Genetic analysis in model organisms such as C. elegans continues to excel as a fundamental approach to discovering how cells, tissues, and organs communicate with each other to control development, and to identifying the signaling components, modulators and mechanisms that govern these processes. Aberrant activity of these same signaling pathways has profound effects on human health, most notably causing cancer, congenital defects, and diverse physiological disorders. Regulating signaling appropriately—in space or cell population, in time, strength or duration, or in combination with other signaling inputs—is thus crucial both for normal development and for understanding human disease, and is the overarching theme of the proposed work. Much of the proposed work concerns Notch, a transmembrane protein that functions as a receptor in cell- cell interactions that control development in all animals. Our overall goal is to elucidate the regulatory logic and mechanisms that regulate Notch activity and that coordinate its activity with other highly conserved major signaling systems to ensure precise and robust cell fate outcomes. C. elegans is amenable to combining traditional genetic analysis with genome engineering using CRISPR/Cas9 to apply many different methods for manipulating and visualizing endogenous genes and their products. These tools, combined with genetically- encoded signaling biosensors, cell-specific transcriptome profiling, and long-term imaging, allow for unprecedented insight and resolution into signaling regulation and integration. Our proposed work will fill three Key Gaps for which we performed foundational work during the current funding period. Key Gap 1 addresses questions about the mechanism by which ligands activate Notch signal transduction, arising from our recent discovery of unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation. Our investigations in this area may prove to be relevant to specific mammalian contexts and to suggest novel strategies for modulating ligand-Notch interactions in clinically-relevant settings. Key Gap 2 concerns a universal feature of animal development, "lateral inhibition" or "lateral specification." In this process, Notch-mediated interactions between initially equivalent cells engage feedback loops that amplify a stochastic, small initial difference between them to resolve their fates. New tools we developed during the current funding period will allow us to delve deeper into the stochastic events and feedback mechanisms in an archetypal case of lateral specification, the Anchor Cell/Ventral Uterine precursor cell fate decision during gonadogenesis. Key Gap 3 concerns how developmental progression of different organs is coordinated, as is necessary to ensure coherent development. We will build on our findings of inter-organ signaling mediated by conserved pathways that control development of the reproductive system. The overall impact of pursuing these area...