The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei, China.
Stem Cell Res Ther. 2022 Feb 22;13(1):78. doi: 10.1186/s13287-022-02749-8.
Commitment of mouse dental papilla cells (mDPCs) to the odontoblast lineage is critical for dentin formation, and this biological process is regulated by a complex transcription factor network. The transcription factor Mycn is a proto-oncogene that plays an important role in tumorigenesis and normal embryonic development. An early study revealed that Mycn is exclusively expressed in dental mesenchymal cells at E15.5, which implies a potential role of Mycn in dentinogenesis. However, the role of Mycn in dentin formation remains elusive. Thus, it is of considerable interest to elucidate the role of Mycn in dentin formation.
Mycn; Osr2 (Mycn) and Mycn; K14 (Mycn) transgenic mice were generated, and micro-CT scans were performed to quantitatively analyse the volumetric differences in the molars and incisors of the mutants and their littermates. Mycn was also knocked down in vitro, and alkaline phosphatase (ALP) and alizarin red staining (ARS) were conducted. Cleavage under targets and tagmentation (CUT&Tag) analysis and dual luciferase assays were performed to identify direct downstream targets of Mycn. Immunofluorescence and immunochemistry staining and western blotting (WB) were performed to analyse the expression levels of potential targets. Quantitative PCR, WB, ALP and ARS were performed to test the rescue efficiency.
Mesenchymal ablation of Mycn (Mycn) led to defective dentin formation, while epithelial deletion (Mycn) had no obvious effects on tooth development. ALP and ARS staining revealed that the commitment capacity of mDPCs to the odontoblast lineage was compromised in Mycn mice. CUT&Tag analysis identified Klf4 as a potential direct target of Mycn, and a dual luciferase reporter assay verified that Mycn could bind to the promotor region of Klf4 and directly activate its transcription. Reciprocally, forced expression of Klf4 partially recovered the odontoblastic differentiation capacity of mDPCs with Mycn knockdown.
Our results elucidated that mesenchymal Mycn modulates the odontoblastic commitment of dental papilla cells by directly regulating Klf4. Our study illustrated the role of Mycn in dentin development and furthers our general comprehension of the transcription factor networks involved in the dentinogenesis process. Thus, these results may provide new insight into dentin hypoplasia and bioengineered dentin regeneration.
鼠牙乳头细胞（mDPC）向成牙本质细胞系的分化是牙本质形成的关键，这一生物学过程受复杂的转录因子网络调控。转录因子 Mycn 是一种原癌基因，在肿瘤发生和正常胚胎发育中发挥重要作用。早期研究表明，Mycn 仅在 E15.5 的牙间质细胞中表达，这表明 Mycn 可能在牙本质形成中发挥作用。然而，Mycn 在牙本质形成中的作用仍不清楚。因此，阐明 Mycn 在牙本质形成中的作用具有重要意义。
生成了 Mycn；Osr2（Mycn）和 Mycn；K14（Mycn）转基因小鼠，并通过 micro-CT 扫描对突变体及其同窝仔鼠磨牙和切牙的体积差异进行定量分析。体外敲低 Mycn 后进行碱性磷酸酶（ALP）和茜素红染色（ARS）。进行切割目标和标签化（CUT&Tag）分析和双荧光素酶测定以鉴定 Mycn 的直接下游靶标。进行免疫荧光和免疫组化染色以及 Western blot（WB）分析以分析潜在靶标的表达水平。进行定量 PCR、WB、ALP 和 ARS 以测试挽救效率。
Mycn 的间质消融（Mycn）导致牙本质形成缺陷，而上皮缺失（Mycn）对牙齿发育没有明显影响。ALP 和 ARS 染色显示 Mycn 小鼠 mDPCs 向成牙本质细胞系的分化能力受损。CUT&Tag 分析鉴定出 Klf4 是 Mycn 的一个潜在直接靶标，双荧光素酶报告基因测定验证了 Mycn 可以结合 Klf4 的启动子区域并直接激活其转录。Mycn 敲低时，强制表达 Klf4 可部分恢复 mDPCs 的成牙本质分化能力。
我们的研究结果表明，间质 Mycn 通过直接调节 Klf4 调节牙乳头细胞的成牙本质细胞分化。我们的研究阐明了 Mycn 在牙本质发育中的作用，并进一步加深了我们对参与牙本质形成过程的转录因子网络的理解。因此，这些结果可能为牙本质发育不全和牙本质生物工程再生提供新的见解。
