ABSTRACT Villi are finger-like projection that line the lumen of the small intestine where they aid in nutrient uptake by increasing the intestinal absorptive surface area. 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, but persistent villus atrophy causes severe complications and patient suffering. Thus, understanding the molecular and mechanical mechanisms underlying villus formation and repair are essential to develop new therapies and for growing and regenerating intestinal tissue for human patients. Villi emerge during development from an initially flat intestinal surface. We recently demonstrated that the forces necessary and sufficient to pattern and fold the epithelium during the initiation of villus morphogenesis derive from a population of Pdgfrahigh cells that differentiate at the interface of the epithelium and mesenchyme. These cells form a motile monolayer that breaks up into compact aggregates that fold the overlying epithelium in a Myosin-II, Hedgehog, MMP and integrin-dependent manner. We named this process “active mesenchymal dewetting.” Following this critical symmetry-breaking event, however, villi must increase their length by at least four-fold. This process coincides with the differentiation of the overlying epithelium into Wnt-responsive proliferative domains in between villi, and non-proliferative domains overlying villi. Finally, villus initiation and extension repeats itself at least four more time in a manner highly reminiscent of branching morphogenesis. Guided by our findings in revealing the mechanism of villus initiation, we propose to test three hypotheses related to the molecular and mechanical processes driving villus extension, villus branching, and epithelial zonation. First, we hypothesize that forces driving villus extension derive from a transition of proliferative, dynamic and narrow epithelial cells, to a non-proliferative, static, and wide morphology as they move into villus domains. Second, we hypothesize that epithelial zonation derives from Pdgfrahigh clusters physically pushing the overlying epithelium away from a critical source of Rspo2 and 3 in the deep mesenchyme. Third, we hypothesize that repeated rounds of villus “branching” occur when the intervillus proliferative epithelium “pushes” into the mesenchyme, causing it to thin, and exposing a population of Pdgfralow cells in the deep mesenchyme to the surface where they come in contact with epithelial hedgehog and differentiate. We will test these hypotheses using an innovative combination of live imaging, mouse genetics, computational modeling, and mechanical perturbations. This project has major implications for our understanding of the developmental of the gut and for tissue engineering because it will provide a new mechanistic blueprint for building villi incorporating both signals and forces. These findings will also lay the groundwork for future studies of regeneration: the adult sub-epithelial mesenchyme retains expression of many of these cell types and many are activated following injury.

PROJECT NARRATIVE Intestinal villi are finger-like projections that are essential for nutrient absorption and become disrupted in inflammatory bowel diseases and following radiation, chemotherapy, and infection. The regenerative process of villi is often impaired, and persistent villus atrophy can cause significant clinical challenges. In this application, we will reveal the forces and molecules that act in the mammalian embryo to pattern and shape villi, which will aid in the design of new strategies to promote villus regeneration in vivo and to build replacement tissue ex vivo.