Early mammalian development is characterized by formation of the blastocyst, composed of the pluripotent inner cell mass surrounded by a one-cell layer of epithelial cells, the trophectoderm. Despite its importance for understanding mammalian development and for stem cell research, the mechanism of blastocyst morphogenesis and patterning has long been elusive and a subject of controversy. The ultimate goal of this project is to understand the molecular mechanism of the lineage establishment in the mouse blastocyst.To this end, functional screens will be performed to identify players essential for the lineage specification. Those gene sets specifically expressed in each cell lineage will be characterized by single-cell gene expression profiling methods. Functional screens will be performed using a subset of siRNA libraries directed against each gene set in order to identify key molecules for the blastocyst differentiation. Here, fluorescent reporter lines established in the host laboratory will serve as a unique reference for the read-out of molecular dynamics during embryonic patterning. Alternatively, among those gene sets, potential candidate molecules will be first identified by focusing to the upstream players and small RNAs in signaling cascades in the lineage specification. Their function will then be analyzed by siRNA-mediated knock-down. Thus, these findings will provide a mechanistic model underlying the lineage establishment in the early mouse embryo. Taken together, the project will provide not only an insight into the pluripotency in vivo but a basis for an interdisciplinary approach for stem cell research and regenerative medicine. It is likely that the techniques established in the project will serve as a unique tool for embryonic development research and will result in a long lasting fruitful collaboration.