PROJECT SUMMARY Effective utilization of organ-specific somatic stem cells to repair injured tissues or to bioengineer organs will revolutionize disease treatment and relieve numerous problems caused by aging and trauma. However, it remains significantly challenging to derive somatic stem cells with precision and expand them with high efficiency for clinical applications. The technical hurdles are in large part due to our incomplete understanding of the genetic regulation that controls fate specification of progenitors during development, as well as cell plasticity in adults. Teeth provide an excellent test case to further understand these aspects and apply clinically, as adult human teeth do not maintain dental epithelial stem cells and lack the capability to regenerate. The mouse tooth is a powerful model system to study both organogenesis and adult stem cell-based regeneration, and amenable for both in vivo genetic studies and ex vivo manipulations to investigate progenitor cell functions. Leveraging this remarkable experimental system and combining it with cutting-edge single cell transcriptomic analysis, this proposal will deliver an in depth understanding of the genetic program and transcriptional changes during the formation of dental epithelial progenitors. This knowledge will allow us to identify a genetic network and critical regulators required to specify the dental fate and provide a blueprint to derive dental progenitors by differentiating pluripotent stem cells along a genetic path. In parallel, this project will test the function and utilization of IRX1/2 and YAP in inducing the formation of dental progenitors through differentiation of oral epithelium and dedifferentiation of ameloblasts respectively. We will compare single cell transcriptomes between induced, embryonic, and adult progenitors to determine whether IRX1/2 and YAP can activate a dental genetic program. Based on these data, we will also be able to address an important question in stem cell biology, which is how different progenitor types in embryos and adults resemble each other transcriptionally. The main innovation of the project arises from the integration of genomic techniques and mouse genetic models to understand the genetic regulation of dental progenitor and stem cell formation, which is understudied. Such knowledge will form the basis of future research and grant applications, and enable developmental principle-driven approaches to tooth bioengineering.