SONIC HEDGEHOG SIGNALING IN ODONTOGENESIS

牙齿发育是一个复杂的、多阶段的过程，包括上皮-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经被详细描述，但基本的调控机制仍然很不清楚。特别是，人们对介导上皮-间充质相互作用的分子的性质、极化生长的方式以及“微小”牙齿生殖细胞群体(如内牙齿上皮相关中间层)的作用知之甚少。在我们下面的初步研究中，我们现在发现：(A)Sonic hedgehog(SHH)在钟状晚期和牙冠阶段的中间层和相关的前成釉细胞中表达，以及(B)在器官培养中用抗SHH或SHH反义寡核苷酸处理牙胚，抑制成釉细胞和成牙本质细胞的成釉器官生长、间充质凝聚和细胞分化。我们首次发现，在牙齿发育过程中，中间层在结构和表型上经历了显著的时空变化，包括瞬时多层和SHH表达；相关的前成釉细胞层发生了相互作用的事件。这些研究和下文详述的其他初步研究将我们引向两个中心假设：(A)SHH是一种信号分子，首先由牙板产生，然后由中间层产生，在牙胚启动和早期形态发生事件中是必需的；以及(B)SHH在成釉细胞终末分化过程中介导中间层和内牙上皮之间的上皮-上皮相互作用。具体地说，我们建议(A)确定SHH信号在牙齿形成过程中的机制，(B)分析中间层结构和表型在牙齿形成过程中的变化，以及(C)确定中间层是否以及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有关形态发生和细胞信号的关键信息，这些信号负责牙齿形成的启动和进展。

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.