Mesenchymal Mycn participates in odontoblastic lineage commitment by regulating Krüppel-like Factor 4 (Klf4) in mice.
Mesenchymal Mycn participates in odontoblastic lineage commitment by regulating Krüppel-like Factor 4 (Klf4) in mice.
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DOI：
10.1186/s13287-022-02749-8
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发表时间：
2022-02-22
影响因子：
7.5
通讯作者：
Bian Z
中科院分区：
文献类型：
--
作者：
Huang Z;Yang R;Li R;Zuo Y;Gu F;He M;Bian Z
小鼠牙乳头细胞（mDPC）对成牙本质细胞谱系的承诺对于牙本质的形成至关重要，并且这一生物过程受到复杂的转录因子网络的调节。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。一项早期研究表明，Mycn 仅在 E15.5 的牙间充质细胞中表达，这意味着 Mycn 在牙本质发生中具有潜在作用。然而，Mycn 在牙本质形成中的作用仍然难以捉摸。因此，阐明 Mycn 在牙本质形成中的作用具有相当大的意义。 Mycnfl/fl; Osr2IresCre (MycnOsr2) 和 Mycnfl/fl；生成K14Cre（MycnK14）转基因小鼠，并进行显微CT扫描以定量分析突变体及其同窝小鼠的臼齿和门牙的体积差异。 Mycn也在体外被敲低，并进行碱性磷酸酶（ALP）和茜素红染色（ARS）。进行靶标切割和标记 (CUT&Tag) 分析以及双荧光素酶测定，以鉴定 Mycn 的直接下游靶标。进行免疫荧光和免疫化学染色以及蛋白质印迹（WB）来分析潜在靶标的表达水平。进行定量PCR、WB、ALP和ARS来测试救援效率。 Mycn（MycnOsr2）的间充质去除导致牙本质形成缺陷，而上皮缺失（MycnK14）对牙齿发育没有明显影响。 ALP 和 ARS 染色显示，MycnOsr2 小鼠中 mDPC 对成牙本质细胞谱系的定型能力受到损害。 CUT&Tag 分析确定 Klf4 是 Mycn 的潜在直接靶标，双荧光素酶报告基因分析证实 Mycn 可以与 Klf4 的启动子区域结合并直接激活其转录。相反，Klf4 的强制表达部分恢复了 Mycn 敲低的 mDPC 的成牙本质细胞分化能力。我们的结果阐明间充质 Mycn 通过直接调节 Klf4 来调节牙乳头细胞的成牙本质细胞定向。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牙本质发生过程中涉及的转录因子网络的总体理解。因此，这些结果可能为牙本质发育不全和生物工程牙本质再生提供新的见解。在线版本包含可在 10.1186/s13287-022-02749-8 获取的补充材料。
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. Mycnfl/fl; Osr2IresCre (MycnOsr2) and Mycnfl/fl; K14Cre (MycnK14) 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 (MycnOsr2) led to defective dentin formation, while epithelial deletion (MycnK14) 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 MycnOsr2 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. The online version contains supplementary material available at 10.1186/s13287-022-02749-8.
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影响因子：
7.6
作者：
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通讯作者：
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作者：
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作者：
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通讯作者：
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作者：
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通讯作者：
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