The roles of Rho GTPases on mouse embryonic stem cell differentiation

• Main Author: Nee, Adina Huey-Fang
• Other Authors: Koh Cheng Gee
• Format: Final Year Project
• Language: English
• Published: 2016
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/67365
• Institution: Nanyang Technological University

Rho GTPases are key regulators of actin cytoskeleton dynamics that influence a diverse array of biological processes. Of which, RhoA, Rac1 and Cdc42 are the most highly conserved and well-characterized Rho GTPases. Notably, RhoA induces actin:mysosin filaments and stress fiber formation, Rac1 induces lamellipodia while Cdc42 induces filopodia. Despite the established roles of Cdc42 in later developmental stages, its roles in mediating lineage specification during early embryogenesis remain elusive. As such, our initial focus was to elucidate the roles of Cdc42 on lineage specification at the mRNA and protein level using RT qPCR and western immunoblot respectively. Through immunoblot analysis, we established that Cdc42 predominantly enhances ectodermal differentiation but inhibits mesodermal and endodermal differentiation. Delving deeper into the mechanism underlying lineage specification, Cdc42 mediating inhibitory phosphorylation on its downstream effector, GSK3β, have been thought to modulate Wnt signaling which, influences cell fate specification. Despite this link being drawn, the role of Cdc42-dependent GSK3β inhibition in mediating germ layer differentiation during early embryogenesis remains unclear. Thereby, the present study mimicked the overexpression Cdc42-dependent GSK3β inhibition using lithium chloride and investigated its effects on lineage specification. Under the overexpression of Cdc42-dependent GSK3β inhibition, only ectodermal Nestin and endodermal FOXA2 differentiation were upregulated.