Doctor of Philosophy
Kelly, Greg M.
The inner cell mass undergoes orchestrated cellular divisions resulting in the formation of the epiblast (EPI) and primitive endoderm (PrE). Understanding the process of cell fate specification is crucial to appreciate the intricacies of proper embryonic development. While the mouse embryo is an excellent model, limitations do exist with number, technical challenges, and accessibility, therefore, in my thesis I employed two cell-based models to recapitulate the EPI-PrE fate in vitro. Many signaling pathways have been implicated in this lineage decision, metabolism and its downstream products have been recently regarded as a driver of lineage commitment. Using various biochemical, molecular, and computational modalities, I sought to understand how metabolism and ROS play a role in XEN differentiation. In Chapter 2, I demonstrated that ROS generated by NOX1 and NOX4, which are regulated by the GATA6 transcription factor, is required to induce XEN-like differentiation. While exogenous ROS supplementation promoted XEN-like differentiation, overexpressing Nox1 and/or Nox4 produced high levels of ROS yet failed to promote XEN-like differentiation. Next in Chapter 3, I examined if mitochondrial ROS played a role in XEN-like differentiation. Strikingly, the metabolic profile of XEN-like cells was glycolytic in nature. Furthermore, these observations were further supported using embryo-derived XEN cells (Chapter 4), which also relied on glycolysis as an energy source. Metabolomic and biochemical analyses showcased how embryo-derived XEN cells maintain high lactate levels by increased LDHA activity and re-routing pyruvate away from the mitochondria. More importantly, modulating pyruvate-lactate fate using pharmacological agents affected XEN differentiation in vitro. Collectively, my results emphasize how metabolism, and its products contribute to XEN differentiation, and more broadly serves to expand our knowledge of mammalian development.
Summary for Lay Audience
One of the earliest cell type decisions that embryos make is the commitment of cells to either the epiblast (EPI), which gives rise to the embryo proper, or the primitive endoderm (PrE) lineage. Often neglected, the PrE supports the EPI by ensuring adequate nutrient and gas exchange, and later its involved in gut organ formation. Many factors play a role in the establishment of these lineages, but of interest to me is metabolism.
Metabolism is a set of chemical reactions that ensure the survival of an organism, specifically, the building or breaking down of energy from food sources. Molecularly, understanding how metabolites and by-products of metabolism, such as reactive oxygen species (ROS), influence this lineage decision is crucial to understand normal embryonic development. Due to the lack of accessibility of EPI and PrE cells from the embryo, I turned to using embryonic stem (ES) cells and embryo-derived extraembryonic endoderm (XEN) cells, which can be maintained in vitro indefinitely under specific culture conditions. In addition, I employed the F9 model, which mimics the transition towards PrE-like lineage when treated with differentiation factors. I hypothesized that both ES and F9 cells have distinct metabolic and Nox (enzymes that generate ROS) expression profiles compared to their XEN counterparts, and that changes in these profiles direct cell fate determination.
I found that GATA6, a PrE lineage inducer, increases Nox1 and Nox4 levels, which are required but not sufficient to form XEN, suggesting that additional mitochondrial ROS sources may be required for XEN differentiation. Surprisingly, XEN-like cells were more reliant on glycolysis than oxidative phosphorylation (OXPHOS), a similar observation was seen in embryo-derived XEN cells. Using cutting edge technologies, I implicated a key metabolite, lactate, as a driver of XEN differentiation in vitro. This thesis is the first to highlight a role for NOX-derived ROS in XEN-like differentiation and implicates lactate in the differentiation towards the XEN lineage. This knowledge helps us understand how factors influence fate decisions, which serves to enhance regenerative medicine therapies.
Gatie, Mohamed, "Metabolic regulation during extraembryonic endoderm differentiation" (2021). Electronic Thesis and Dissertation Repository. 8259.
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