Follistatin与连续性牙齿（Successional Teeth）发育的遗传学控制

Mammals including human replace their teeth once in life, belonging to successional teeth. However, the molecular control of successional teeth development still remains elusive. Our preliminary studies demonstrated that the deletion of mouse Follistatin gene result in the formation of successional tooth germ lingual to the molar, the kidney capsule implantation of such Follistatin ntooth germ redulted in the formation of differentiated tooth structure. In addition, the level of phosphorylated Smad-1/5/8 in Follistatin-/- increases dramatically, in comparison with the wild type control. Thus, these preliminary data lead to the hypothesis that Follistatin acts by antagonizing BMP and/or Activin to inhibit the successional teeth formation in wild type mouse. To confirm this hypothesis, we first propose to investigate the developmental mechanism of successional tooth germ in Follistatin-/- by deciding the gene expression pattern of critical tooth marker genes, the cell proliferation, and cell apotosis. Pattern. We also pursue the question of how and to what extent the successional tooth germ in the absence of mouse Follistatin undergo the differentiation and morphogenesis, by tissue-specific ablation of Follistain in dental lamina and underlying neural crest derived mesenchyme with K14-Cre/Osr2-IERS-Cre at E11.75, which might be a way to overcome the neonatal lethality of Follistatin straight deletion. Next question we propose to study in this project is the developmental ability and molecular nature of dental lamina in Follistatin-/- mutant. We will perform the tracing of Sox2 cell lineages in successional teeth budding from the dental lamina in Follistatin-/- mutant carrying Sox2-ki-EGFP allele. Using K14Cre, we will also delete Sox2 in dental lamina and oral epithelium in Follistatin-/- mutant to determin whether the successional teeth budding results from the stem precursors in dental lamina. We also propose the genetic investigation of whether the supernumary teeth is induced in the dental lamina where the beta-catenin is activated by K14-Cre in Follistatin-/- carrying beta-catenin3ex allele. Finally, we propose to investigate the mechanism by which the Follistatin inhibits the successional teeth formation in mouse by antagonizing the BMP/or Activin signaling. We will examine the levels of BMP and Activin βA signaling by checking the phorsphorylation of Smad1/5/8 and Smad2/3 by immunohistovchemistry, respectively. We then will access the mechanism investigation by overexpression of Noggin, the secreted antagonist to BMP protein, in Follistatin-/- tooth germ organ culture. In a paralleled experiment, we propose to knockdown the Activin-βA by expression of si-Activin βA in the tooth germ organ culture. We expect to approach the molecular control of successional tooth initiation and formation development by elucidating the role of Follistatin in control of successional tooth budding from dental lamina in mouse gene modification model system.

模式动物小鼠没有牙齿替换特征。对于替换牙齿形成的分子控制仍不很明确。我们发现敲除小鼠Follistatin导致牙板在磨牙舌侧连续形成超数牙胚；pSmad1/5/8水平在突变牙胚上调。提示Follistatin可能拮抗BMP或Activin通路抑制野生小鼠牙板的连续性生长。为验证上述设想，本项目首先利用原位杂交，细胞增殖，细胞凋亡测定对Follistatin基因突变中连续性牙胚进行发育机理研究。还利用双Cre活性在E11.5同时敲除口腔上皮与间充质Follistatin来研究连续性牙齿生长分化程度。同时利用Sox2tm2Hoch /Follistatin-/-对牙板上皮Sox2细胞系进行追踪，及功能验证。并检测突变牙板对由于β-catenin激活形成超数牙胚的反应以解析牙板连续发育能力。最后，通过拮抗BMP水平；敲低Activin表达等验证野生小鼠中Follistatin的遗传性抑制功能。

Follistatin(Fst)基因在哺乳动物牙齿发育过程中起着重要的控制作用。本项目研究发现：Fst基因的RNA 转录产物在牙胚发育中同时存在于上皮组织和间充质组织中；利用Cre/Lox系统的条件性基因敲除手段同时从胚胎口腔上皮组织及牙胚间充质组织敲除控制Fst基因（Fstfx/fx；K14-Cre；Wnt1-Cre）导致形成超数的上切牙。利用Micro-CT确定这种牙齿表型有别于其他研究者利用在牙向上皮组织过表达Fst所发现的在舌侧形成异常位置的釉质的表型不同。一种可能性是在Follistatin缺失下，切牙舌侧口腔上皮可能获得了与牙向诱导相关的能力；二是Follistatin缺失导致牙间充质中与已知与BMP4信号相关的与牙向发育能力关联的信号通路或反馈信号回路的激活。我们利用组织学及牙胚标志性基因Shh的原位RNA分子表达分析发现， FST缺失的上切牙牙胚在E16.5其基部上皮明显可以分辨为两个牙颈环区域。成年小鼠切牙的不断生长的细胞来源及动力来自牙颈环区的具有干细胞性质的细胞，因而这些组织学分析结果也意味着Follistatin与牙颈环区的发育调节相关。我们对牙颈环上皮的干细胞标志基因Sox2 的表达进行了进一步分析。原位杂交与Sox2蛋白质都显示，E14.5 Fstfx/fx；K14-Cre；Wnt1-Cre突变胚胎中Sox2在上切牙牙胚出现超数的上皮表达区域表达数量增多，并且CyclinD1的免疫荧光结果显示在该部位的细胞增殖显著增强。这可能说明超数形成的上切牙牙胚会发育成切牙牙齿结构，未来超数牙的牙颈环结构具有保证了使得切牙保持不断连续生长提供牙源干细胞。由于FST fx/fx；K14-Cre；Wnt1-Cre的超数门牙表型提示超数发生的牙齿可能反映了FST通过对Smad1/5/8或Smad2/3的BMP或TGF-β信号的正负调节完成。我们通过对野生型和FollistatinK14-Cre;Wnt-Cre突变小鼠的上切牙进行免疫荧光发现在FollistatinK14-Cre;Wnt-Cre牙齿中，TGF-β信号通路被过度激活，表现为p-Smad2的水平显著增强。RNA-Seq分析也表明突变牙齿中的基因表达发生显著变化，这也提示牙胚发育中Fst通过TGF-beta信号调节牙颈环干细胞提供对小鼠门牙生长的支持。

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