Objective: To investigate the role of WW domain containing E3 ubiquitin protein ligase 1 (WWP1) in enamel development of mice. Methods: Single-cell RNA sequencing data of incisor tissues of postnatal day 7 (P7) mice and mandibular first molar tooth germs of P3.5 mice were used to analyze the expression of WWP1 in dental epithelial cells. Immunohistochemistry was performed to observe the distribution and expression levels of WWP1 in the epithelium of mouse incisors and mandibular first molar tooth germs. Wwp1 knockout (Wwp1 KO) mice were generated and collected with their control littermates at P1, P7, three mice per group, as well as at P14, P28, 2 months (2M), and 3M, six mice per group. The enamel volumes of molars and incisors were analyzed using micro-CT. Scanning electron microscopy was employed to examine the enamel cross-sections of Wwp1 KO and control mice. Energy dispersive spectroscopy (EDS) was used to analyze the calcium and phosphorus content of the enamel rod of incisors. Immunofluorescence was performed to detect the expression of amelogenin (AMELX) in the ameloblasts of Wwp1 KO and control mice. Additionally, LS-8 ameloblast-like epithelial cells were cultured, and Wwp1 siRNA or overexpression plasmids were transfected to knock down or overexpress WWP1. The protein levels of AMELX were then assessed by Western blotting. Results: Single-cell sequencing result showed a high Wwp1 mRNA expression level in the epithelial cells of mouse incisors and mandibular molar tooth germs. Immunohistochemistry revealed the expression of WWP1 in presecretory, secretory, transitional, and mature ameloblasts. Wwp1 KO mice exhibited enamel developmental defects. The enamel volumes of molars and incisors in Wwp1 KO mice [(0.155±0.016), (0.300±0.017) μm3] were reduced by 23.95% (P<0.001) and 28.31% (P<0.001) compared with the control group [(0.203±0.062), (0.418±0.023) μm3] respectively. Scanning electron microscopy showed disorganized enamel structures in Wwp1 KO incisors and molars. EDS results showed the weight percent of calcium in the enamel rod of incisors decreased in Wwp1 KO mice [(20.74±0.91)%] compared with the control group [(30.30±3.83)%] (P<0.001), and the calcium-to-phosphorus ratio decreased in Wwp1 KO mice (1.93±0.01) compared with the control group (2.02±0.01) (P<0.001). Immunofluorescence showed weaker AMELX expression in ameloblasts of mandibular first molar tooth germs from P1 and P7 Wwp1 KO mice compared with the control group (P<0.001, P<0.001). In LS-8 cells, Wwp1 knocked-down led to a decrease of AMELX protein expression, while WWP1 overexpression resulted in an increased AMELX protein level. Conclusions: WWP1 promotes ameloblast differentiation and enamel matrix mineralization, playing a critical role in enamel formation.
目的： 研究含有WW结构域的E3泛素蛋白连接酶1（WWP1）在小鼠牙釉质形成中的作用。 方法： 对出生后（P）7 d（P7）小鼠切牙和P3.5小鼠下颌第一磨牙牙胚的单细胞RNA测序数据进行可视化，分析Wwp1在小鼠牙上皮中的整体表达情况。采用免疫组化染色观察WWP1在不同时期小鼠切牙和下颌第一磨牙牙胚上皮中的表达。构建Wwp1基因敲除（Wwp1 KO）小鼠。分别收集P1、P7 Wwp1 KO及同窝对照小鼠各3只，P14、P28、2个月（2M）、3M的Wwp1 KO及同窝对照小鼠各6只。采用显微CT分析小鼠磨牙及切牙的牙釉质形成量。采用扫描电镜观察小鼠切牙和磨牙牙釉质断面结构。能谱分析检测切牙断面釉柱表面钙磷含量。采用免疫荧光检测小鼠釉原蛋白（AMELX）表达。培养类成釉上皮细胞LS-8，转染Wwp1 siRNA或过表达质粒，采用蛋白质印迹法检测AMELX的表达。 结果： 单细胞测序数据显示，Wwp1在小鼠切牙和下颌第一磨牙牙胚上皮细胞中高表达。免疫组织化学染色显示，小鼠切牙和磨牙牙胚中WWP1高表达于分泌前期、分泌期、过渡期和成熟期的成釉细胞。不同于对照小鼠，Wwp1 KO小鼠下颌切牙呈现不透明白垩色。Wwp1 KO小鼠磨牙和切牙牙釉质体积［分别为（0.155±0.016）、（0.300±0.017）μm3］较对照小鼠［分别为（0.203±0.062）、（0.418±0.023）μm3］显著降低23.95%（P<0.001）和28.31%（P<0.001）。扫描电镜观察显示，Wwp1 KO小鼠釉柱低平、松散、结构紊乱。能谱分析结果显示，Wwp1 KO小鼠切牙牙釉质断面钙元素的质量百分比［（20.74±0.91）%］和钙磷比（1.93±0.01）均显著低于对照组［分别为（30.30±3.83）%和2.02±0.01］（均P<0.001）。免疫荧光结果显示，P1和P7的Wwp1 KO小鼠下颌第一磨牙牙胚成釉细胞中AMELX表达均显著低于对照组（均P<0.001）。在LS-8细胞中敲低或过表达WWP1分别使AMELX蛋白表达下降或上升。 结论： WWP1对小鼠牙釉质有促进成釉细胞分化和釉基质矿化的作用，从而可以促进牙釉质的正常发育。.