Project Summary We are requesting funds to purchase a fluorescence-based cell sorter, the BioRad S3E, for the purpose of screening nanobody libraries and identifying and isolating high affinity binders to cell surface proteins that transduce Wnt signals. Wnt proteins represent an important class of developmental signals with diverse functions across all metazoan species. Deregulation of Wnt signaling can have catastrophic consequences, including embryonic lethality, birth defects, and disease. Specific and selective targeting of Wnt signaling has great therapeutic potential in regenerative medicine and the treatment of cancer. Additionally, with their diverse and potent activities in development and stem cells, Wnt proteins hold great promise as potent tools in cell and tissue engineering. The long-term objective of our research is to gain a better understanding of how Wnt proteins and their downstream signaling events influence cell fate decisions, and thereby advance technologies and treatments that specifically target Wnt signaling. To this end, we have developed an innovative technology that utilizes engineered Wnt agonists, called Wnt mimetics, which exhibit superior biochemical properties compared to native Wnt proteins. Using our previously developed and published Wnt mimetic, called F7L6, as a guide, we propose to develop a collection of such Wnt agonists, each designed to engage and activate distinct types of Wnt receptors. The requested instrument will enable the identification and isolation of novel high affinity binders and accelerate the development of novel Wnt mimetics. The proposed research will significantly advance the field of stem cell research and tissue engineering by establishing new tools and methods to manipulate Wnt signaling in vitro and in vivo. With its abundant roles in human disorders and diseases, a better understanding of Wnt signaling is essential for the development of novel therapies for currently incurable diseases.