Human with bi-allelic WNT10A mutations and epithelial Wnt10a knockout mice present enlarged pulp chamber and apical displacement of the root furcation of multi-rooted teeth, known as taurodontism; thus, indicating the critical role of Wnt10a in tooth root morphogenesis. However, the endogenous mechanism by which epithelial Wnt10a regulates Hertwig’s epithelial root sheath (HERS) cellular behaviors and contributes to root furcation patterning remains unclear. In this study, we found that HERS in the presumptive root furcating region failed to elongate at an appropriate horizontal level in K14-Cre;Wnt10a^fl/fl mice from post-natal day 0.5 (PN0.5) to PN4.5. EdU assays and immunofluorescent staining of cyclin D1 revealed significantly decreased proliferation activity of inner enamel epithelial (IEE) cells of HERS in K14-Cre;Wnt10a^fl/fl mice at PN2.5 and PN3.5. Immunofluorescent staining of E-Cadherin and acetyl-α-Tubulin demonstrated that the IEE cells of HERS tended to divide perpendicularly to the horizontal plane, which impaired the horizontal extension of HERS in the presumptive root furcating region of K14-Cre;Wnt10a^fl/fl mice. RNA-seq and immunofluorescence showed that the expressions of Jag1 and Notch2 were downregulated in IEE cells of HERS in K14-Cre;Wnt10a^fl/fl mice. Furthermore, after activation of Notch signaling in K14-Cre;Wnt10a^fl/fl molars by Notch2 adenovirus and kidney capsule grafts, the root furcation defect was partially rescued. Taken together, our study demonstrates that an epithelial Wnt10a-Notch signaling axis is crucial for modulating HERS cell proper proliferation and horizontal-oriented division during tooth root furcation morphogenesis.

具有双等位基因WNT10A突变的人类和上皮Wnt10a基因敲除小鼠会出现牙髓腔扩大和多根牙根分叉的根尖移位，称为牛牙症；因此，表明Wnt10a在牙根形态发生中的关键作用。然而，上皮Wnt10a调节 Hertwig 上皮根鞘 (HERS) 细胞行为并促进根分叉模式的内源机制仍不清楚。在这项研究中，我们发现K14-Cre;Wnt10a ^fl/fl小鼠从出生后第 0.5 天 (PN0.5) 到 PN4.5，推定根分叉区域的 HERS 未能在适当的水平水平上伸长。 EdU 测定和细胞周期蛋白 D1 的免疫荧光染色显示，在 PN2.5 和 PN3.5 的K14-Cre;Wnt10a ^fl/fl小鼠中，HERS 内釉质上皮 (IEE) 细胞的增殖活性显着降低。 E-钙粘蛋白和乙酰-α-微管蛋白的免疫荧光染色表明，HERS 的 IEE 细胞倾向于垂直于水平面分裂，这损害了K14-Cre 假定根分叉区域中 HERS 的水平延伸；Wnt10a ^fl/fl老鼠。 RNA-seq和免疫荧光显示K14-Cre;Wnt10a ^fl/fl小鼠的HERS IEE细胞中Jag1和Notch2的表达下调。此外，Notch2腺病毒和肾被膜移植物激活K14-Cre;Wnt10a ^fl/fl磨牙中的Notch信号后，根分叉缺陷得到部分修复。 综上所述，我们的研究表明，上皮 Wnt10a-Notch 信号轴对于调节牙根分叉形态发生过程中 HERS 细胞的适当增殖和水平分裂至关重要。