The role of inherited determinants and embryonic-extraembryonic cell interactions in models of mammalian embryogenesis
Mammalian embryogenesis begins with the fusion of an oocyte and a sperm to create the first cell of the newly developing embryo called the zygote. The mechanisms by which this single cell generates a fully formed organism have long been the subject of study of experimental biologists. Initially, embryonic development is reliant on maternally inherited determinants (e.g., RNA, proteins) generated in the oocyte. Overtime, maternal products are degraded and development is controlled by the zygotic genome. Zygotic genome activation (ZGA) is critical for successful embryogenesis, and yet the precise mechanisms of its regulation remain elusive. The first aim of this thesis was to assess the contribution of inherited cell cycle regulators to the onset of ZGA in the early mouse embryo. Specifically, the role of the maternal cell cycle regulator cyclin B3 was examined through zygote microinjections with mRNA combined with live imaging and pharmacological approaches. The findings presented here show a novel link between cyclin B3 activity and the expression of ZGA-related genes Zscan4, Eif1a and Sp110 in the mouse embryo. Overexpression of a degradation-resistant cyclin B3 resulted in the elongation of the S-phase at the expense of the G2 phase in the 2-cell embryo and a suppression of ZGA-related genes. This effect was partially rescued when embryos were forced through the cell cycle by exposure to a Chk1 inhibitor. Conversely, elongation of the G2 phase through Cdk inhibition resulted in an upregulation of ZGA-related genes, which suggested a close link between the cell cycle progression and ZGA regulation in the early embryo. As embryogenesis proceeds, cells are gradually restricted in potency, and morphogenesis shapes the formation of the tissues and organs in the developing body. Complex signalling interactions between embryonic and extraembryonic tissues are indispensable for successful development. The study of early mammalian post-implantation development is challenging due to a lack of accessibility to the embryo as it grows in the uterus. Recent work has focused on establishing models to study and manipulate developmental stages that are equivalent to post-implantation development in the mouse. Yet, extraembryonic tissues are not appropriately represented in current in vitro models and as such these are incapable of recapitulating the true morphogenetic events and architecture of the embryo developing in vivo. The second aim of this thesis was to extend development of an integrated stem cell model of post-implantation embryogenesis that includes an extraembryonic component. When subjected to culture conditions capable of sustaining natural embryo development beyond gastrulation, iETX synthetic embryos were capable of recapitulating the architecture and morphogenesis of the natural embryo and developing to organogenesis stages in the absence of external signalling cues. Specifically, iETX embryos underwent morphogenesis of headfold structures, a neural tube, somite pairs, specification of primordial germ cells, and generated mesodermal tissues including a beating heart. Furthermore, iETX embryos captured extensive development of the extraembryonic lineages, including the formation of a yolk sac with blood islands, and tissues resembling the chorion and allantois. Finally, iETX embryos generated with Pax6 knockout embryonic stem cells were able to recapitulate known phenotypes of Pax6-/- mouse embryos, such as the expansion of the ventral progenitor domain in the neural tube. Overall, these results highlighted that iETX embryos form a complete in vitro model of the mouse embryo that can be used to study development and disease at stages that were previously inaccessible, particularly to shed light on signalling interactions between the embryo, the yolk sac and the placenta.
embryology, gastrulation, neurulation, organogenesis, stem cell differentiation
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