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Molecular Mechanisms Regulating Extraembryonic Endoderm Lineage Potential


The separation of embryonic and extraembryonic lineages is a process that has evolved in amniotes to adapt for fetus survival on land. One of the major roles of the extraembryonic tissues is to provide nutritive support sourced from the maternal environment, since development occurs in utero. The initial differentiation of the extraembryonic lineages occurs first prior to embryonic cell differentiation to prioritize cell survival. Thus past studies have extensively focused on understanding these lineage segregation mechanisms which take place in three major waves of lineage differentiation events. The key commonality of these events begins with a bipotent progenitor that co-expresses lineage-specific transcription factors (TFs), which is resolved by mutual inhibition of TFs and key signaling pathways. During the first lineage breaking event, differential Hippo signaling activation and cross-antagonism of POUF51(OCT4) (ICM TF) and CDX2 (TE TF) forms the trophectoderm (TE) and the inner cell mass (ICM). Likewise, in the second lineage breaking event, differential Fgf signaling activation and cross-antagonism of NANOG (Epi TF) and GATA6 (PrE TF) regulates the ICM bifurcation to the epiblast (Epi) and primitive endoderm (PrE). Next, the extraembryonic trophectoderm differentiate to facilitate the implantation process. Concurrently, during this rapid and dynamic event, the PrE differentiates into the parietal endoderm (PE) and the visceral endoderm (VE). However, less is known about the regulation of these extraembryonic endoderm (ExEn) lineages partly due to inaccessibility of these cells during implantation. Therefore, the molecular basis of PrE cell fate divergence to PE or VE and the concerted actions of TFs that moderate PE- versus VE-cell gene regulatory programs remains to be elucidated. To investigate the regulative behavior of ExEn cells, we analyzed scRNA-seq datasets of E4.5 mouse embryos. We delineated PrE, PE and VE cell states and resolved TFs associated with PE or VE. Comparative motif analysis of the enhancer repertories of VE cells and PE cells suggests that GATA6, SOX17, and FOXA2 are core TFs in the ExEn gene regulatory network. To test this model, we performed transcriptome analyses on cXEN cells using degron tagged GATA6 and SOX17 combining with FoxA2 knockout revealed that PE development requires positive GATA6 and SOX17 inputs, whereas VE development requires FOXA2 to activate VE gene program suppressed by GATA6 or SOX17. Next, we found that BMP signaling cues instruct the PE-VE lineage decision. These data reveal a core gene regulatory module that underpins PE and VE cell fate choice. Lastly, we compare GATA4 and GATA6 functional activities in PE cells, which suggest they have overlapping functions in ExEn development. Overall, this dissertation will present a characterization of the ExEn gene regulatory network (GRN), while also proposing new models and regulators that govern ExEn development.

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