Project Summary Effective utilization of somatic stem cells to repair injured tissues or to bioengineer organs is an important goal in regenerative medicine. However, clinically-proven application of stem cells in therapies remains limited in medicine today. The translational hurdles are in large part due to our lack of ability to precisely control stem cell proliferation and differentiation, which is critical for safe and effective clinical use. To overcome this challenge, we must first deepen our knowledge of normal stem cell regulation in organs. In addition to biochemical signals, tissue mechanical forces exerted by cell pulling and pushing can in theory serve as a signaling mechanism to regulate gene expression and various cellular processes in adult stem cells. However, the modulation and influence of these force signals within a 3D tissue are dramatically understudied, leaving open questions around how stem cells sense and interpret forces. We and others have demonstrated the mouse incisor as a powerful model system to study adult epithelial stem cells and we have previously shown that the transcription co-factor Yes-associated protein (YAP) and chromatin repression are important for regulating incisor epithelial stem cells. Our initial studies indicate that both mechanical deformation of cells and the cell geometry associated with dense packing can influence the expression of YAP and repressive chromatin marks in the incisor stem cell niche. The mouse incisor thus provides a valuable in vivo platform to study how cellular organizations coordinate mechanical signals to control stem cell functions via YAP and chromatin. In this application, we propose to test the hypothesis that dense cell packing modulates the effect of tissue forces on nuclear deformations, which in turn regulate YAP nuclear entry and H3K27me3-mediated transcriptional repression in the dental epithelial stem cells. To test this: Aim 1 will characterize the force patterns, magnitude, and nuclear stiffness in wild type incisors, specifically in the densely packed dental epithelial stem cells and the more loosely packed transit amplifying cells. Aim 2 will study how changes in the cell geometry and packing affect tissue force patterns, nuclear deformations, YAP localization, and chromatin states. We will perform mechanical rescue experiments to test the role of forces. Aim 3 will address the functional role of lamin A in regulating nuclear stiffness and heterochromatin formation in the dental epithelial stem cells, as well as its scaling response to cell packing. Together, these studies will deliver a mechanistic understanding of how tissue forces control dental stem cells and yield findings that will be of general interest to both dental researchers and to the stem cell and regenerative medicine communities.