Project Summary: It is becoming increasingly clear that one of the ways that cells interpret and encode information into multiple cell fates is by multiplexing information through dynamic encoding. This is especially true for the MAPK/Erk pathway, that governs many cell processes including cell proliferation, differentiation and migration. For years, how such diverse outcomes were controlled by the same pathway remained elusive, but the advent of single cell studies and optogenetics has elucidated the many ways in which Erk activity can be interpreted into distinct cell fates, and even more recently, the role of dynamics in these complex decisions. The role of developmental Erk dynamics in determining and coordinating human gastrulation, however, has not yet been investigated. We will combine live cell kinase activity reporters and optogenetic control over intracellular signaling pathways to probe the role of ERK dynamics in positioning and coordinating the three germ layers. Additionally, we will uncover whether RASopathy causing mutations influence human gastrulation to pace the way for potential therapeutic intervention. This proposal brings together recent advances in stem cell and molecular engineering. We utilize advances in 2D micropatterning, cellular optogenetic control, live cell kinase activity reporters and CRISPR Cas9 genome editing. Together, these technologies give us unprecedented control over and visualization of microengineered models of human gastrulation, thereby enabling us to investigate the principles of dynamic information transmission. Our platform does not face the same ethical barriers that have limited human embryo research, allowing us to provide otherwise unavailable information about human embryonic development. In this proposal we focus on the role of Erk signaling dynamics in coordinating the three germ layers, as well as uncover impact of RASopathy mutations. In Aim 1 we will image and quantify Erk signaling dynamics using the Erk kinase translocation reporter during the process of gastrulation and determine which features of signaling dynamics are predictive of germ layer fate. Aim 2 will allow us to identify which of these dynamical features are sufficient to determine the cell fate outcome using cellular optogenetics. Finally, in Aim 3 we will uncover whether RASopathy mutations lead to gastrulation defects and investigate whether these are linked to disruption to Erk dynamical signatures using CRISPR Cas9 gene editing and our 2D gastruloid model. Approaching this cell biological question from a systems level perspective, using reproducible precisely controllable tools that are otherwise unavailable without optogenetics and microengineered platforms, has the potential to shine new light on the field.