INVESTIGATING THE ROLE OF GENOME MAINTENANCE DURING THE PLACENTAL DEVELOPMENT
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Replication stress (RS) and DNA damage critically threaten genomic stability, particularly during early embryogenesis, where precise genome maintenance underpins proper stem cell differentiation and organogenesis. Formation of the placenta – an organ that is essential for fetal growth, nutrient supply, endocrine signaling, and maternal-fetal immune tolerance, relies on the integrity and proliferation of trophoblast stem cells (TSCs). However, the mechanisms by which RS-induced DNA damage influences trophoblast lineage specification and placental morphogenesis remain incompletely understood.Using the “Chaos3” mouse model harboring a hypomorphic mutation in Mcm4—a critical subunit of the DNA replication helicase—we established a genetically controlled system to elucidate how chronic RS and consequent DNA damage impact trophoblast progenitor function and placental architecture. Homozygous Chaos3 (Mcm4Chaos3/Chaos3) TSCs displayed hallmark phenotypes of chronic RS, including elevated markers of DNA breaks (γH2AX foci), diminished DNA synthesis, and increased polyploidy, accompanied by the activation of the ATR–CHK1–P53–P21 signaling pathway. These stress responses collectively induced cell cycle arrest, premature differentiation, and apoptosis, significantly reducing TSC self-renewal capacity and derivation efficiency. In vivo analyses revealed broader placental consequences beyond replication licensing impairment. Chaos3 embryos exhibited significant placental hypoplasia, characterized particularly by severe depletion of progenitor-rich junctional zone (JZ) constituents responsible for endocrine functions, including spongiotrophoblasts and glycogen trophoblast cells. Transcriptomic analysis confirmed extensive downregulation of key pathways associated with trophoblast proliferation, differentiation, and hormone biosynthesis, implicating RS-induced DNA damage as a fundamental disruptor of trophoblast developmental programs. Furthermore, immunohistochemical characterization identified a marked reduction in proliferation of progenitor cells within the placental JZ, implicating a causal linkage to genomic instability. Remarkably, partial alleviation of RS through genetic modulation restored TSC proliferative capacity and partially rescued JZ compartment morphology, underscoring the necessity of balanced DNA replication fidelity for placental organogenesis. Collectively, this dissertation underscores the critical impact of RS and DNA damage on trophoblast stem cell function, delineating a generalizable mechanism through which genomic instability disrupts trophoblast proliferation, lineage specification, and placental endocrine function. These insights offer broader implications for understanding how environmental and genetic perturbations compromising genome integrity may contribute to placental insufficiency, pregnancy complications, and developmental disorders.