Evaluating Placental Influence on the Development of the Brain, Heart, and Face

Abstract

The placenta is the sole source of nutrient and gas supply to the growing embryo during pregnancy. While deficiencies in the function of this vital organ lead to fetal growth retardation, and in severe cases, embryonic lethality, the placenta also has a critical role in the formation of specific organ systems beyond nutrient supply. This is evident in mouse mutants where placental function is disrupted, frequently leading to defects in cardiovascular, neural, and facial development. The age of the maternal uterine environment may also be detrimental for placental health. Advanced maternal age correlates with an increased frequency of heart and neurodevelopmental defects in offspring. Despite the reliance of gestational outcome on placental function, this organ is often overlooked as a potential cause of embryonic defects, and the molecular pathways underlying the developmental link between placenta, heart, brain, and face are largely unknown. In this thesis, I investigated the impact of advanced maternal age on the placenta, heart, brain, and face at embryonic day (E)10.5, and uncovered, as well as functionally characterized, a key transcriptional regulator, Peg3, which is highly expressed in all four tissues. The transcriptomic profiles of the placenta, heart, brain, and face of embryonic day (E) 10.5 conceptuses developed in young and aged female mice were analyzed using RNA-seq, which identified that the brain is the tissue most profoundly affected by advanced maternal age. The embryonic brains of conceptuses from aged mouse females had the most differentially expressed genes, displayed transcriptional evidence for a slight developmental delay, and exhibited decreased expression of neurodevelopmental disorder genes. These transcriptomic changes are likely mediated through the placenta, whose trophoblast portion was grossly underdeveloped, and are most likely due to defective signaling cues emanating from the aged uterine stroma. In an attempt to identify putative master regulators of shared gene networks between the four tissues of interest, the RNA-seq data from E10.5 placenta, heart, brain, and face revealed a key gene of interest, Peg3, which is a recruiter of epigenetic modifiers that build repressive chromatin. To investigate its role in the placenta, I used trophoblast stem cells (TSCs), generated Peg3 KO TSC clones, and performed cell assays and sequencing analyses. Deletion of Peg3 resulted in the disruption of mitosis, most likely due to increased DNA damage, which created anaphase bridges and produced “donut-shaped” nuclei. Furthermore, Peg3 KO cells exhibited proliferation defects that may be tied to errors in karyokinesis. These results were corroborated by the gene ontology terms enriched for DNA repair and mitosis amongst downregulated genes. As might be predicted, there was also a global reduction in DNA methylation, which was mainly localized to CpG islands (CGIs). This hypomethylation resulted in the mis-expression of lineage inappropriate genes and contributed to aberrant TSC behaviour. Therefore, PEG3 is required for proper mitosis and differentiation of TSCs, as well as to help orchestrate the DNA methylation landscape.