Mechanisms of epiblast and primitive endoderm segregation

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Abstract Embryonic development is a fascinating process during which different cells types are made and organized into complex and functional structures, ultimately building an entire organism. If we watch embryos develop, patterns emerge with remarkable reproducibility. However, if we zoom in to the level of individual cells, the scene, especially during mammalian development, often seems chaotic. Cells move around, change shape, contact different neighbors and show fluctuations in their gene expression patterns. Yet from this apparent chaos, robust and reproducible patterns emerge. How are the right cell types made at the right time and place in correct proportions? In this proposal we will use a crucial cell fate decision in the preimplantation mouse embryo, when the inner cell mass (ICM) lineage segregates into the epiblast (EPI) and the primitive endoderm (PE) lineages, the cell types that will give rise to most of the fetus and extraembryonic cell types, respectively, as a model system to reveal fundamental insights into the molecular and cellular mechanisms that ensure robust and reproducible lineage formation. While studies employing genetic and pharmacological approaches have established the necessary molecular players involved in EPI and PE cell fates, these are limited in providing temporal information on an inherently dynamic process. Additionally, ICM cells differentiate into EPI and PE cell types in a seemingly random pattern, which varies from one embryo to the next, complicating the interpretation of analysis performed only at fixed time points. Here we develop novel genetically encoded fluorescent reporter mouse lines for key factors involved in EPI and PE cell fates, which allow us to visualize and probe the mechanisms of cell fate acquisition with unprecedented spatial and temporal resolution, using a combination of cutting-edge live imaging, computational image processing, and genomics approaches. Using these powerful tools, we will investigate the mechanism driving initial symmetry-breaking in the ICM to initiate EPI/PE lineage differentiation and determine whether purely stochastic fluctuations or more predictable systematic variations present in the embryo are responsible for initiating this fate decision. Next, we will determine how cell fates are propagated over space and time to achieve reproducible cell-type proportions, by uniquely visualizing multiple nodes in cell-cell communication signaling. Finally, we will use recently developed genomics techniques to probe transcription factor occupancy and chromatin accessibility to observe how cell type-specific transcriptional programs are set up by key factors during EPI/PE lineage segregation. This work will uncover fundamental mechanisms by which variable developmental process can lead to robust and reproducible developmental patterning in the early mammalian embryo.