Methodologies for Following EMT In Vivo at Single Cell Resolution

Abstract

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.

Keywords: Epithelial-mesenchymal transition; Sea urchin; Single cell RNA-sequencing; Tissue morphogenesis.

Figure 1:

General scRNAseq pipeline Figure adapted from and inspired by the single cell RNA sequencing course (Kiselev et. al 2019). Bioconductor is a repository that houses toolkits for sequencing and cell quality control, analysis, visualization, exploration, and more. Common packages used for each step in the pipeline are included. Using these methods, each gene’s expression during EMT can be quantitatively measured in single cells, allowing for a deeper understanding of the underlying mechanistic structure of EMT.