PROJECT SUMMARY/ABSTRACT Tissues are sculpted into 3D shapes through coordinated force generation during embryogenesis. Force generation is mediated by the cytoskeletal protein, F-actin, and the motor protein, myosin, which can be coupled across cells through adherens junctions to form supracellular actomyosin networks. A range of tissues use supracellular actomyosin networks to facilitate a diversity of folding events, adjusting the network composition and structure satisfy the mechanical needs of the tissue. The modularity of actomyosin networks is evident in vertebrate neural tube closure (NTC). All vertebrates undergo NTC in an actomyosin-dependent manner, but the mechanism of closure varies substantially across organisms. Additionally, failures in NTC lead to congenital malformation in humans. However, it remains to be determined if there are generalizable mechanisms of actomyosin-mediated tissue folding or what mechanisms fail and prevent NTC. The mechanisms of mouse NTC are unclear, due to the large tissue size and prolonged folding time. Using a computational workflow that we developed to create two-dimensional shell projections from curved three- dimensional samples, we reconstructed the apical actomyosin network of the entire mouse neural tube (NT) at subcellular resolution. Using preliminary analysis of these reconstructions, we propose a lateral tension model for cranial mouse NTC, where enrichment of actomyosin and apical constriction introduces apical tension in the lateral neural folds. Combined with concurrent tissue thickening, the neural folds stiffen and efficiently elevate when pushed by adjacent tissues, promoting NTC. To test this model, we will: 1) Quantify key features of the actomyosin networks of wild-type mouse NTs which inform on the mechanical tissue properties, including protein levels, actomyosin cable orientation, and cellular morphologies. A comparative analysis with mutant mice whose networks are perturbed will allow us to identify features of the actomyosin network and regions of the NT critical to NTC. 2) Experimentally determine the presence differential tissue tension. We will perform laser ablation in live mouse embryos to demonstrate the presence and directionality of tension throughout the mouse NT. We will also use this live-imaging platform to quantify the dynamic changes in the actomyosin networks throughout NTC. This work will be primarily conducted in the lab of Prof. Adam C. Martin at the MIT Department of Biology with laser-ablation training and experiments occurring in the lab of Prof. Gabriel Galea at the University College London, UK. The proposed work will illuminate paradigms in actomyosin-mediated tissue folding during development and build an intellectual foundation from which to understand congenital malformations, ultimately leading the design of preventative therapies and treatments. The applicant will also receive advanced training that will support their pursuit of an academic career.