Regulation of death and differentiation of embryonic stem cells
Creators: name: LeBlanc, Lucy
Publisher: name: The University of Texas at Austin
Year: 2021
Subjects: Embryonic stem cells
Pluripotency
Differentiation
Hippo signaling
Wnt signaling
Yap1
Apoptosis
Beta-catenin
Esrrb
Cell density

description: Embryonic stem cells (ESCs) possess two notable traits: self-renewal, the ability to proliferate without senescence or quiescence, and pluripotency, the capacity to differentiate into nearly any cell type in the body. Whereas the transcriptional regulatory circuitry of self-renewal has been extensively characterized, the mechanisms underlying proper differentiation are less well known. My dissertation focuses on two pressing questions regarding the molecular regulation of cell fate. First, during differentiation, ~30% of ESCs undergo apoptosis, likely to cull cells that resist differentiation and thus allow the overall cell population to differentiate successfully. However, upstream regulators that control the endogenous rate of apoptosis are not known. Second, although it has been observed qualitatively that selecting appropriate seeding densities for cell culture is critical for proper differentiation, the effects of seeding density on global gene expression and cell fate specification have not yet been quantitatively measured. To address these questions, first, I investigate the dependence of mouse ESCs (mESCs) on the Hippo transcriptional effector Yap1 during differentiation. I show that genetic ablation of Yap1 substantially augments the rate of apoptosis during differentiation, but not self-renewal, regardless of intended cell fate. Mechanistically, I demonstrate that Yap1 regulates the transcription of anti- and pro-apoptotic genes. The absence of Yap1 results in heightened mitochondrial priming, leading to elevated activation of the endogenous apoptotic cascade. Finally, I demonstrate that ectopic expression of pro- and anti-apoptotic factors as well as the Yap1 paralog Taz allow for modulation of apoptosis during differentiation. To quantitatively address the effect of seeding density on global gene expression, I perform transcriptomic analysis of mESCs during both self-renewal and differentiation over a gradient of seeding densities. I show that a simple change in seeding density is sufficient to alter the expression of thousands of genes, including those involved in cell fate specification. I demonstrate that low seeding density facilitates more efficient mESC differentiation during LIF withdrawal due to enhanced upregulation of lineage markers. Notably, I uncover three transcriptional regulators (β-catenin, Tcf7l1, and Esrrb) that activate or repress many of these density-sensitive genes. Perturbation of a single density regulator is sufficient to shift gene expression patterns from a low density profile to a high density-like profile, and vice versa. My findings in this dissertation highlight the contributions of several transcription factors and co-regulators to fate specification in the context of programmed cell death and seeding density. Taken together, this work illustrates several molecular mechanisms of the regulation of mESC differentiation