The basic architecture of animal bodies is established during early embryogenesis. Compared to other vertebrate systems, little is known about how a mammalian egg creates the basic body plan. The major goal of this study is to elucidate the molecular mechanisms responsible for cell fate determination and body axis formation in the mouse embryo. The first specific aim of this study is to identify cytoplasmic and membrane components in the egg that are involved in the formation of the inner cell mass (ICM) and the trophectoderm (TE). Formation of the ICM and TE is the first cell differentiation event in mouse development. We will use lineage-specific molecular markers to identify the differentiation of these two distinct cell types in embryos treated with various inhibitors of the cytoskeleton, membrane trafficking and cell adhesion. We will also investigate the role of egg cytoplasmic components in the ICM and TE development. For this study, we will exploit the centrifugation techniques, which have been successfully used to identify developmentally important factors in the egg of other vertebrates and chordates.
The second aim i s to analyze the function of localized molecules in the egg, namely Stat3 and Leptin proteins. Since both Stat3 and Leptin act as regulators of cell differentiation and growth in many systems, we hypothesize that the localized distribution of Stat3 and Leptin in the egg plays an important role in the establishment of the body plan. We will test this hypothesis by overexpressing Stat3 and Leptin in the mouse egg to disturb their normal distribution. We will also use constitutive active and dominant negative constructs of Stat3 to examine the function of the localized Stat3 in more detail.
The third aim i s to define the role of the Wnt/b-catenin signaling pathway in body axis formation in the embryo. Although the signaling pathway is suggested to be involved in axis formation in the mouse embryo, its mode of action is not as clear as in other vertebrate systems, specifically the frog. We will define the role of the Wnt/b-catenin signaling pathway in the mouse axis formation in more detail by introducing inhibitory constructs of the signaling pathway, and by locally activating the signaling pathway in early embryos. The studies proposed in this application should enable us to better understand the molecular mechanisms of the earliest critical events in mammalian body plan formation, and provide clues to resolve many problems associated with human birth defects as well as biomedical technology.
