PROJECT SUMMARY The goal of this proposal is to contribute new engineering technologies that gain control over cell collective phenomena responsible for driving epithelial organogenesis in vivo. Tightly packed epithelial sheets and tubules are crucial to the function of a diverse group of organs including the lung, kidney, mammary gland, and prostate. Congenital defects and adult diseases such as cancer that occur during the development and maintenance of such organs are prevalent, creating a significant disease burden. This has driven innovation in tissue engineering, which aims to create artificial tissues that serve as disease and therapy screening models. However, modern tissue engineering approaches fail to harness several hallmarks of epithelial organ formation, limiting the complexity of synthetic tissues and creating an urgent need for innovation. Rather than being assembled from constituent cells all at once, processes like branching morphogenesis progressively ramify, sculpt, and pack together functional tubule structures in vivo. The long- term goal of the Hughes lab is to study and engineer missing tissue-building principles at play in organ development to fill ongoing gaps in tissue engineering. This goal builds upon ongoing work in the Hughes lab from the previous period of R35 funding beginning in 2019. This includes 1) precision ssDNA-based cell and tissue interface micropatterning technologies to 2) control the size, cell composition, and differentiation trajectory of epithelial organoids; 3) shape-changing ‘kinomorph’ tissue scaffolds for guiding tissue building behaviors in cell collectives; and 4) mechanically manipulating epithelial progenitor niche regulation. Now our overall objectives are to further advance three research visions across kidney and lung model systems. We firstly seek to map the relationship between tissue mechanics and cell-cell signaling on epithelial morphogenesis via orthogonal optogenetic ‘handles’. This will allow us to better control organoid cell composition. We secondly seek to gain control over epithelial tubule shape and elongation by engineering anisotropic collective cell forces through planar cell polarity pathways. We thirdly seek to combine tubule construction and microfluidics approaches to engineer axial tubule polarity. Together these missing tissue- building modules will contribute innovative enabling technologies for ‘higher-order’ tissue construction relevant to the general medical sciences. The Hughes lab is uniquely suited to tackle our research vision due to our expertise in stem cell engineering and optogenetics, live imaging, microfluidics, and advanced tissue assembly strategies. We expect our proposed contributions to have significant positive impact in the areas of fundamental biological discovery, tissue construction, and drug target screening.