Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Developmental Biology

Supervisor

Dr. Gregory M. Kelly

Abstract

Mouse F9 cells differentiate into primitive endoderm (PrE) when treated with retinoic acid (RA) and into parietal endoderm (PE) following subsequent treatment with dibutyryl cAMP. Wnt6 is up-regulated in PrE cell, and although it is sufficient to induce differentiation by signaling through the canonical WNT/β-catenin pathway, the mechanism by which the Wnt6 gene is regulated is not known. In addition to WNT signaling, PrE differentiation is accompanied by an increase in reactive oxygen species (ROS). ROS have been implicated in regulating the canonical WNT/β-catenin signaling pathway through Nucleoredoxin (NRX), but whether they are sufficient to induce extraembryonic endoderm in vitro is not known. In F9 cells the overexpression of Gata6 or Foxa2, which are two integral members responsible for patterning extraembryonic endoderm, induces biochemical and morphological markers of PrE by directly up-regulating the expression of Wnt6, and activating the canonical WNT/β-catenin signaling pathway. Treating cells with H2O2, or knocking down the expression of Nrx also activates canonical WNT/β-catenin signaling leading to the induction of these markers. Treating cells with antioxidants, however, impedes the ability of RA to induce PrE. Furthermore, and regardless as to how F9 cells are induced, these PrE cells remain competent and differentiate into PE when treated with db-cAMP. Together, these results indicate that Gata6 and Foxa2 are responsible for initiating the canonical WNT/β-catenin pathway in F9 cells and ROS, impinging on NRX, regulate the pathway necessary for PrE differentiation.

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