Foxi3 Suppresses Signaling Center Fate and is Necessary for the Early Development of Mouse Teeth

Tooth morphogenesis is regulated by ectodermal-mesenchymal interactions which are controlled by reiteratively used, evolutionarily conserved pathways. How these signals translate into different cellular behaviors is beginning to be understood. Embryonic cell behaviors are regulated by signaling centers that define organ position, size, and shape. The recently identified initiation knots (IKs) and the well-known enamel knots (EK) are tooth epithelial signaling centers that drive proliferation of the neighboring cells and are involved at different stages of morphogenesis, from the early epithelial thickening to the final formation of shape. Signaling center cell fate and maintenance can be regulated by numerous factors. Here, we studied the early stages of murine incisor and molar epithelial morphogenesis and overcame the previous shortage of early-stage mutant models to functionally manipulate the initiation processes of tooth morphogenesis. We achieved the early conditional knock down of the forkhead-box transcription factor Foxi3 during tooth initiation and used imaging approaches to explore cellular and molecular disease mechanisms, specifically those related to signaling center dysfunction in tooth dysplasia. We show that early deficiency of Foxi3 in incisors and molars frequently arrests growth at bud stage, whereas later knockdown of Foxi3 affects tooth downgrowth and shape. Cell-level analyses revealed a decrease in epithelial proliferation and the ectopic appearance of cells with hallmarks of signaling centers: quiescent cell state and canonical Wnt activity. However, the distribution of these cells was wider all over the tooth buds with abnormal decrease of apoptosis. We postulate that, depending on Foxi3 expression levels, the bud cells shift commitment into signaling center fate, ultimately leading to growth arrest or growth/shape changes, implicating Foxi3 as a regulator of cell fates between the signaling centers and proliferating bud cells.