Abstract (Project Summary) A major goal of developmental biology is to understand the detailed molecular progression of all embryonic cell lineages, from pluripotency to adulthood, and how signalling pathways control lineage choices at every step of differentiation. Such an understanding addresses several fundamental questions in developmental biology, while having practical implications for re-programming cells in disease, and for in vitro differentiation for cell therapy. Recently, we developed droplet microfluidic single cell RNA-Sequencing (scRNA-Seq) technology, which allows profiling the transcriptome of tens of thousands of single cells at low cost, and we additionally developed a method to combine droplet scRNA-Seq with lineage tracing, and the computational methods required to reconstruct time series of differentiation from scRNA-Seq and lineage data. In preliminary work, we applied these tools to generate a comprehensive map of cell state trajectories in zebrafish development through the first 24 hours post fertilization. In this proposal, we will extend our map of zebrafish development, combining scRNA-Seq with clonal analysis to track every cell state in the developing zebrafish embryo up to 7 days post-fertilization. We will then use staged, acute perturbations of seven major signaling pathways, followed by scRNA-Seq, to define which signaling pathways control the flow of cells down different trajectories throughout development, as well as their context dependent and invariant gene targets. Focusing deeply on neural tube patterning, we will then dissect the transcription factor networks that integrate signaling pathways, by CRISPR/Cas9 perturbation coupled to scRNA-Seq. This proposal builds on a multi-year collaboration between two labs with strong and synergistic expertise -- the Megason lab in the use of zebrafish for developmental systems biology and the Klein lab in single cell genomics and analysis.