Abstract: In the adult intestine, an organized array of finger-like projections called villi increase the surface area by 10- fold to allow maximum absorptive function. Villi are covered by an endodermally-derived absorptive epithelial layer, and contain a mesodermally-derived core of supporting tissues, including a tightly coupled vascular network. Loss of villi by reduction in intestinal length or villus atrophy results in malabsorption resulting in death in severe cases. Because villus development is primarily a fetal stage event, there are currently no therapeutic strategies to increase absorptive surface area other than transplantation of donor intestine. Promising advances have been made in engineering intestinal tissue using organoids, however these organoids fail to form villi unless they are transplanted into a vascular rich environment of a host, such as the kidney capsule (tHIO; transplanted human intestinal organoid), suggesting that vasculature may provide a key signal to promote villus development. During fetal villus development, villus emergence (beginning at E14.5 from an initially flat epithelium) requires Hedgehog (Hh) signaling for the formation of a patterned array of mesenchymal clusters, which act as signaling centers. Clusters, which are composed mainly of PDGFRa+ cells, remain attached to the tips of emerging villi, becoming part of the villus core. New preliminary data demonstrate that vascular tip cells interact with PDGFRa+ mesenchymal cells during cluster formation and that perturbing vasculature with vasculature-specific small molecule inhibitors leads to a loss of cluster formation. Furthermore, broadly inhibiting Hh signaling with small molecules perturbs the vasculature as well as the clusters. Collectively, these data suggesting that endothelial cells play a central role in villus formation lead to the following hypothesis: epithelial Hh ligands signal directly to endothelial cells to drive mesenchymal cluster formation. This hypothesis will be tested and key gaps in knowledge addressed by 1) identifying the Hh responsive cells within the cluster, 2) determining whether Hh signaling is necessary or sufficient within the cluster cells and/or endothelial cells, 3) testing whether endothelial cells impact the generation of human intestinal villi, and 4) defining the mechanisms that underlie the regulation of villus morphogenesis by Hh signaling in the human intestine and human intestinal organoids. These studies, which exploit high resolution microscopy, lineage tracing, genetic modulation of signaling and transcriptional profiling in both in vitro and in vivo models of intestinal development, will expand our understanding of how villi are generated, reveal new cell-cell interactions that control villus development and potentially lead to new therapeutic strategies for villus growth in organoid systems.