Project Summary In the first two weeks of development, the human embryo breaks symmetry twice, transforming itself from a uniform ball of cells into a highly-patterned, spatially organized set of tissues. Although decades of elegant genetics and biochemistry in non-human model organisms have uncovered many of the essential signaling proteins and pathways for embryonic patterning at this early stage, a lack of tools for directly manipulating the signaling in time and space, as well as limitations to working with human embryos, have limited our ability to understand how these early patterning events arise in humans. Thus, my laboratory seeks to develop engineering strategies to (1) understand what patterns of signaling activity encode extracellular information, and (2) determine how these patterns are decoded at the tissue level to drive high fidelity collective cell fate decisions in human embryonic stem cells. This proposal brings together many recent advances in stem cell and molecular engineering to decode how spatiotemporal signaling and embryo size instruct tissue fate patterning. We leverage advances in 2D micropatterning, 3D bioprinting, cellular optogenetic control over developmental signaling pathways, and CRISPR-based reporters of cell signaling pathways. Together, these technologies give us unprecedented control over and visualization of microengineered models of human gastrulation, thereby enabling us to investigate the principles of environmental information transmission and potential mechanisms of pregnancy loss and developmental anomalies that arise with a surprisingly high frequency (10-20% by some estimates) in the early human embryo. In addition, our platform does not face the same ethical barriers that have limited human embryo research, allowing us to ascertain how physical and information-bearing parameters of the embryo lead to stereotyped patterning of the germ layers during human gastrulation. In this proposal, we focus on the role of canonical developmental signaling pathways by dissecting the effects of spatiotemporal signaling and variance of the Wnt pathway on germ layer fate positioning in 2D (Aim 1); examining the role of Erk signaling on positioning, dynamics, and coordination of cells during the development of the mesoderm/trophectoderm boundary (Aim 2); and examining the effect of cell number and tissue size in 3D gastrulating models of the human epiblast and amniotic sac (Aim 3).