Developing organoid model to study active folding in a human genetic context

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ABSTRACT Organ shape is critical for the organism to function properly. For the example neural tube, early formation defects may have devastating consequences to the body. A fundamental building principal in embryogenesis is first organizing cells into simple structures, such as a closed sheet surrounding a lumen. Cells then undergo well-concerted programs to reconfigure tissue shape, or fold out of the plane of the sheet. In plane flows elongate one axis while shortening another during convergent extension. Folding in contrast dramatically changes tissue form, to endow the organ with its characteristic shape. In neural tube closure, an initially flat sheet of cells undergoes two sequential bending steps to fold up into a cylindrical tube surrounding a single lumen. A large body of work uncovered fundamental insights on fate determination. Yet, mechanisms controlling shape, particularly folding in the context of early human development, remain poorly understood. At the cellular level genetic patterning coordinates behaviors over long distances to instruct active forces that underlie folding. Before distilling a physical picture of how forces fold tissue, we need to identify behaviors leading to folding, and characterize coordination. Organoids harbor promise for studying early development, disease and regeneration in tightly controlled environments and human genetic background. However, progress is hampered by technical limitations, due to irregular patterning, and shape. This proposal seeks to develop a new strategy for designing robust models to study basic mechanisms of human organogenesis. Using micro patterning we successfully generated highly reproducible organoids with set size, shape, and formation kinetics. Our approach features flat rectangular shaped sheets that spontaneously fold out of the plane, and seamlessly close along the short dimension, generating a tube. The resulting platform is high throughput, compatible with live imaging, and well suited for quantitative data analysis strategies. This setup recapitulates major steps during neural tube closure in animal model systems, such as hinge formation and a zippering mechanism with actomyosin cable accompanying closure. Here we aim to adapt existing protocols to our platform to 1) induce formation of neuroepithelium and more closely model neural tube formation. We then aim to 2) assemble a quantitative atlas of cell behaviors observed during closure. Finally, we aim to 3) enter new territory and generate anatomical axes spanning the organoid tube using microfluidics. Our approach opens up a new path towards controlled models of human organogenesis, using the neural tube as example.