Epithelial Hedgehog signaling is essential for tooth patterning and enamel formation.

Supernumerary teeth are common congenital dental anomalies resulting from disrupted epithelial-mesenchymal interactions during tooth development. Although Hedgehog signaling is known to regulate tooth development, its temporal requirement for tooth number determination remains elusive. To investigate the role of early epithelial Hedgehog signaling, we conditionally deleted Smo in the dental epithelium using Pitx2-Cre, which initiates recombination at E10.5, prior to the onset of K14-Cre activity. SmoPitx2Cre mutant mice developed two supernumerary mandibular incisors together with defective ameloblast differentiation and disrupted enamel formation. BrdU labeling revealed significantly increased cell proliferation in the lingual cervical loop, with no changes in apoptosis. In situ hybridization showed reduced expression of Wnt inhibitors Dkk1 and Sostdc1, accompanied by upregulation of the Wnt target gene Lef1, indicating aberrant activation of the Wnt/β-catenin pathway in SmoPitx2Cre incisors. Treatment with SHH in vitro upregulated Dkk1 and Sostdc1 expression, confirming that Hedgehog signaling positively regulates these Wnt inhibitors. Moreover, the SmoPitx2Cre mutant mice exhibited decreased expression of Fgf3, Sp6, Dlx2, and Msx2, genes implicated in ameloblast differentiation and dental patterning. Together, these findings demonstrate that epithelial Hedgehog signaling is required during early odontogenesis to establish normal tooth patterning through coordinated regulation of Wnt signaling and epithelial-mesenchymal communication, while simultaneously promoting ameloblast differentiation.