Electronic Thesis and Dissertation Repository


Master of Science




Dr. Gregory M. Kelly


Mouse F9 cells differentiate into primitive extraembryonic endoderm (PrE) with retinoic acid (RA) treatment, resulting in up-regulation of Gata6, which when translated directly activates Wnt6. Canonical Wnt signalling is required for PrE differentiation, and this, like most developmental processes, requires input from one or more additional pathways, including hedgehog (Hh). The Hh pathway is regulated by GATA6, and crosstalks positively/negatively with Wnt signalling. Ihh up-regulation in F9 cells accompanies PrE induction, but a role for GATA6 or Hh pathway activation in obligatory Wnt/ß-catenin signalling is not known. To address this, I show that RA induces Ihh and altered expression of Hh targets. Overexpressing Gata6 alone significantly induced Ihh. When Hh signalling was blocked with cyclopamine, RA-induced differentiation, as analyzed with TROMA-1 and DAB-2 signal, was significantly reduced, demonstrating requirement of the Hh pathway for PrE differentiation. Interestingly, each of SAG, BIO, and GLI3A overexpression failed to induce markers of PrE differentiation. These results indicate a regulatory role for Hh in PrE differentiation, as the pathway is required, but is not independently sufficient for this event.