Date of Award
Master of Science (MSc)
In addition to its roles in developmental and cancer-related processes, emerging evidence suggests that the canonical Wnt pathway is also involved in regulating stem cell self-renewal and differentiation. The ubiquitous serine/threonine kinase, Glycogen Synthase Kinase-3 (GSK-3) is a key regulator of this pathway. Genetic ablation of the two GSK-3 genes α and β (referred to as DKO, for double knockout) in mouse embryonic stem cells (mESCs), renders them incapable of efficiently differentiating into the three germ layers. In an attempt to rescue the differentiation blockade of DKO mESCs, we expressed dominant-negative forms of the TCFs (TCF1, LEF1, TCF3 and TCF4), the final effector molecules of the canonical Wnt pathway. The expression of dominant-negative TCF1, LEF1 or TCF4 was tolerated and resulted in attenuated expression of β-catenin/TCF target gene expression in cells that continued to self-renew and express pluripotency markers. By contrast, the expression of dominant-negative TCF3 resulted in cell differentiation/death. Thus, TCF3 appears to play a unique role in the regulation of mESC properties. To clarify the underlying mechanism of TCF3’s action in mESCs, we examined the effects of expressing a dominant-negative TCF3 (TCF3DN) and full-length TCF3 (TCF3FL) in DKO and wild-type mESCs using an inducible doxycycline-regulated system. We found that expression of either TCF3DN or TCF3FL in DKO and wild-type mESCs down-regulates TCF activity and Nanog expression. However, expression of TCF3DN or TCF3FL in DKO mESCs was unable to rescue the neuroectoderm blockade, but may have directed cells to differentiate into the endoderm lineage. Moreover, we have established a potential role for β-catenin in regulating the expression levels of TCF3 itself. The information obtained from these studies provides new insights into the mechanisms through which TCF3 regulates mESC self-renewal and differentiation.
Ng, Deborah, "Delineating the Role of TCF3 in Mouse Embryonic Stem Cell Self-Renewal and Differentiation" (2012). Open Access Dissertations and Theses. Paper 6724.
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