The Role of FAM20B-catalyzed Proteoglycans in Tooth Development

项目摘要/摘要 多余的牙齿会导致广泛的牙科并发症。当额外的齿形成在 现有的牙列，解开多生牙齿形成的分子机制不仅有助于 制定这种疾病的治疗策略，同时也为牙齿再生提供见解。尽管 在理解形态致病因子、生长因子和 转录因子在多生牙齿形成中的作用，对细胞外的作用知之甚少 在这一病理过程中的一些成分，如蛋白多糖。我们最近的研究表明，灭活 新发现的糖胺多聚糖(GAG)必需的木糖激酶--牙上皮细胞FAM20B 组装，会导致小鼠多出门牙。我们的初步研究表明，老年人缺乏胃口。 牙齿上皮导致WNT信号的异位激活，而SOX2的异位表达是 位于同一区域，通常应在E14.5之后从该站点消失。我们的体外研究 结果表明，某些FAM20B催化的蛋白多糖上的GAG抑制WNT信号转导，但促进WISE- 介导对WNT的抑制作用。相反，在突变胚胎中应用WNT抑制剂可以拯救 某些情况下的牙齿表型。这些数据引导我们形成了我们的中心假设，即某些FAM20B- 催化蛋白多糖通过负调控介导干细胞更新来调节牙齿更新 关于牙齿上皮细胞中WNT信号的研究。为了验证这一假设，我们提出了以下三个具体的假设 目的：(1)确定FAM20B催化的蛋白多糖是否通过负调控介导牙齿更新 关于WNT信号，以及IF-GAG介导的对WNT的明智抑制是多生牙的基础 队形。我们将通过在牙齿上皮细胞中过表达Dkk1或Wise来进行拯救实验 抑制K14Cre/+；Fam20Bfl/fl小鼠WNT信号的过度激活。(2)决定FAM20B是否- 催化蛋白多糖通过调节牙齿上皮中干细胞的更新来调节牙齿的更新。 我们将进行谱系追踪和诱导性基因敲除实验来确定干细胞的作用。 GAG缺乏引起的多生牙的更新，以及FAM20B催化的PGs是否介导了茎 牙齿上皮细胞以细胞自主的方式更新。(3)鉴定FAM20B酶 负责多余牙齿形成的蛋白多糖。我们将确定表达模式 FAM20B催化的蛋白多糖在牙齿中的表达，并确定那些负调控WNT信号的分子。这个 候选蛋白多糖的生物学功能将通过基因敲除和器官培养来确定 方法：研究方法。这项研究的完成将促进我们对分子机制的理解 形成多余牙齿的基础，并有助于为牙齿再生奠定基础。AS FAM20B和蛋白多糖在许多组织中广泛表达，由此获得的知识 这项研究也可能为蛋白多糖在其他组织中介导的信号传递提供有价值的见解。

Project summary/Abstract Supernumerary teeth can cause a broad range of dental complications. As extra teeth are formed on existing dentition, unraveling the molecular mechanism of supernumerary tooth formation will not only help develop the therapeutic strategy for this disease but also provide insights into tooth regeneration. Despite the significant progress in understanding the regulatory role of morphogens, growth factors, and transcriptional factors in supernumerary tooth formation, little is known about the role of extracellular components such as proteoglycans in this pathological process. Our recent studies show that inactivation of dental epithelial Fam20B, a newly discovered xylose kinase essential for glycosaminoglycan (GAG) assembly, leads to supernumerary incisors in mice. Our pilot study reveals that GAG deficiency in the dental epithelium leads to ectopic activation of WNT signaling, and that an ectopic Sox2 expression is located in the same area, which normally should disappear from this site after E14.5. Our in vitro study shows that GAGs on certain FAM20B-catalyzed proteoglycans suppress WNT signaling but facilitate Wise- mediated inhibition on WNT. Conversely, administering WNT inhibitor to the mutant embryos rescued the tooth phenotype in some cases. These data led us to form our central hypothesis that certain FAM20B- catalyzed proteoglycans regulate tooth renewal by mediating the stem cell renewal via negative regulation on WNT signaling in the dental epithelium. To test this hypothesis, we propose the following three specific aims: (1) To determine if FAM20B-catalyzed proteoglycans mediate tooth renewal via negative regulation on WNT signaling, and if GAG-mediated Wise inhibition on WNT underlies the supernumerary tooth formation. We will perform "rescue" experiments by overexpressing DKK1 or Wise in the dental epithelium to inhibit the overactivated WNT signaling in K14Cre/+;Fam20Bfl/fl mice. (2) To determine whether FAM20B- catalyzed proteoglycans regulate tooth renewal by mediating the stem cell renewal in the dental epithelium. We will perform lineage tracing and inducible knockout experiments to determine the role of stem cell renewal in GAG deficiency-caused supernumerary teeth, and if the FAM20B-catalyzed PGs mediate stem cell renewal in the dental epithelium in a cell-autonomous manner. (3) To identify the FAM20B-catalyzed proteoglycans responsible for the supernumerary tooth formation. We will determine the expression pattern of FAM20B-catalyzed proteoglycans in tooth and identify those negatively regulating WNT signaling. The biological function of candidate proteoglycans will be determined by gene knockdown and organ culture methods. The completion of this study will advance our understanding about the molecular mechanism underlying supernumerary tooth formation and help in laying the groundwork for tooth regeneration. As FAM20B and proteoglycans are extensively expressed in many tissues, the knowledge gained from this study may also provide valuable insights into the proteoglycan-mediated signaling in other tissues.