A GPI-anchored protein Lypd1 regulates odontoblast differentiation in tooth development

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. They are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports on the specific effects of lipid rafts on organogenesis have been reported. Here, we focused on the odontoblast differentiation that secretes the dentin matrix and generates dentin. We found the GPI-AP, lymphocyte antigen-6 (Ly6)/Plaur domain-containing1 (Lypd1), specifically expressed in the dental papilla, especially preodontoblasts, using single-cell RNA sequence (scRNA-seq). Depletion experiments of lipid rafts and Lypd1 using ex vivo culture and cell cultures showed that differentiation of tooth germ and dental mesenchymal cells was inhibited. Furthermore, the C-terminal structural domain containing the omega site, which is necessary to localize the plasma membrane, of Lypd1 is necessary for odontoblast differentiation and the morphological change like odontoblasts. Stimulating downstream Bone morphogenetic protein (Bmp) signaling by BMP2 caused it to activate the phosphorylation of Smad1/5/8 and promote the expression of odontoblast differentiation markers such as Pannexin 3 (Panx3), Alkaline phosphatase (Alp), and Dentin sialophosphoprotein (Dspp). In contrast, suppression of Lypd1 inhibited the phosphorylation of Smad1/5/8. In addition, the expression of Lypd1 predates any other marker of odontoblast differentiation previously described. That implies that Lypd1 could be employed as a novel earlier differentiation marker of odontoblasts. These results suggest that Lypd1 as GPI-AP of lipid rafts is important for tooth organogenesis and plays a pivotal role in odontoblast differentiation by regulating phosphorylation of Smad1/5/8. Examination of epithelial and mesenchymal cells in mouse embryonic day 16 molars.脂筏（Lipid rafts）是富含胆固醇、鞘磷脂、糖基磷脂酰肌醇锚定蛋白（GPI-APs）及受体的膜微区。它们定位于质膜，对信号转导与器官发生至关重要。然而，目前关于脂筏对器官发生的具体调控作用的研究报道较少。本研究聚焦于分泌牙本质基质、生成牙本质的成牙本质细胞分化过程。通过单细胞RNA测序（scRNA-seq），我们发现糖基磷脂酰肌醇锚定蛋白淋巴细胞抗原6（Ly6）/Plaur结构域包含蛋白1（Lypd1）特异性表达于牙乳头，尤其是前成牙本质细胞中。利用离体培养与细胞培养开展脂筏耗竭及Lypd1敲除实验，结果显示牙胚与牙间充质细胞的分化受到抑制。进一步研究发现，Lypd1中定位于质膜所必需的含ω位点的C端结构域，对于成牙本质细胞分化及成牙本质细胞样形态改变不可或缺。通过骨形态发生蛋白2（BMP2）激活下游骨形态发生蛋白（BMP）信号通路，可促进Smad1/5/8的磷酸化，并上调成牙本质细胞分化标志物如连接蛋白3（Panx3）、碱性磷酸酶（ALP）及牙本质涎磷蛋白（DSPP）的表达。反之，抑制Lypd1的表达则会削弱Smad1/5/8的磷酸化水平。此外，Lypd1的表达早于此前已报道的所有成牙本质细胞分化标志物。这表明Lypd1可作为一种新型的成牙本质细胞早期分化标志物。本研究结果提示，作为脂筏上的糖基磷脂酰肌醇锚定蛋白，Lypd1对牙齿器官发生具有重要作用，并通过调控Smad1/5/8的磷酸化在成牙本质细胞分化中发挥关键功能。本研究还对小鼠胚胎第16天磨牙的上皮细胞与间充质细胞进行了检测。