Alx1 is required for enamel knot formation during incisor morphogenesis.

INTRODUCTION: Mutations in ALX1 cause frontonasal dysplasia type 3 (FND3), a congenital craniofacial disorder characterized by severe abnormalities of the frontonasal and midfacial structures. However, the role of ALX1 in tooth development remains unclear. METHODS: We investigated the function of Alx1 during mouse incisor morphogenesis using a neural crest-specific conditional knockout model. Histological, molecular, and transcriptomic analyses were performed to assess incisor morphogenesis, enamel knot formation, odontogenic gene expression, and signaling pathway activity. RESULTS: Mutant embryos exhibited complete agenesis of maxillary incisors and impaired development of mandibular incisors. Although tooth initiation was preserved, mutant maxillary incisors failed to progress through the bud-to-cap transition and lacked a discernible enamel knot. Transcriptomic profiling of E13.5 dental mesenchyme revealed alterations in odontogenic gene programs, including genes associated with BMP and Wnt signaling pathways. Consistent with these findings, expression of Bmp4 and Msx1 was markedly reduced, accompanied by impaired enamel knot-associated Shh expression. In parallel, canonical Wnt signaling activity, as indicated by active β-catenin and LEF1, was attenuated in mutant incisors. DISCUSSION: These findings identify Alx1 as an essential regulator of enamel knot formation and incisor morphogenesis, demonstrate a critical role for Alx1 in maintaining odontogenic signaling during early tooth development, and provide new mechanistic insight into the dental abnormalities associated with ALX1 deficiency.