Pluripotent cells have the potential to create any cell type, and therefore represent a source of healthy cells by which to replace diseased cells in regenerative therapies. Thus, understanding how pluripotent cells are established and regulated is a major milestone in developing safe and effective regenerative therapies. In the mammalian embryo pluripotent cells emerge through two rounds of cell fate decisions that in addition to establishing pluripotent cells, establish two differentiated cell lineages. The molecules and signaling pathways that regulate these cell fate decisions represent key players in the establishment and regulation of pluripotent cells. In this thesis, I use the mouse embryo as a system in which to gain insight into how pluripotency is established and regulated. Cdx2, Oct4 and Sox2 are transcription factors that are important regulators of pluripotency in vitro, but their role in regulating the cell fate decisions that establish pluripotency in vivo is less clear. By using a loss-of-function approach, I define the roles of each of these factors in regulating the cell fate decisions that establish pluripotency in the early mouse embryo. First, by eliminating maternal and zygotic Cdx2 I demonstrate that Cdx2 is not a maternal determinant for pluripotency or differentiation during early embryo cell fate decisions. Second, I eliminate both maternal and zygotic Oct4 and demonstrate that the initial specification of pluripotent and non-pluripotent lineages in the early mouse embryo is independent of Oct4, and that instead, Oct4 cell-autonomously regulates both formation of pluripotent cells, and differentiation of a non-pluripotent lineage, the primitive endoderm. Third, I demonstrate that in contrast to Oct4, Sox2 promotes differentiation of primitive endoderm non-cell autonomously. These results support the model that pluripotency is acquired through these early embryonic cell fate decisions, rather than being determined during early embryogenesis. Furthermore, analysis of the roles of Oct4 and Sox2 in these early embryonic cell fate decisions highlights distinct roles for these factors in establishing pluripotency and suggests that although in vitro Oct4 and Sox2 appear to have overlapping roles, these master regulators of pluripotency have distinct cell type specific roles in regulating the cell fate decisions that establish pluripotency in the early mouse embryo. Taken together, the research presented in this thesis greatly increases our understanding of the regulation of the cell fate decisions that establish pluripotency in the embryo and provides important insights into the biology of key regulators of pluripotency in vitro and in vivo.
