Summary/Abstract The vertebrate intestine develops through a common series of events prior to maturation of the adult form. Comparison of different species reveals that as the tube forms, proliferation occurs throughout the unfolded epithelium. As the epithelium begins to fold, epithelial proliferation becomes restricted to the base of the developing folds (zebrafish) and villi (mouse). As proliferation is restricted to the villi base, stem cells begin to form. While the developing stem cells express some of the genes of their adult counterparts, expression is significantly lower suggesting that these cells are immature. Intestinal stem cells also decrease levels of extracellular matrix components as they mature. While signaling pathways such as Wnt play a role in driving proliferation of these stem cells, there is likely to be novel signaling and different interactions with unique cell types to complete growth and maturation of the immature stem cells. Details about the state of the immature stem cells has been investigated but less is known about signals and cells in the surrounding niche that regulate growth during the period before adult stem cells form. In this proposal, we will investigate the roles of novel cell types in regulation of epithelial proliferation and how they interact with other signaling pathways between restriction of proliferation to maturation of the stem cell niche. Here we use the advantages of external development and transparency of the zebrafish model system to visualize the developing stem cell niche following manipulation of transgenic lines. In Aim 1 we determine how Notch signaling interacts with other pathways in regulating epithelial proliferation. In Aim 2 we will ablate cells receiving Notch signaling to analyze their role in epithelial proliferation. Finally, in Aim 3 we will determine which Notch receptors participate in these signaling events. Together, these studies will identify new features of the network of signals controlling growth of the immature stem cell niche as it matures to the adult form. Understanding the role of regulatory cells and signals within the developing stem cell niche will provide information about the commonalities of how stem cell niches develop across different tissues and species. Understanding these commonalities will aid in approaches to grow new or reactivate aged stem cell niches.