Chapter 23
Methodologies for Following EMT In Vivo at Single Cell
Resolution
Abdull J. Massri, Geoffrey R. Schiebinger, Alejandro Berrio, Lingyu Wang,
Gregory A. Wray, and David R. McClay
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.
Key words Epithelial-mesenchymal transition, Single cell RNA-sequencing, Sea urchin, Tissue
morphogenesis
1 Introduction
Most embryonic mesoderm cells are initially specified when they
reside in an epithelium. An epithelial-to-mesenchymal transition
(EMT) then removes them from the epithelial layer and they
adapt a mesenchymal phenotype. In some cases, these cells again
become epithelial and go through additional EMTs. This process of
leaving the epithelium also occurs with carcinoma cells. Whether
the two EMTs share mechanistic components of the process is a
question that has often been asked. Literature reports indicate that
they do indeed share multiple properties: they tend to use the same
controlling transcription factors (twist, snail, and zeb1), though not
always. They appear similar in behavior (the cells become motile,
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_23,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
303
Methodologies for Following EMT In Vivo at Single Cell
Resolution
Abdull J. Massri, Geoffrey R. Schiebinger, Alejandro Berrio, Lingyu Wang,
Gregory A. Wray, and David R. McClay
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.
Key words Epithelial-mesenchymal transition, Single cell RNA-sequencing, Sea urchin, Tissue
morphogenesis
1 Introduction
Most embryonic mesoderm cells are initially specified when they
reside in an epithelium. An epithelial-to-mesenchymal transition
(EMT) then removes them from the epithelial layer and they
adapt a mesenchymal phenotype. In some cases, these cells again
become epithelial and go through additional EMTs. This process of
leaving the epithelium also occurs with carcinoma cells. Whether
the two EMTs share mechanistic components of the process is a
question that has often been asked. Literature reports indicate that
they do indeed share multiple properties: they tend to use the same
controlling transcription factors (twist, snail, and zeb1), though not
always. They appear similar in behavior (the cells become motile,
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_23,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
303