Visualizing Mouse Embryo Gastrulation Epithelial-Mesenchymal Transition Through Single Cell Labeling Followed by Ex Vivo Whole Embryo Live Imaging.

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.

Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.

Mitochondrial AIF loss causes metabolic reprogramming, caspase-independent cell death blockade, embryonic lethality, and perinatal hydrocephalus.

OBJECTIVES: Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction. METHODS: We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown. RESULTS: AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF+/- females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF+/- females by supplying dams and newborns with an antioxidant in drinking water. CONCLUSIONS: In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies.

Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo.

In mouse embryo gastrulation, epiblast cells delaminate at the primitive streak to form mesoderm and definitive endoderm, through an epithelial-mesenchymal transition. Mosaic expression of a membrane reporter in nascent mesoderm enabled recording cell shape and trajectory through live imaging. Upon leaving the streak, cells changed shape and extended protrusions of distinct size and abundance depending on the neighboring germ layer, as well as the region of the embryo. Embryonic trajectories were meandrous but directional, while extra-embryonic mesoderm cells showed little net displacement. Embryonic and extra-embryonic mesoderm transcriptomes highlighted distinct guidance, cytoskeleton, adhesion, and extracellular matrix signatures. Specifically, intermediate filaments were highly expressed in extra-embryonic mesoderm, while live imaging for F-actin showed abundance of actin filaments in embryonic mesoderm only. Accordingly, Rhoa or Rac1 conditional deletion in mesoderm inhibited embryonic, but not extra-embryonic mesoderm migration. Overall, this indicates separate cytoskeleton regulation coordinating the morphology and migration of mesoderm subpopulations.

Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis.

The heart arises from distinct sources of cardiac progenitors that independently express Mesp1 during gastrulation. The precise number of Mesp1 progenitors that are specified during the early stage of gastrulation, and their clonal behavior during heart morphogenesis, is currently unknown. Here, we used clonal and mosaic tracing of Mesp1-expressing cells combined with quantitative biophysical analysis of the clonal data to define the number of cardiac progenitors and their mode of growth during heart development. Our data indicate that the myocardial layer of the heart derive from ∼250 Mesp1-expressing cardiac progenitors born during gastrulation. Despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. These results provide insights into the number of cardiac progenitors and their mode of growth and open up avenues to decipher the clonal dynamics of progenitors in other organs and tissues.