PROJECT SUMMARY Organogenesis is a process in which biochemical signals and mechanical cues transform embryonic germ layers into organs during fetal development. With recent advancements in stem cell biology, it has become possible to differentiate human pluripotent stem cells into expandable 3D tissues that contain many of the cellular and functional characteristics of fetal organs. These so-called organoids hold a tremendous potential to answer longstanding questions of human organogenesis and to one day serve as a renewable source of patient-specific human tissues. However, human organoids still only approximatively recapitulate organogenesis as they rely on spontaneous tissue self-organization and the establishment of signaling gradients in unpredictable ways. Furthermore, available organoid protocols focus on single organs and do not focus on morphogenesis. We be- lieve that making a meaningful progress in the field demands 1), gaining a deeper understanding of the coupling between signaling networks, tissue-specific transcriptional signatures, and tissue morphogenesis and 2), devel- oping novel cross-disciplinary tools that control spatiotemporal signaling to accurately mimic organogenesis in vitro. This proposal aims precisely to advance these gaps in our knowledge. We focus on the organogenesis of the gut tube, the embryonic structure on which many adjacent organs form, from thymus and lungs to the colon. Breaking away from the current organoid paradigms, which heavily rely on self-organization and the establish- ment of often uncontrolled internal gradients, we will combine tissue micropatterning and microfluidics to gen- erate precise signaling gradients so to reproducibly mimic both the signaling and the morphogenesis of gut tube organogenesis in vitro. Combined with live-cell microscopy, CRISPR editing, and single cell transcriptomics our organoids will reveal a detailed hierarchy of fate choices cells make from pluripotency to regionally specialized tissues in the gut tube, and they will allow us to make crucial connections between signaling, transcriptional regulation, and tissue morphogenesis. Our work will shed light on the largely unknown regulatory mechanisms by which the complex signaling pathways interact to create asymmetric patterns along the body axes. For the first time, our approach will recapitulate the formation of multiple adjacent gut tube progenitors from an em- bryonic germ layer, providing a unique window into one of longstanding questions in developmental biology: how do continuous signals create discretely separated organs along body axes? Finally, in collaboration with CRISPR experts, we will generate a pipeline that bypasses traditional morphogen-driven differentiation of plu- ripotent cells, but instead uses inducible genetic circuits to mimic fate decisions. This novel system will have the capacity to generate highly precise human tissues on the fly, in an approach that can be termed synthetic organ- ogenesis...