SONIC HEDGEHOG SIGNALING IN ODONTOGENESIS

牙齿发育是一个复杂的多阶段过程，涉及上皮-间充质相互作用、极化生长、两侧对称性的建立、形态发生运动和细胞分化。 这些复杂的发育事件已被详细描述，但基本的调控机制仍不清楚。特别是，很少有人知道介导上皮间充质相互作用的分子的性质，极化生长的模式，以及“小”牙胚细胞群的作用，如内牙上皮相关的中间层。在我们下面的初步研究中，我们现在表明：（a）Sonic hedgehog（SHH）在钟状晚期和冠状期的中间层和相关的成釉细胞中表达，和（B）用抗SHH或SHH反义寡核苷酸的抗体处理器官培养物中的牙胚抑制成釉器官生长、间充质凝聚以及成釉细胞和成牙本质细胞的细胞分化。我们第一次表明，中间层经历了显着的时空变化的结构和表型在牙发育过程中涉及短暂的多层和SHH的表达;互惠事件发生在相关的成釉细胞层。这些和下面详细描述的额外的初步研究使我们得出两个中心假设：（A）SHH是首先由牙板和其后的中间层产生的信号分子，其是牙胚起始和早期形态发生事件所需的;和（B）SHH在后期介导中间层和内牙上皮之间的上皮-上皮相互作用，其是成釉细胞终末分化所需的.具体而言，我们建议（a）确定SHH信号在牙发育过程中的机制，（B）分析中间层的结构和表型如何在牙发育过程中的变化，和（c）确定中间层是否和如何诱导和调节成釉细胞分化。为了实现这些目标，我们将利用器官培养、组织移植、原位杂交、免疫学方法、重组蛋白制备和逆转录病毒驱动的异位基因表达。该项目的结果将提供关键信息的形态发生和细胞信号负责的启动和发展的牙齿。

Tooth development is a complex, multistage process that involves reciprocal epithelial-mesenchymal interactions, polarized growth, establishment of bilateral symmetries morphogenetic movements, and cytodifferentiation. These complex developmental events have been described in detail, but the underlying mechanisms of regulation remain largely unclear. In particular, little is known about the nature of the molecules mediating epithelial-mesenchymal interactions, the modes of polarized growth, and the roles of "minor" tooth germ cell populations such as the inner dental epithelium-associated stratum intermedium. In our Preliminary Studies below, we now show that (a) Sonic hedgehog (SHH) is expressed in stratum intermedium and associated preameloblasts at late-bell and crown stages, and (b) treatment of tooth germs in organ culture with antibodies against SHH or SHH antisense oligonucleotides inhibits enamel organ growth, mesenchymal condensation, and cytodifferentiation of ameloblasts and odontoblasts. We show for the first time that stratum intermedium undergoes striking spatio-temporal changes in structure and phenotype during odontogenesis involving transient multilayering and SHH expression; reciprocal events occur in the associated preameloblast layer. These and additional Preliminary Studies detailed below lead us to two central hypotheses: (A) SHH is a signaling molecule produced by the dental lamina first and stratum intermedium thereafter, which is needed for tooth germ initiation and early morphogenetic events; and (B) SHH mediates epithelial- epithelial interactions between stratum intermedium and inner dental epithelium at later stages, which are needed for ameloblast terminal differentiation. Specifically , we propose to (a) determine the mechanisms of SHH signaling during odontogenesis, (b) analyze how stratum intermedium structure and phenotype change during odontogenesis, and (c) determine whether and how stratum intermedium induces and regulates ameloblast cell differentiation. To achieve these goals, we will make use of organ cultures, tissue transplantations, in situ hybridization, immunological approaches, recombinant protein preparation, and retrovirally-driven ectopic gene expression. The results of the project will provide key information on morphogenetic and cellular signals responsible for initiation and progression of odontogenesis.