Elucidating the Role of the Cell Cycle in Regulating Early Post Implantation Mammalian Embryonic Development

Abstract

Following implantation of the murine embryo into the maternal uterine endometrium, the embryo undergoes significant morphogenetic changes that are essential for proper development. Polarization cues from the surrounding basement membrane direct the morphogenesis of embryonic pluripotent epiblast cells into an apical-basal polarized cup-shaped epithelium. During this process the proamniotic cavity, an essential embryonic structural element involved in embryonic growth and patterning, is formed. Proamniotic cavity formation is coincident with the time the embryo is known to exhibit cell number regulation to ensure a proper cell number prior to gastrulation. Following the successful formation of the proamniotic cavity, gastrulation occurs, where the three mammalian germ layers (ectoderm, mesoderm, and endoderm) are specified from the one-dimensional epithelialized layer of epiblast cells, with the mesoderm emerging first from the primitive streak. The post implantation epiblast is a highly proliferative tissue prior to and during gastrulation and previous studies have implicated the cell cycle in both cell number control and in “licensing” germ layer specification in an in vitro model. However, these studies have been limited by the inability to accurately measure cell cycle progression in live embryos or stem cell models. Thus, our mechanistic understanding of how the cell cycle might impinge on these processes is incomplete. This study uses in vitro models of the post implantation epiblast and a recently published live reporter of the cell cycle, PIP-FUCCI, to study the role of the cell cycle in regulating early post implantation development. In this thesis, I first confirmed the ability to recreate the pluripotent state of the post implantation epiblast in vitro. Next, I cloned plasmids that would be used in a CRISPR experiment to verify the role of PIP-FUCCI as an accurate reporter of the cell cycle in a pluripotent cell line. Using a three-dimensional in vitro culture system of pluripotent stem cells that mimics events in the post implantation epiblast concurrent with when size regulation occurs, I identified a time window of rosette formation that resembles the epithelialization of the epiblast and proamniotic cavity formation. This system will be used to investigate the relationship between cell cycle progression and morphogenesis. Finally, using the novel PIP-FUCCI reporter, I found that cells in the G1 phase of the cell cycle upon initiation of primitive streak-like differentiation were more likely than cells in the S or G2 phases to differentiate into the mesoderm germ layer. The results of the work in this thesis will contribute to future studies of the mechanisms of embryonic size control and expand the field’s understanding of cell fate acquisition during gastrulation.