Follistatin regulates tooth root furcation morphogenesis

Follistatin (Fst), an extracellular inhibitor of the TGF-β signaling pathway, plays a critical role in tooth development, particularly in crown morphogenesis and enamel patterning. While molar root number is conserved within species and between upper and lower jaws, the molecular mechanisms governing root patterning remain largely unknown. To investigate the role of Fst in root formation, we analyzed K14-Cre-mediated Fst knockout mice. During embryonic and early postnatal stages, Fst was dynamically expressed in the dental epithelium including the lingual epithelium, and primary and secondary enamel knots, as well as in the dental mesenchyme at the base of the dental papilla. However, during root formation, Fst expression became restricted to the outer layer of Hertwig's epithelial root sheath (HERS). Micro-CT analysis revealed that mutant mice exhibited molar root abnormalities, including enlarged pulp chambers and partial furcation defects on the lingual side of the mandibular molars. Histological analysis showed that, while HERS initially exhibited buccolingual asymmetry - with the buccal-side HERS extending more prominently in both control and mutant mice - control mice eventually established bucco-lingual symmetry in HERS extension, ensuring proper root furcation formation. In contrast, mutant mice displayed delayed lingual-side HERS extension, resulting in defective root furcation. Molecular analysis revealed that Gli1 expression was upregulated, whereas Cdkn1a and Wnt signaling molecules (Wnt4, Lhx6, and Notum) were significantly downregulated in the delayed lingual HERS of mutant mice. These findings suggest that Fst regulates tooth root morphogenesis by modulating Shh and Wnt signaling to control lingual epithelial proliferation and HERS extension. This study offers novel insights into the molecular mechanisms governing molar root number and morphology determination.