PROJECT SUMMARY/ABSTRACT The tooth root is a critical component of the tooth, anchored to surrounding alveolar bones through the periodontal ligament (PDL). Appropriate formation of the root and its surrounding structure is essential not only for fundamental functions of the tooth in mastication for nutrition intake, but also for growth and development of the lower face. Prevalent dental diseases such as caries and periodontal diseases are the etiology for tooth loss, which can be treated only by prostheses lacking functional structures, leading to compromised long-term prognosis. An effective approach to regenerate a functional periodontal attachment apparatus is needed to achieve a breakthrough in dental regenerative therapies. During tooth root formation, the dental follicle (DF) provides precursor cells for cementoblasts, periodontal ligament (PDL) cells and alveolar cryptal bone osteoblasts to establish the functional periodontal attachment apparatus, the periodontium. Currently, how diverse populations of dental root mesenchymal progenitor cells work together and generate highly functional tooth roots remains unknown. In this proposal, we will define how concerted actions of distinct classes of dental root mesenchymal progenitor cells are essential for proper formation and regeneration of the tooth root accompanied by a functional periodontal attachment apparatus. In Aim 1, we will define how Hedgehog-Fox pathway regulates DF mesenchymal progenitor cells. We hypothesize that the Forkhead (Fox) transcription factors play a key role in regulating physiological functions of Hedgehog-responsive DF mesenchymal progenitor cells. We will first reveal the diversity of Hedgehog-responsive Gli1+ DF cells using single cell RNA- seq analyses, and determine their relationships with PTHrP+ DF cells and other precursor cell populations. Second, we will determine the function of Gli1+, PTHrP+ and Runx2+ cells in periodontium formation using inducible cell ablation experiments, and further test the function of Foxf1 and Foxf2 in periodontium formation using their floxed alleles. In Aim 2, we will identify Wnt-mediated roles of apical root mesenchymal progenitor cells in tooth root formation. We hypothesize that chemokine (C-X-C motif) ligand 12 (CXCL12)+ mesenchymal progenitor cells in the apical root area orchestrate formation of the tooth root and the apical periodontium in a canonical Wnt signaling-mediated manner. We will determine the relationship between CXCL12+ cells and stem cells for apical papilla (SCAP) using ex vivo culture system, and define molecular mechanisms underlying a Wnt-mediated cell fate choice of mesenchymal progenitor cells by a comparative RNA-seq analysis followed by in situ validation. In Aim 3, we will determine actions of dental root mesenchymal progenitor cells in periodontal regeneration. We hypothesize that distinct classes of dental root mesenchymal progenitor cells contribute to regeneration in a concerted manner. We will ...