REGULATION OF PITX2 IN DENTAL DEVELOPMENT

牙齿发育是一个复杂的形态发生过程，既涉及内在遗传信号，也涉及表观遗传环境信号。最近的研究已经开始揭示一些牙齿形态发生的调节因子，包括同源结构域转录因子和骨形态发生蛋白（BMPs）。本应用的重点是一个新的同源结构域基因，是与里格尔综合征。里格综合征的特征是形态发生异常，包括牙齿发育不全。里格基因，现在被称为Pitx2，是类bicoid家族的成员，具有交互激活活性。它是已知最早的牙齿原基标记，被认为在早期牙齿形态发生中起关键作用。然而，调控pitx2表达和功能的机制尚不清楚。例如，我们关于Pitx2转激活的初步数据表明，c端结构域具有新的抑制作用。此外，Pitx2的表达需要一个未知的间质信号，并且明显需要其他因子才能完全激活。这个应用程序的一个主要目标是回答这个问题，什么是调节Pitx2功能的因素和信号？我们建议首先建立Pitx2泛函域的工作模型。在这种情况下，我们将问C末端区域如何抑制反式激活以及自然发生的Rieger突变如何影响Pitx2功能？然后我们将研究控制Pitx2表达和功能的机制。在第一组实验中，我们将通过信号分子检查Pitx2的控制。由于牙齿的形成依赖于上皮-间质相互作用，我们提出在外植体培养中验证细胞外信号BMP-4和Sonic hedgehog （Shh）调节Pitx2表达的假设。我们还将测试这些药物是否能激活模型细胞系中的Pitx2启动子活性。在第二组实验中，我们将询问Pitx2活性是否受到与口腔上皮转录因子的物理相互作用的控制，正如我们使用Pit-1蛋白的初步研究所表明的那样。我们将测试与牙上皮中与Pitx2共表达的已知候选蛋白的相互作用，例如Msx2同源结构域蛋白。这种方法还将使我们能够确定新的相互作用的伙伴。然后，我们将测试这些蛋白质相互作用对Pitx2功能很重要的假设。这些研究将为更好地理解早期牙齿发育的分子调控提供基础。

Tooth development is a complex morphogenetic process that involves both intrinsic genetic cues and epigenetic environmental signals. Recent studies have begun to reveal some of the regulators of tooth morphogenesis, including homeodomain transcription factors and bone morphogenic proteins (BMPs). The focus of this application is a novel homeodomain gene that is linked to Rieger syndrome. Rieger syndrome is characterize by morphogenetic abnormalities that include dental hypoplasia. The Rieger gene, now termed Pitx2, is a member of the bicoid-like family and has transactivation activity. It is the earliest known marker in the tooth primordia and is believed to play a key role in early tooth morphogenesis. However, the mechanisms that regulated Pitx 2 expression and function are not known. For example, our preliminary data on Pitx2 transactivation suggests a novel inhibitory role for the C-terminal domain. Furthermore, Pitx2 expression requires an unknown mesenchymal signal and additional factors are clearly needed for full transactivation activity. A major objective of this application will be to answer the question, what are the factors and signals that regulate Pitx2 function? We propose to first establish a working model of Pitx2 functional domain. In this context we will ask how does the C- terminal region inhibit transactivation and how do the naturally occurring Rieger mutations affect Pitx2 function? We will then examine the mechanisms controlling Pitx2 expression and function. In the first set of experiments we will examine control of Pitx2 by signaling molecules. Since tooth formation relies on epithelial-mesenchymal interactions, we propose to test the hypothesis that the extracellular signals BMP-4 and Sonic hedgehog (Shh) regulate Pitx2 expression in explant cultures. We will also test whether those agents activate Pitx2 promoter activity ion a model cell line. In the second set of experiments, we will ask if Pitx2 activity is controlled by physical interactions with oral epithelial transcription factors, as suggested by our preliminary studies using the Pit-1 protein. We will test interactions with known candidates that are co-expressed with Pitx2 in the dental epithelia, such as the Msx2 homeodomain protein. This approach will also enable us to identify novel interacting partners. We will then test the hypothesis that these protein interactions are important for Pitx2 function. These studies will provide the foundation for a better understanding of the molecular control of early tooth development.