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Abstract

In contrast to the majority of animals, mammalian embryonic development is highly regulative, beginning from one or several functionally-identical cells and sequentially acquiring increasing complexity over a relatively short period of time. During this period, the embryo also undergoes significant changes in morphology so as to accommodate the increase in complexity, especially during the landmark developmental event of gastrulation. How these morphological changes are enacted, coordinated, and controlled at the supra-cellular scale is yet unclear. The study of this question, and indeed this scientifically interesting period of development, has been historically difficult. This is partially due to the fact that unlike in other animals such as the zebrafish or the fruit fly, most mammalian embryos – including that of mice, the most common model organism in which such studies are conducted – are undergoing or have already undergone implantation into the uterine tissues during this period. To facilitate the study the peri-implantation development of mouse embryos, I have developed a 3D ex vivo culture system that is compatible with long-term light-sheet imaging, jointly with colleagues and collaborators. I characterised the development of embryos in this culture system and optimised it so that it supported the physiological growth and morphogenesis of embryonic and several extra-embryonic lineages. After validating this culture system, I used it to investigate the cell- and tissue-scale changes that occurred in the embryonic tissues during the peri-implantation period. In the mouse embryo, this period is associated with significant growth and differentiation of both embryonic and extra-embryonic tissues. For example, the epiblast (an embryonic cell lineage) adopts a pseudo-stratified epithelium organisation, while a subpopulation of the visceral endoderm (an extra-embryonic cell lineage) specialises into a distinct lineage that patterns the underlying embryonic ectoderm to lay down the first embryonic body axis. I demonstrated that the 3D ex vivo culture system supports live imaging at sufficient spatial and temporal resolution to visualise these processes. In addition, using an automatic 3D segmentation pipeline developed by colleagues and collaborators, I showed that the culture system can be used to study cell- and tissue-scale dynamics. The regulative nature of early mammalian development is also evident in the fact that the early mammalian embryo can tolerate drastic deviations in tissue size and cell number during development and correct these deviations so that at birth, embryos are once again within the stereotypical range of sizes for the species. The early mouse embryo can tolerate at least 2 four-fold changes in cell number, brought about by removal or addition of blastomeres; however, these size deviations are reportedly resolved by the time gastrulation is initiated, with embryos all undergoing gastrulation with the epiblast cell number at a certain threshold. I observed discrepancies between my findings and those previously reported, demonstrating that this process is still poorly understood despite decades of study, and identify cell- and tissue-scale parameters that may play a role in the sensing and correction of size deviations in the embryo.