Eomes Directs the Formation of Spatially and Functionally Diverse Extra-Embryonic Mesoderm Derived Tissues
During mouse embryonic development, the first mesodermal cells to originate at the posterior primitive streak will give rise to the extra-embryonic mesoderm (ExEM). These cells will contribute to key tissues supporting the development of the embryo, including the amnion, chorion, yolk-sac, and allantois. Together, they protect the embryo and play key roles in the supply of nutrients and oxygen, waste management, haematopoiesis, and establishing the maternal-foetal interface. Embryonic stem cell (ESC)-derived in vitro differentiation systems serve as an invaluable resource for the study of embryogenesis, with many systems being tailored to recapitulate specific aspects of development. However, in vitro models of the murine ExEM compartment are mostly designed to mimic yolk-sac haematopoiesis, while lacking the remaining ExEM derivatives. While the allantois can be cultured ex vivo, experiments ultimately still require embryos to acquire the initial starting material. In Chapter 2 of this thesis, I perform in-depth single-cell characterization of a previously established in vitro system, identifying that it simultaneously recapitulates both yolk-sac and allantois development. This allows for the identification of molecular dynamics that distinguish the early progenitors of these lineages. As such, I uncover gene expression and chromatin accessibility dynamics that are differentially regulated between the yolk-sac and allantois, revealing both known and novel differences in transcription factors and signalling pathway members. One of the genes showing specificity for early yolk-sac progenitors, is the T-box transcription factor Eomesodermin (Eomes). Eomes is expressed early in gastrulation and plays known roles in various extra-embryonic and embryonic tissues, such as the trophectoderm, visceral and definitive endoderm, and various anterior mesodermal tissues. Additionally, Eomes has been implicated to be an important regulator of yolk-sac haematopoiesis, but was thought not to play a role in the development of yolk-sac endothelium. In Chapter 3 of this thesis, I profile the immediate and knock-on defects occurring upon EOMES knock-down (KD) in the yolk-sac and allantois in vitro differentiation system. This revealed that Eomes is critical for the earliest fate decision towards yolk-sac differentiation, resulting in a complete lack of yolk-sac haematopoiesis and endothelium upon EOMES-KD. Instead, EOMES-KD early progenitors are biased to differentiate into allantoic mesoderm. As such, the formation of the allantois, including its endothelium, is not impacted by EOMES-KD. These results are further confirmed by single-cell sequencing and imaging of chimeric embryos generated via injection of wild-type blastocysts with Eomes knock-out ESCs. Together, unbiased in vitro and in vivo profiling of Eomes disruption reveals a broader role for Eomes in the yolk-sac than previously thought. Finally, in Chapter 4 of this thesis, I perform high-resolution molecular profiling of EOMES-KD in the establishment of the anterior primitive streak (APS) and its derivatives. While Eomes is known to be a crucial factor in establishment of the APS-derived definitive endoderm and notochord, its molecular role in this context remains underexplored. Using single-cell sequencing of wild-type and EOMES-KD APS in vitro differentiation, I identify specific genes and enhancers that regulate the establishment of the APS in an Eomes dependent manner. Together, this thesis provides new insights into the molecular regulation of extra-embryonic mesoderm development and the critical role of Eomes in shaping early embryonic lineage decisions. Using in vitro differentiation systems, gene perturbations, and single-cell sequencing, I reveal key transcriptional and epigenetic mechanisms governing yolk-sac, allantois, and APS formation.
allantois, anterior primitive streak, bio-informatics, chimera, embryonic development, Eomesodermin, extra-embryonic mesoderm, in vitro differentiation, single-cell multi-omics, stem cell biology, transcription factor, yolk-sac
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