Investigating the molecular regulation and morphological dynamics of the Visceral Endoderm

Cell migration is of huge importance in the field of development, where the correct positioning of signaling centers and functional structures is of paramount importance. An example of this occurs within the Visceral Endoderm (VE), an extra-embryonic epithelium that is involved in patterning the embryo. A specialized subset of VE cells known as the anterior visceral endoderm (AVE) is induced at the distal tip of the egg∼cylinder, and is responsible for specifying anterior embryo pattern. AVE cells migrate in a stereotypic manner towards the prospective anterior. Failure of this migration leads to dramatic mis-patterning of the primary body axis and renders the embryo unviable. The main aim of my D.Phil is to investigate cell movements throughout the VE, in order to understand the behaviour of both the AVE and non-AVE cells and to give insight into the global cell dynamics of reorganizing epithelial tissues during development. After using DIC and fluorescence microscopy to visualize migratory processes I then applied cell-tracking analysis to the resultant data. Confirming and furthering work that quantified cell movement on the anterior face of the embryo, I performed time∼lapse microscopy to visualize the entire surface of the embryo as the AVE migrated, in higher temporal and axial resolution than has been demonstrated to date. Cell∼tracking analysis of this data indicates that these areas exhibit different cell behaviour to that previously quantified for the anterior side of the embryo. Further to this, I have developed a novel method of imaging the distal tip of the egg∼cylinder embryo. Using this technique, I have generated high definition time∼lapse sequences of induced AVE cells prior to migration, allowing previously uncharacterized behavior to be described. In order to fully understand the cell dynamics of the VE, I took extremely high spatial resolution fluorescence microscopy of the VE from live embryos in culture and manually segmented the tissue in 3D. This allowed the description of regional differences of cell shape within the tissue. The Wnt/PCP pathway has been implicated in coordinating AVE migration. The second major aim of my project is to investigate how Planar Cell Polarity (PCP) signaling controls the behaviour of cells in the VE. I investigated the PCP core component Vangl2 using mice that contain a dominant negative version of this protein, and determined that there was no overt AVE migration defects in mutant embryos. In order to investigate the role of the Vangl2-binding PCP component Dvl-2, specifically examining whether localized recruitment of Dvl-2 in the VE is obligatory for normal AVE migration, I have generated genetic tools for use in transient transgenics. Further to this, I have demonstrated a role for the Dvl-2-binding protein β- arrestin2 in AVE migration, using mice null for this protein.