Project abstract Maintaining the junctional epithelium (JE) is of primary importance if preservation of the cementum, periodontal ligament (PDL), and alveolar bone is to be achieved. New insights into JE barrier functions came with our discovery of a Wnt- responsive stem cell niche in the JE. In this proposal, our goal is to characterize the cellular players, niche signals, and regulatory mechanisms that control and maintain the JE stem cell niche in health, and after damage or disease. AIM 1 experiments will first test whether Wnt/β-catenin signaling is necessary for JE maintenance. Pathway inhibition will be achieved in Axin2LacZ/+ mice using adenovirus expressing the soluble Wnt inhibitor Dkk1; controls will receive adenovirus encoding the Fc portion of immunoglobulin G. At defined intervals, quantitative analyses will assess endogenous Wnt/β- catenin signaling via Xgal staining; JE hemidesmosomal gene and protein distribution via quantitative immunohistochemistry (qIHC); JE and GE cell cycle kinetics by EdU/BrdU labeling; and inflammatory cell infiltration in connective tissues underlying the JE and GE by FACS. Second, whether Wnt/β-catenin signaling is necessary for JE regeneration will be determined by subjecting Wnt lineage tracer e.g., Axin2CreERT2/+;R26RmTmG/+ mice to partial gingivectomy followed by Ad-Dkk1/Ad-Fc delivery. Lineage-tracing and quantitative analyses will establish a relationship between Wnt- responsive cell progeny, cell cycle kinetics, hemidesmosomal gene and protein distribution, and regeneration of JE barrier functions. AIM 2 experiments will evaluate the ability of a stabilized formulation of WNT protein to regenerate a functional JE. In one injury-repair model the JE will be surgically excised; in a second model, JE breakdown will be triggered via a ligature-induced periodontitis; both will be carried out in Axin2LacZ/+ and Axin2CreERT2/+;R26RmTmG/+ mice. Delivery of the WNT therapeutic will be followed at multiple timepoints by quantitative analyses to assess re-establishment of JE barrier functions. AIM 3 experiments will characterize Wnt-responsive JE stem cells and their progeny. Wnt-responsive stem cell pools from adjacent gingival epithelium (GE) will serve as control. Axin2CreERT2/+;R26RmTmG/+ mice will be exposed to tamoxifen, followed by harvest of JE and GE tissues at defined timepoints. GFP+ cells will be sorted by flow cytometry. Gene expression profiling of GFP+ cells will focus stem cell and differentiation markers. Fluorescent in situ hybridization will confirm gene expression patterns using RNA probe libraries corresponding to stem cell markers, components of Wnt/β-catenin, Notch, and Bone Morphogenetic Protein (BMP) pathways. Collectively, this proposal promises to provide important new insights into the requirement for Wnt/β-catenin signaling in maintaining the JE stem cell niche; regulating JE and GE cell proliferation and differentiation; and influencing hemidesmosomal-mediated attachment to the t...