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Abstract

Mammalian embryo development is highly regulative in nature, with cell fate being reversible to compensate for changes in the nascent environment during early stages. In such regulative paradigms, the complex interactions between cellular processes to accomplish precision in patterning of tissues present exciting open questions in biology. In this thesis, I explore the patterning mechanisms underlying blastocyst maturation during pre-implantation development of the mouse embryo. Development of the mammalian blastocyst involves the progressive segregation of the inner cell mass comprising the embryonic epiblast and the extra-embryonic primitive endoderm, and the expansion of a fluid-filled cavity. A gene regulatory network between fibroblast growth factor signalling and cell fate-specific transcription factors establishes the characteristic salt-and-pepper distribution of epiblast and primitive endoderm cells in the early blastocyst. Following the establishment of the two precursor populations, cells spatially segregate into distinct domains within the embryo. The primitive endoderm forms an epithelial layer at the surface of the fluid cavity, with the pluripotent epiblast enclosed between the trophectoderm and the primitive endoderm. Symmetry-breaking in the blastocyst has been the focus of much research in the past decades. However, a coherent mechanism of blastocyst pattern emergence coordinating cell fate specification, sorting and morphogenesis is lacking. Using reduced systems and advanced live-image analysis, I established a method to characterise cell sorting during fate segregation. Further, combined with biophysical measurements and perturbations, I investigate the interplay between cytoskeletal dynamics, cell migration, and polarity in driving fate segregation. I demonstrate that cell sorting in the inner cell mass is characterised by active migration of primitive endoderm cells towards the cavity surface. The primitive endoderm cells in the early blastocyst display autonomous acquisition of apical polarity, a defining feature essential for their proper segregation into a single epithelial layer enveloping the epiblast. Moreover, in contrast to epiblast cells, apical polarity in primitive endoderm cells facilitates the lowering of surface tension upon encountering the cavity surface, thus being sufficient for cell positioning. Lastly, I provide evidence that cell fate plasticity is lost in late blastocysts due to which the fixed lineage composition of the inner cell mass is optimal exclusively for a particular embryo size. Altogether, the findings presented in this work provide mechanistic insights into segregation of cell fates in the mammalian blastocyst and put forth a novel understanding of robust patterning during embryonic development.