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.