背景:了解细胞因子在牙齿发育中的作用对于推进牙齿组织工程至关重要。成纤维细胞生长因子9(FGF9)是唯一在整个牙齿上皮组织中一致表达的FGF,从牙芽形成开始到牙齿成熟。然而,缺乏Fgf9(Fgf9-/-)的小鼠在牙齿发育中没有明显的异常,建议其他FGFs的潜在补偿。在这里,我们报告了Fgf9S99N突变小鼠模型的发现,具有显性负效应的功能丧失突变。我们的研究表明,Fgf9对牙齿上皮干细胞(DESC)存活和牙釉质形成至关重要。
方法:为了剖析Fgf9在牙齿发育中的作用,我们做了显微CT,S99N突变小鼠和胚胎的组织形态学分析和基因表达测定。此外,我们通过DESC球体形成试验和牙齿外植体培养评估了FGF9对DESC存活和牙齿上皮分化的影响。细胞/组织培养方法,基因表达分析,特异性抑制剂,和抗体阻断分析用于探索Fgf9如何通过直接和间接机制调节牙釉质分化和DESC存活。
结果:小鼠的Fgf9S99N突变导致成釉细胞减少,牙釉质形成受损,和增加的细胞凋亡在宫颈环(CL)。DESC球体培养实验表明,FGF9通过激活ERK/CREB信号促进DESC存活,不影响细胞增殖。此外,体外组织培养实验表明,FGF9促进釉质形成的方式依赖于间充质的存在。有趣的是,FGF9刺激抑制了分离的釉质上皮和DESC球体中的釉质形成。进一步的研究表明,FGF9通过MAPK/ERK信号通路刺激牙间充质细胞中FGF3和FGF10的分泌,支持DESC存活并促进牙釉质生成。
结论:我们的研究表明,Fgf9对于DESC存活和牙釉质形成至关重要。Fgf9作为牙釉质上皮的双向调节器,不仅抑制DESC分化为成釉细胞以保持DESC的干性,而且还通过上皮-间质相互作用促进成釉细胞分化。
BACKGROUND: Understanding the role of cytokines in tooth development is critical for advancing dental tissue engineering. Fibroblast growth factor 9 (FGF9) is the only FGF consistently expressed throughout dental epithelial tissue, from the initiation of tooth bud formation to tooth maturation. However, mice lacking Fgf9 (Fgf9-/-) surprisingly show no obvious abnormalities in tooth development, suggesting potential compensation by other FGFs. Here we report findings from an Fgf9S99N mutation mouse model, a loss-of-function mutation with a dominant negative effect. Our study reveals that Fgf9 is crucial for dental epithelial stem cell (DESC) survival and enamel formation.
METHODS: To dissect the role of Fgf9 in tooth development, we performed the micro-CT, histomorphological analysis and gene expression assay in mice and embryos with S99N mutation. In addition, we assessed the effect of FGF9 on the DESC survival and dental epithelial differentiation by DESC sphere formation assay and tooth explant culture. Cell/tissue culture methods, gene expression analysis, specific inhibitors, and antibody blockage analysis were employed to explore how Fgf9 regulates enamel differentiation and DESC survival through both direct and indirect mechanisms.
RESULTS: The Fgf9S99N mutation in mice led to reduced ameloblasts, impaired enamel formation, and increased apoptosis in the cervical loop (CL). DESC sphere culture experiments revealed that FGF9 facilitated DESC survival via activating ERK/CREB signaling, without affecting cell proliferation. Furthermore, in vitro tissue culture experiments demonstrated that FGF9 promoted enamel formation in a manner dependent on the presence of mesenchyme. Interestingly, FGF9 stimulation inhibited enamel formation in isolated enamel epithelia and DESC spheres. Further investigation revealed that FGF9 supports DESC survival and promotes amelogenesis by stimulating the secretion of FGF3 and FGF10 in dental mesenchymal cells via the MAPK/ERK signaling pathway.
CONCLUSIONS: Our study demonstrates that Fgf9 is essential for DESC survival and enamel formation. Fgf9 performs as a dual-directional regulator of the dental enamel epithelium, not only inhibiting DESC differentiation into ameloblasts to preserve the stemness of DESC, but also promoting ameloblast differentiation through epithelial-mesenchymal interactions.