的分子细节，因为正在发生 EMT 的细胞在胚胎细胞群体中只占少数，并且在大多数情况下无法分离该群体。在这里，我们提供了一种方法，可以在 EMT 之前、期间和之后检查基因的表达情况，重点关注那些经历了转化的细胞。单细胞 RNA 测序（scRNA-seq）技术的发展使得在体内、在没有其他细胞群体背景噪声的情况下，专门研究感兴趣的细胞中的基因表达轨迹成为可能。海胆骨骼生成细胞仅占胚胎中总细胞数的 5%，但通过 scRNA-seq，有可能在没有背景噪声的情况下研究这些细胞表达的基因。尽管这种方法并不完美，但它为揭示这种细胞类型 EMT 的机制增加了一种新工具。
