Correct tissue shape is essential for proper tissue function and morphogenetic dysregulation results in many common congenital disorders. Yet, how groups of thousands or even hundreds of cells coordinate to yield stereotypic shape change through large-scale movements is still poorly understood. One way for cells to interact is through mechanical coupling. In fact, largescale networks of actomyosin connections between cells span developing tissues across various model organisms. The highly reproducible developmental program and powerful genetic tool-kit of Drosophila makes the Drosophila ventral furrow an ideal system for studying such networks. During furrow formation cells coordinate pulsed constrictions to yield tissue-wide bending. The tissue possesses a dynamic myosin network which fully forms prior to folding. Little is known, however, how mechanical information in the network guides collective constriction. This proposal will address how a network of mechanical connections is established to drive tissue folding. First, how a 2D network of intercellular connections promotes epithelial folding will be established. A novel approach, adapting methods from both astronomy and the mathematics of network theory will map the previously unquantifiable myosin network across hundreds of cells in a developing tissue. Preliminary studies have identified an initial growth phase and a subsequent contractile phase in the network. Growth Phase: We hypothesize that persistent network connections are established when neighboring cells simultaneously undergo a pulsed myosin accumulation. To test this hypothesis the position and timing of myosin recruitment will be coupled with the creation or reorganization network connections. Embryo injections inhibiting myosin pulsing will test the requirement of pulsing for network formation. Contractile Phase: We hypothesize that signatures in network geometry guide stereotypic tissue folding. Tissue-wide connectivity will be correlated with regions of coordinated cell constriction. Laser cutting will test the importance of connectivity patterns for tissue folding by selectively severing configurations in the network. Our approach could identify a novel unit of cooperation between the cell and the tissue scale over which cells synchronize. Second, how RhoA signaling influences cell interactions across a tissue will be investigated. Rho-associated coiled-coil kinase (ROCK) can activate myosin directly through phosphorylation or indirectly via inhibitory phosphorylation of myosin phosphatase (MP). To test the hypothesis that the balance between ROCK and MP dictates myosin network connectivity, MP will be constitutively activated at varying levels uncoupling its activity from ROCK regulation. This technique yields a phenotypic regime whereby the network is disrupted with varying severity. The global MP to ROCK activity required for myosin network regulation, as well as the local role of ROCK in shielding myosin filaments from disassembly, will be addressed. Taken together the two aims will form a foundational framework to understand general rules that govern how cells interact to reproducibly change tissue shape.