ABSTRACT Villi are finger-like projection that line the lumen of the small intestine. Villi play a critical role in nutrient uptake by increasing the intestinal absorptive surface area by several orders of magnitude. Loss of this absorptive surface through villus atrophy causes major digestive complications and nutrient malabsorption. Abnormalities in villi are found in many gastrointestinal maladies, such as inflammatory bowel and celiac diseases, and are also side effects of radiation, chemotherapy, and infection. Degenerated villi can sometimes fully reform, yet in other situations regeneration is impaired, resulting in persistent villus atrophy and patient suffering. Villi emerge during development from an initially flat intestinal surface. The mechanisms underlying villus formation and repair remain poorly described, and an understanding of these processes is essential to develop new therapies. The long term goal of this proposal is to build an understanding of the molecules and forces that sculpt the villus during development and regeneration so as to improve strategies for growing and regenerating the intestine for human patients. Recent data from our labs suggest that the mesenchyme plays a central role in sculpting the architecture of the villus. Specifically, our preliminary data implicate a specialized population of self- organizing sub-epithelial mesenchymal cells that condense immediately below the forming villus as the source of the physical forces necessary to pattern and fold the overlying epithelium into villi. We investigated the molecular mechanisms leading to condensation of these cells using single cell RNAseq and found they also express a unique transcriptional program. This program has an unusual overlap with genes regulating Ca2+ mediated contractility in smooth muscle cells but without expressing smooth muscle actin. We provide evidence that inhibition of key proteins in this program results in a loss of mesenchymal condensation and villus evagination. Guided by these preliminary data, we propose to test two hypotheses related to the complementary physical and molecular aspects of villus formation. We combine quantitative measurements and computational modeling to test the hypothesis that the formation of villus condensates occurs analogously to phase separation phenomena studied extensively by physicists and material scientists, and that condensates exert physical forces on the overlying epithelium initiating its folding. We then test the hypothesis that Endothelin released from the epithelium triggers increased calcium signaling in the subepithelial mesenchyme, which are synchronized through gap junctions to drive cell contractility leading to phase separation and condensation. This project has major implications for our fundamental understanding of the developmental of the gut and for tissue engineering of intestinal tissue, as we do not know how mammalian intestinal villi are built. Thus, these findings will be impact...