The Role of Hippo Signalling in Cell Fate Decisions in Mouse Embryonic Stem Cells and Pre-Implantation Development

Abstract

The Hippo signalling pathway is a conserved kinase cascade involved inthe regulation of tissue growth and organ size. Activation of the Hippopathway through multiple upstream inputs results in phosphorylation andinactivation of the transcriptional co-activator, Yes associated protein(Yap). While the role of Yap as an oncogene and an effector of the Hippopathway is well established, its role in cellular differentiation is less wellknown. In this present work I have utilised quantitativeimmunofluorescence analysis to examine the expression of Yap in thedifferentiation of mouse embryonic stem cells (mESCs). I show thatdifferentiation of mESCs is accompanied by an initial increase in nuclearYap expression. Furthermore, I show that this increase in nuclear Yapexpression is associated with differentiation towards the primitiveendoderm lineage (PrE). Moreover, small molecule inhibition of Yap wasable to decrease the proportion of cells differentiating towards PrE.Following on from these in vitro studies, I examine the expression of Yapin vivo in the corresponding differentiation event in mouse preimplantationembryos. I show that increased nuclear Yap expression isassociated with expression of the PrE-specific transcription factor Gata6during specification and eventual sorting of the PrE. Culturing embryos inthe presence of small molecule inhibitors of Yap resulted in decreasedexpression of Gata6 and a reduction in trophectoderm cell number.These studies demonstrate that Yap is involved in the process of cellulardifferentiation and is associated with specification of the PrE lineage.Finally I attempt to create an inducible knockout of Yap in mESCs using aserial targeting strategy, with the intention of creating a model system inwhich to examine the role of Yap in mESCs.

Date of Award	25 Apr 2016
Original language	English
Awarding Institution	University of Bath
Supervisor	Silvia Munoz-Descalzo (Supervisor) & David Tosh (Supervisor)