Project Summary/Abstract The colonic epithelium is the site of a host of human diseases, and therefore understanding its development and function is essential for human health. The colon epithelium is separated into morphologically and functionally distinct domains, with proliferative stem cells being housed at the bottom of the crypts of Lieberkühn, whereas differentiated cells populate the epithelium closest to the lumen. The maintenance of epithelial homeostasis in the crypt is dictated in large part by spatially patterned signals from the underlying mesenchyme. The mesenchymal cells involved in adult colonic crypt homeostasis have been of recent interest. However, little is known about the molecular mechanisms underlying colonic crypt formation during development. Our proposal will determine how mesenchymal cell populations and signals interact with the developing epithelium to drive colonic crypt morphogenesis and define stem cell dynamics during development and regeneration. We will accomplish this work through our three aims: First, we will map mesenchymal-epithelial interactions over the course of colonic crypt development using single cell RNAseq and histological approaches. We will then use genetically modified mouse models to investigate the specific role of mesenchymal WNT signals in three stages of colonic crypt development: invagination, elongation, and fission of colonic crypts. Second, we will investigate the potential re-activation of an embryonic program in both the epithelium and mesenchyme following injury induced by DSS treatment. We will test the ability of embryonic or injury-associated mesenchyme to stimulate plasticity, defined as de-differentiation of committed progenitors to a more multipotent state, in the adult epithelium. Third, we will take a synthetic approach to reconstruct patterned mesenchymal signals around colon spheroids, and we will use this new model to test the sufficiency of polarized WNT signals to drive colonic crypt formation in vitro. Overall, our project leverages a combination of in vivo studies, in vitro models, synthetic engineering and bioinformatic approaches. This comprehensive scope is made possible through a highly collaborative environment at UCSF and the productive partnership of the two principal investigators carrying out this project. The findings from this proposal will significantly advance our basic knowledge of colon development and regeneration, highlighting the dynamics of mesenchymal-epithelial crosstalk. This will have implications beyond our fundamental understanding of colon biology and will help pave the way for discovery of therapeutic strategies to treat and modulate colonic disease.