Mechanisms of endo-mesodermal lineage choice

Acquisition of definitive cell identity during gastrulation is a crucial event during embryogenesis. However, we still lack a comprehensive understanding of the factors and genetic networks involved in making and regulating the cell fate decision that establish definitive lineage identity. Here, we will specifically focus on the lineage decisions resulting in the formation of definitive endoderm. A detailed understanding of endo-mesodermal lineage choice will have important implications for regenerative medicine approaches to establish cell types of endo-mesodermal origin, such as insulin producing beta cells or heart tissues. We propose that only by understanding the key molecular steps of cell fate choice, we will be able to experimentally control cell fate. The overall goal of this project is to identify factors regulating endo-mesodermal lineage choice, and to understand their mode of action. To this end, we have developed and will perform robust and precise genetic screens, using a cell line reporting on a continuum of cell states from the initiation of endo-mesoderm via cells at the point of decision making between mesoderm and endoderm to cells of definitive endoderm identity. To identify the key regulators of endo-mesodermal differentiation, we will use monolayer-based screens, but will also use a novel clonal 3D-based screening system. This platform will be able to form hundreds of thousands of clonal embryoid bodies (EBs), 3D model systems for complex early differentiation processes. Combining this high-throughput compatible 3D model with the availability of a large particle sorter we will be able to fraction EBs based on fluorescent parameters in a manner compatible with high-throughput genetic screens. In sum, this project will provide fundamental insights into the decision machinery that regulates definitive lineage choice.