Epithelial <i>Edar</i> Is Responsible for Dental Anomalies in HED

Hypohidrotic ectodermal dysplasia (HED), primarily caused by mutations in EDA and EDAR , is characterized by tooth agenesis and other dental anomalies, including smaller teeth, conical or peg-shaped teeth, and taurodontism. However, the specific role of EDAR in this process remains unclear, and suitable animal models for functional studies are lacking. In this study, we generated both global and epithelium-specific Edar knockout mice, demonstrating that epithelial Edar is essential for ectodermal organ development. Loss of Edar in the epithelium led to multiple developmental dental anomalies, including third molar agenesis, reduced tooth size, fused roots, decreased cusp number, taurodontism-like molars, and enamel hypoplasia, which could mimic most of the phenotypes seen in global Edar deletion and HED. Single-cell RNA sequencing further revealed that Edar deletion alters the normal developmental progression of preameloblast lineage cells toward later ameloblast states. This alteration may underlie the enamel defects observed upon Edar loss and is further supported by the finding that Edar knockdown impaired mineralization of ameloblast lineage cells in vitro. Notably, Dlx3 was downregulated in ameloblasts at various stages in Edar -deficient mice, and Dlx3 overexpression in Edar -knockdown ameloblast lineage cells could rescue the mineralization function. Chromatin immunoprecipitation–quantitative polymerase chain reaction revealed that p65 binds to the Dlx3 promoter, linking Edar to Dlx3 transcriptional activation via NF-κB signaling. Collectively, these findings reveal a critical role of epithelial EDAR in tooth development and, for the first time, provide evidence that its downregulated expression can lead to enamel formation defects, offering new insights into the pathogenic mechanisms of EDAR -related HED.