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. Author manuscript; available in PMC: 2021 Jul 21.
Published in final edited form as: Methods Mol Biol. 2021;2179:7–12. doi: 10.1007/978-1-0716-0779-4_2

Perspective on Epithelial-Mesenchymal Transitions in Embryos

David R McClay 1
PMCID: PMC8294114  NIHMSID: NIHMS1681480  PMID: 32939708

Abstract

The epithelial-mesenchymal transition (EMT) is a key process required for building the early body plan of metazoa. It involves coordinated and precisely timed changes in multiple cell processes such as de-adhesion, motility, invasion, and cell polarity. While much has been learned about how embryos deploy epithelial-mesenchymal transitions since Betty Hay named the process decades ago, a number of things are still not well understood. Here I will discuss some of the big questions that remain, including how is all of this controlled, how does each of the cell biological events work, and how are they so nicely coordinated with one another?

Keywords: Gastrulation, Epithelial-mesenchymal transition, Morphogenesis, Sea urchin

1. Introduction

Morphogenetic movements at gastrulation reshape the embryos of multicellular animals. Among those movements, an epithelial-mesenchymal transition (EMT) removes mesoderm cells from an epithelium and places them in between the ectoderm and endoderm. Other embryonic cell types also go through EMTs at various stages of development in many animals. The EMT process is thus essential for building the early body plan of metazoa, and because of this it is important to learn how the process works at a molecular level.

A number of properties of developmental EMTs are not well understood. As part of the EMT, the cells become motile while still in the epithelium. That motility results in cell shape changes, helps the cell penetrate through the basement membrane, and mechanically contributes to the loss of adhesion as the cell leaves the adherens junction. The invasion through the basement membrane involves at least a partial remodeling of the basement membrane plus a determined polarized movement of the cell. The cell de-adheres from the adherens junction. Movies of the process show that to de-adhere, the cells stretch, mechanically, pulling away from the adherens junction before that junctional connection is lost. Release from the adherens junction occurs late in the EMT, and as soon as the cell “tail” is released, the membrane containing cadherin is endocytosed while membrane bearing mesenchymal determinants are inserted via exocytosis. The cell, now in the interstitial spaces, rapidly demonstrates a mesenchymal phenotype and expresses very different cell surface markers relative to its earlier state as an epithelial cell. The big questions then are to ask how all of this is controlled, how does each of the cell biological events work, and how they are so nicely coordinated with one another. The approach of my lab over the years has changed with the technologies available but with each iteration, the question has always been: How does it work?

Our first observation was made 35 years ago. We had begun a study of sea urchin skeletogenic cells which can be seen to undergo an EMT beginning about 9 h after fertilization. The sea urchin embryo is transparent, and the skeletogenic cells are the first cells to engage in gastrulation movements so the EMT is easy to see. Methods had been developed to separate the skeletogenic cells from the other cells of the embryo, and we noticed that at 9 h post fertilization the skeletogenic precursor cells, now in culture, began to move. They changed shape and began to crawl on the substrate. In other words, they behaved as if they were still part of the embryo and they began those movements autonomously, at the same time as skeletogenic cells in vivo started EMT. We developed a quantitative adhesion assay using a centrifuge to measure the force needed to remove the cells from a substrate. Rachel Fink, now a professor at Mt. Holyoke, and I observed that during the 45 min period of the EMT the skeletogenic cells lost their affinity for other cells and gained an affinity for extracellular matrix [1]. In other words, the EMT was accompanied by a dramatic and quantifiable adhesion change.

The next obvious question was to ask what molecules were responsible for those adhesion changes? At the time the field was busy identifying cell-cell and cell substrate adhesion molecules. Masatoshi Takeichi had discovered cadherins [2], and Richard Hynes, Clayton Buck, and others had identified integrins [3, 4], so our efforts shifted to identifying as many of those molecules as possible. The field also was embracing molecular biology so all of my graduate students from that time onwards became familiar with molecular technologies. We identified cadherins, integrins, a number of basement membrane proteins and put each of them to the test in an effort to learn which molecules participated in the EMT change. Those efforts were productive and showed that as the skeletogenic cells went through EMT they lost an adhesive affinity to cadherin and gained an affinity for extracellular matrix components via integrins. Cadherin was removed from the cell surface as the skeletogenic cell went through EMT and was immediately endocytosed [5, 6].

While these efforts were productive, we were bothered by the approach. It seemed as if we were stuck playing hunches about molecules and it did not seem like we were gaining a broad understanding of how the EMT process was coordinately regulated. We did not understand the detailed cell biology behind the changes, and we did not understand what was directly controlled by the regulatory apparatus. Productive genetic approaches were being taken in other embryonic systems, most notably by Maria Leptin in Drosophila [7], but the sea urchin lacked the use of genetics as a tool for discovery. So, my choices were either to switch to a more tractable genetic model, or to find a way to move forward using the sea urchin system.

At about that time new discoveries in the lab sent us down a different path. We began identifying genes that regulated developmental specification. Because of that, Eric Davidson at Caltech, with a longstanding focus on mechanisms of gene regulation, asked if I might be interested in joining him and his lab in identifying the gene regulatory network (GRN) that established early specification of the sea urchin embryo. I thought about it overnight and realized that if we were able to identify the regulatory network governing specification we would also be generating the regulatory apparatus controlling the EMT. With that goal in mind I called Eric back and eagerly entered into an entirely new direction for the lab. Other members of the sea urchin community quickly joined in and over the next 10 years our understanding of the complexity of early specification grew enormously. The skeletogenic cell lineage led the way with a detailed regulatory sequence [8].

With a fairly detailed GRN model in hand we returned to the EMT problem. Could we use the GRN to understand how the EMT is regulated and coordinated? Our strategy was fairly simple. Since we knew when the EMT began we started by asking what regulatory events occurred in the 2 h prior to launching the EMT. We systematically knocked down each of the transcription factors in the GRN that were activated within that two-hour time period and asked whether, in the absence of that transcription factor, was the EMT crippled? To assess the outcome, we designed several simple assays, and used time-lapse microscopy to watch the cell behavior during the EMT. To focus on the EMT only, the assays employed fluorescently tagged skeletogenic cells in embryos that were unlabeled. We could also assess whether each EMT event was cell autonomous or if there might be a non-autonomous component to the process. Lindsay Saunders, a graduate student, and I did the analysis [9]. We learned that at least 10 transcription factors were involved in the EMT regulation. Some regulated only the motility. Others regulated the invasive process, and still others regulated the de-adhesion component. No single transcription factor was involved in all of the EMT behaviors we scored. Some of those behaviors were most interesting. For example, we found that FoxN2/3 was involved in acquisition of motility. FoxN2/3 KO cells remained in the epithelium but continued to remodel the basement membrane as part of the invasive function. Without motility, however, the FoxN2/3 KO cells failed to take advantage of their own remodeled matrix.

With at least a partial understanding of the EMT transcriptional control, the next goal was to ask what genes were controlled by each contributing transcription factor. Those downstream effector genes, we hypothesized, encoded the proteins that initiated and conducted the EMT mechanics. We knew this strategy would not yield all proteins involved in the EMT. For example, actin and myosin are present constitutively in the cell and surely are participants in the motility component, but other proteins, controlled by the motility transcriptional sub-circuit, we hypothesized, were the drivers of actin and myosin cytoskeletal contractions. Those proteins were our targets. We decided to first use RNA-seq and did a temporal profile starting with sampling 2 h before, then during and after the EMT. We also did the same profile with embryos in which twist or snail had been knocked down. The database was huge, but we could eliminate constitutively expressed RNAs since we wanted to find the genes activated by twist and snail. We were able to segment that population of genes into clusters that were activated at the predicted times relative to the EMT, and we could identify which of those genes were putative targets of Twist or Snail regulation. That allowed us to narrow the search from over 16,000 genes to fewer than 50, a manageable group to then work with. Upon further narrowing we arrived at a small number of proteins that we could functionally confirm as participants in the EMT.

While we were successful in pulling out a few genes involved in the de-adhesion phase of the EMT, and those studies continue, we wanted to access proteins in all component processes. A handicap with the RNA-seq profiles, however, is noise. Even though the skeletogenic cells went through EMT at a similar time, they were not perfectly synchronous, and also there were a number of cells in the database that were not involved in the EMT. With improvements in single cell-sequencing (sc-RNAseq) it is now possible to interrogate each cell of an embryo. For that reason, we launched a sc-RNAseq project, the methods of which are described in this Methods book. It still is too early to fill in the details about that approach, but it offers a way to identify a substantial fraction of the RNAs present in a cell at any given time and has the very nice advantage of being able to computationally project a temporal sequence of RNA appearance and disappearance. This along with the known GRN has enabled us to pry much more deeply into the workings of the EMT than we ever thought possible. Still, there are many questions to address.

The future holds many remaining questions. Along with gaining details of the de-adhesion, motility, invasion, and cell polarity changes, it is important to know how uniform or diverse the EMT process is in different tissues. As morphogenesis progresses in the sea urchin, five different cell types undergo an EMT hours apart from one another. In time-lapse movies, those EMTs look similar but from what little we have learned, each EMT is quite different from the others at a molecular level. What is needed is an in-depth analysis of many of these EMTs to learn whether in fact each EMT is indeed unique in its molecular control, both transcriptionally and with effector proteins, or, are there proteins that are universally and uniquely involved in EMTs? That universality seems to be the target of cancer EMT studies because it would provide a focal point for attempts to inhibit that EMT that initiates metastasis. In embryos, such a protein would also be valuable for many reasons, ranging from evolutionary mechanisms to targeted perturbations in studies of morphogenesis.

Another big question in morphogenesis is how the EMT is timed and coordinated. As indicated above, five different EMTs occur during gastrulation. Each starts at a fairly precise time, and each is coordinated such that all the cell biological changes of that EMT occur harmoniously. An ability to follow events in single cells as outlined above will help uncover these mechanisms. From the time-lapse movies of the process it appears as though there are a number of possibilities for mechanical sensing, and if that is the case there will be opportunities for gaining an understanding of how such sensing relays information to other components of the EMT.

Even though the remaining questions are quite numerous, much has been learned about how embryos deploy epithelial-mesenchymal transitions since Betty Hay named the process decades ago. Since then progress has paralleled advances in molecular technologies. With many new technologies being introduced at a rapid pace, the next decade should be rich with discoveries about how EMTs work in embryos.

Acknowledgments

Thanks to the many students and postdocs in the McClay lab who contributed so much over the years to advance our understanding of the EMT. Thanks also to the NIH for supporting this work: RO1 HD14483 (to DRM) and PO1 HD 37105 (Project 2 to DRM).

References

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