Monitoring of canonical BMP and Wnt activities during postnatal stages of mouse first molar root formation

Abstract Objective This study aimed to explore the precise temporospatial distributions of bone morphogenetic protein (BMP) and Wnt signaling pathways during postnatal development of mammalian tooth roots after the termination of crown morphogenesis. Methodology A total of two transgenic mouse lines, BRE-LacZ mice and BAT-gal mice, were undertaken. The mice were sacrificed on every postnatal (PN) day from PN 3d up to PN 21d. Then, the first lower molars were extracted, and the dissected mandibles were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and fixed. Serial sections at 10 µm were prepared after decalcification, dehydration, and embedding in paraffin. Results We observed BMP/Smads and Wnt/β-catenin signaling activities in the dental sac, dental pulp, and apical papilla with a certain degree of variation. The position of activation of the BMP/Smad signaling pathway was located more coronally in the early stage, which then gradually expanded as root elongation proceeded and was associated with blood vessels in the pulp and developing complex apical tissues in the later stage. However, Wnt/β-catenin signaling was highly concentrated in the mesenchyme below the cusps in the early stage, gradually expanded to regions around the root in the transition/root stage, and then disappeared entirely in the later stage. Conclusions These results further confirmed the participation of both BMP and Wnt canonical signaling pathways in tooth root development, as well as formed the basis for future studies on how precisely integrated signaling pathways regulate root morphogenesis and regeneration.

摘要 研究目的：本研究旨在探究牙冠形态发生完成后，哺乳动物牙根出生后发育过程中骨形态发生蛋白（Bone Morphogenetic Protein, BMP）与Wnt信号通路的精确时空分布特征。
研究方法：本研究共计采用两种转基因小鼠品系，即BRE-LacZ小鼠与BAT-gal小鼠。分别于出生后（postnatal, PN）第3天至第21天的每个时间点处死小鼠，摘除下颌第一磨牙，分离下颌骨后采用5-溴-4-氯-3-吲哚-β-D-半乳糖苷（5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, X-gal）进行染色并固定。经脱钙、脱水、石蜡包埋后，制备厚度为10 μm的连续切片。
研究结果：我们在牙囊、牙髓与根尖乳头中均观察到BMP/Smads及Wnt/β-连环蛋白（Wnt/β-catenin）信号通路的激活活性，且该活性存在一定程度的差异。早期阶段，BMP/Smad信号通路的激活位点更靠近牙冠侧，随着牙根伸长进程逐渐扩展，后期则与牙髓内血管及发育中的根尖复合组织相关联。而Wnt/β-连环蛋白信号通路在早期阶段高度集中于牙尖下方的间充质中，在过渡期/牙根发育期逐渐扩展至牙根周围区域，后期则完全消失。
研究结论：本研究结果进一步证实了BMP经典信号通路与Wnt经典信号通路均参与牙根发育过程，同时为后续精准解析整合信号通路如何调控牙根形态发生与再生奠定了研究基础。