Chapter 12
Visualizing Mouse Embryo Gastrulation
Epithelial-Mesenchymal Transition Through Single Cell
Labeling Followed by Ex Vivo Whole Embryo Live Imaging
Wallis Nahaboo, Bechara Saykali, Navrita Mathiah, and Isabelle Migeotte
Abstract
Epithelial-mesenchymal transition (EMT) is often studied in pathological contexts, such as cancer or
fibrosis. This chapter focuses on physiological EMT that allows the separation of germ layers during
mouse embryo gastrulation. In order to record individual cells behavior with high spatial and temporal
resolution live imaging as they undergo EMT, it is very helpful to label the cells of interest in a mosaic
fashion so as to facilitate cell segmentation and quantitative image analysis. This protocol describes the
isolation, culture, and live imaging of E6.5–E7.5 mouse embryos mosaically labeled in the epiblast, the
epithelium from which mesoderm and endoderm layers arise through EMT at gastrulation.
Key words EMT, Mouse embryo, Live imaging
1 Introduction
The passage from an epithelial to a mesenchymal cell organization
(epithelial-to-mesenchymal transition or EMT) is essential for gastrulation, a key morphogenetic event that establishes the three
layers of the animal body plan (ectoderm, mesoderm, and endoderm). In this context, EMT renders cells competent for motility,
but is also coupled to a switch of cell fate. In amniotes, it occurs at a
transient organizing center called the primitive streak [1–3]. In
mouse, the overlap of multiple signaling gradients coming from
embryonic epiblast, as well as extraembryonic ectoderm and visceral endoderm, creates a permissive zone in the posterior epiblast
for primitive streak establishment at around embryonic day (E) 6.
Prior to the onset of gastrulation, the epiblast consists of a
columnar pseudostratified epithelium sitting on a basal lamina.
EMT involves degradation of basal membrane proteins, downregulation of cell–cell junction proteins, loss of polarity, and acquisition of motility. Live imaging showed that epiblast cells in the
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_12,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
135
Visualizing Mouse Embryo Gastrulation
Epithelial-Mesenchymal Transition Through Single Cell
Labeling Followed by Ex Vivo Whole Embryo Live Imaging
Wallis Nahaboo, Bechara Saykali, Navrita Mathiah, and Isabelle Migeotte
Abstract
Epithelial-mesenchymal transition (EMT) is often studied in pathological contexts, such as cancer or
fibrosis. This chapter focuses on physiological EMT that allows the separation of germ layers during
mouse embryo gastrulation. In order to record individual cells behavior with high spatial and temporal
resolution live imaging as they undergo EMT, it is very helpful to label the cells of interest in a mosaic
fashion so as to facilitate cell segmentation and quantitative image analysis. This protocol describes the
isolation, culture, and live imaging of E6.5–E7.5 mouse embryos mosaically labeled in the epiblast, the
epithelium from which mesoderm and endoderm layers arise through EMT at gastrulation.
Key words EMT, Mouse embryo, Live imaging
1 Introduction
The passage from an epithelial to a mesenchymal cell organization
(epithelial-to-mesenchymal transition or EMT) is essential for gastrulation, a key morphogenetic event that establishes the three
layers of the animal body plan (ectoderm, mesoderm, and endoderm). In this context, EMT renders cells competent for motility,
but is also coupled to a switch of cell fate. In amniotes, it occurs at a
transient organizing center called the primitive streak [1–3]. In
mouse, the overlap of multiple signaling gradients coming from
embryonic epiblast, as well as extraembryonic ectoderm and visceral endoderm, creates a permissive zone in the posterior epiblast
for primitive streak establishment at around embryonic day (E) 6.
Prior to the onset of gastrulation, the epiblast consists of a
columnar pseudostratified epithelium sitting on a basal lamina.
EMT involves degradation of basal membrane proteins, downregulation of cell–cell junction proteins, loss of polarity, and acquisition of motility. Live imaging showed that epiblast cells in the
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_12,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
135