Embryonic stem cell differentiation: A key to unraveling the cellular mechanisms of patterning

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The complexity, intricacy and efficacy of the process that orchestrates the changes in form and function of the fertilized egg as it develops to the embryo presents one of the most fascinating and fundamental problems of biology. Over the past decades, the genetic networks and signaling pathways that are key to the development of the early embryo have been identified and described in detail. However, the unmitigated complexity of the individual pathways, in particular their interaction and dependence on time have so far prevented attempts to unambiguously attribute temporal signaling cues to a specific behavior of a cell. This proposal presents a strategy for closing this gap by suggesting a dedicated set of experiments to which a quantitative model of signaling pathway activity and cell fate in cell cultures of human embryonic stem cells (hESC) is to be fitted. The project builds on two recent findings of the host laboratories of Profs. Siggia and Brivanlou at Rockefeller University, New York, USA. In recent experiments, they could show that uniform application of growth factors to circularly confined stem cell colonies is sufficient to recreate the spatial patterning into the germ layers of the human embryo, highlighting the importance of self-organization through cell-to-cell communication and the geometrical shape of the colonies. On the theoretical side, Prof. Siggia's group has recently developed a framework for fitting a parsimonious, yet quantitative model to experimental data from Caenorhabditis elegans in order to describe cell fate decision making as a function of two signaling pathways. This approach circumvents the molecular details of the pathways and describes their effective action with a minimal number of aggregate variables. We propose a set of experiments that will be amenable to quantification within the framework described above. Human embryonic stem cells will be grown in different confining geometries and the activity of pathways, in particular TGF-ß and Wnt, as well as the cell fates will be determined at cellular resolution using immunofluorescent staining and live fluorescent reporters of cell fate and pathway activity. This data will provide a mapping of the signaling pathway activity to cell fates in a space and time resolved manner and will also provide unprecedented information about the cell-to-cell communication that leads to a particular fate pattern. A quantitative mathematical model of cell fate adapted to the case of embryonic stem cells will then be fitted to the data. The proposed research not only will provide unique insight into early mammalian embryogenesis, in particular in the human. Because of the characterization of pathways highly conserved across species and relevant for a wide range of cellular processes in the adult, the impact of the proposed research extends from embryogenesis to topics as diverse as organogenesis, regenerative medicine, and cancer.