Date of Award


Degree Type


Degree Name

Master of Science


Developmental Biology


Dr. Greg Kelly


Development of extraembryonic endoderm is essential for proper placenta formation and mammalian embryogenesis. Formation of primitive endoderm from cells of the inner cell mass is one of the earliest cell fate decisions made in the developing embryo. However, the signals and mechanisms involved in orchestrating this developmental process are not completely understood and are difficult to study in vivo. The mouse embryonic carcinoma (F9) cell line can be chemically re-programmed to mimic cells of the inner cell mass, thereby serving as an in vitro model to study extraembryonic endoderm differentiation. F9 cells treated with retinoic acid (RA) differentiate into primitive endoderm Similarly, activation of the Wnt-β-catenin pathway is also sufficient to induce differentiation. Reactive oxygen species (ROS), including H202 have been identified as key regulators of normal cell physiology. In the present study, a sustained increase in the levels of ROS was found when F9 cells were treated with RA. In addition, an increase in TCF-LEF transcriptional activity, a read out of Wnt-β-catenin signaling, was also seen in response to exogenous H202 treatment. Immunoblot and immunofluorescence analyses using molecular markers of differentiation confirmed that H202-treated F9 cells had formed primitive endoderm. In contrast, differentiation was blocked when RA-treated

cells were exposed to the antioxidants N-Acetyl Cysteine or Trolox. In a survey to identify the source of the ROS, a candidate gene encoding the NADPH oxidase NOX4,

was identified as being up-regulated in response to RA. Furthermore, diphenyleneiodonium chloride a general NADPH oxidase inhibitor, was shown to attenuate the RA-induced differentiation, implicating NADPH oxidases and possibly


NOX4, as the source of H2O2. Together, these results strongly suggest that ROS impinge on the Wnt-β-catenin signaling that is required for the cell fate decision making

process(es) that lead to the formation of extraembryonic endoderm.



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