DataSheet1_A potential role of p75NTR in the regulation of circadian rhythm and incremental growth lines during tooth development.ZIP|牙齿发育数据集|生物钟调节数据集

Objective: Tooth morphogenesis and the formation of hard tissues have been reported to be closely related to circadian rhythms. This study investigates the spatiotemporal expression and relationship of p75NTR with core clock genes, mineralization-related or odontogenesis-related genes, and aims to derive the potential role of p75NTR in regulating circadian rhythm and incrementality growth line formation during tooth development.Materials and methods: The dynamic morphology of the rat dental germ was observed at seven stages (E14.5 d, E16.5 d, E18.5 d, P.N. 4 d, P.N. 7 d, P.N. 10 d, and P.N. 15 d). Next, the expressions of p75NTR and other target factors were traced. The ectomesenchymal stem cells (EMSCs) were isolated from the E18.5d rat dental germs and synchronized using 50% of fetal bovine serum. Then, they were cultured in light/light (L.L.), dark/dark (D.D.), and light/dark (L.D.) conditions for 48 h. The total RNA was collected every 4 h, and the circadian rhythm dynamics of target factors were observed. To reveal the mechanism further, p75NTR was down-regulated in p75NTRExIII−/− mice and up-regulated in immortalized mouse dental apical papilla progenitor cells. The change tendencies of other target factors were also detected.Results: The clock genes Bmal1, Clock, Per1, and Per2 were all expressed in tooth germs before the formation of dental hard tissues and demonstrated a regular oscillating expression pattern in EMSCs from dental germs. Their expression was affected by the L.D. stimulus, and most of them were promoted by D.D. conditions. p75NTR presented a similar expression pattern and a positive or negative relationship with most clock genes, mineralization-related and odontogenesis-related factors, such as brain and muscle ARNT-like protein-1 (Bmal1), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), MSH-like 1 (MSX1), dentin matrix acidic phosphoprotein 1 (Dmp1), and dentin sialophosphoprotein (Dspp). Moreover, the arrangement, morphology, and even boundary in pre-odontoblast/pre-ameloblast layers were disordered in the p75NTRExIII−/− mice.Conclusion: Circadian rhythm was found to affect tooth development. p75NTR might play a crucial role in regulating clock genes in the mineralization and formation of the dental hard tissues. p75NTR is actively involved in the odontoblast-ameloblast junction and cell polarity establishment during tooth morphogenesis.

研究目标：牙齿形态发生以及硬组织的形成与昼夜节律密切相关。本研究旨在探究p75NTR与核心时钟基因、矿化相关基因和牙生成相关基因的空间-时间表达及其相互关系，并探讨p75NTR在调节昼夜节律和牙齿发育过程中生长线形成过程中的潜在作用。研究材料与方法：观察了大鼠牙胚在七个阶段（E14.5天、E16.5天、E18.5天、P.N. 4天、P.N. 7天、P.N. 10天和P.N. 15天）的动态形态。随后，追踪了p75NTR和其他目标因子的表达。从E18.5天的大鼠牙胚中分离出外胚层间充质干细胞（EMSCs），并使用50%的胎牛血清进行同步培养。随后，在光/光（L.L.）、暗/暗（D.D.）和光/暗（L.D.）条件下培养48小时。每4小时收集一次总RNA，观察目标因子的昼夜节律动态。为进一步揭示机制，通过p75NTRExIII−/−小鼠下调p75NTR的表达，并在永生化小鼠牙乳头祖细胞中上调p75NTR的表达，检测其他目标因子的变化趋势。研究结果：时钟基因Bmal1、Clock、Per1和Per2均在牙齿硬组织形成前在牙胚中表达，并在EMSCs中表现出规律性的振荡表达模式。其表达受到L.D.刺激的影响，其中大多数在D.D.条件下得到促进。p75NTR呈现出与大多数时钟基因、矿化相关和牙生成相关因子（如脑和肌肉ARNT样蛋白-1（Bmal1）、runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、MSH样1（MSX1）、牙本质基质酸性磷酸蛋白1（Dmp1）和牙本质唾液磷酸蛋白（Dspp））相似的表述模式，并与其呈现正相关或负相关关系。此外，在p75NTRExIII−/−小鼠中，牙本质前成牙细胞/前成釉细胞层的排列、形态甚至边界均出现紊乱。研究结论：昼夜节律被发现会影响牙齿发育。p75NTR可能在调节矿化和牙齿硬组织形成中的时钟基因中发挥关键作用。p75NTR在牙齿形态发生过程中，积极参与成牙细胞-成釉细胞结合部和细胞极性的建立。