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.

Understanding How Cells Make Decisions During Early Development The development of a complex organism begins with a small group of unspecialized cells. As development progresses, these cells must make a series of important decisions that determine what types of tissues and organs they will eventually become. Understanding how these decisions are made is one of the central challenges in modern biology. This project investigated the mechanisms that guide one of the earliest and most important cell fate decisions during embryonic development: the choice between endoderm and mesoderm. These two cell populations give rise to many essential organs and tissues. Endodermal cells eventually form organs such as the liver, pancreas, lungs, and digestive tract, while mesodermal cells contribute to the heart, muscles, blood, and other tissues. Although scientists have identified many molecules involved in these developmental processes, a comprehensive understanding of how cells decide between alternative developmental paths has remained elusive. To address this challenge, the project developed innovative genetic screening approaches that allow researchers to systematically identify the genes and molecular pathways controlling these decisions. A major achievement of the project was the establishment of advanced experimental platforms that recreate key stages of early development in the laboratory. These include both conventional cell culture systems and a novel three-dimensional screening platform capable of generating and analyzing hundreds of thousands of miniature cell aggregates. By combining these models with fluorescent markers and high-throughput sorting technologies, researchers can track cell identity and efficiently identify factors that influence developmental choices. The newly developed methods provide powerful tools for studying how cell identities are established and maintained. They also enable the investigation of complex genetic networks that govern the formation of specific cell types. By revealing the molecular machinery that controls lineage choice, the project contributes fundamental knowledge about how embryos develop and how cells acquire their specialized functions. Beyond its importance for basic research, this work has significant implications for regenerative medicine. A deeper understanding of cell fate decisions may ultimately help scientists produce medically relevant cell types, such as insulin-producing cells for diabetes treatment or cardiac cells for heart repair, with greater efficiency and precision. Overall, the project provides important new tools and insights for understanding early development and brings us closer to the long-term goal of controlling cell fate for biomedical applications.