Small molecule control of Wnt signal transduction

在完整的动物体内通过失活来研究特定蛋白质的功能提出了几个挑战。基因删除或“敲除”技术完全消除了一种蛋白质，但由于这种蛋白质可能在不同的组织或不同的发育阶段发挥作用，敲除小鼠可能无法存活到所需的成熟阶段。基于药物的方法是有吸引力的替代方法，因为小分子可以用来抑制基因正常动物的蛋白质功能，它们可以在特定时间给药和移除，因此是可逆的，并且它们通常为药物开发提供有吸引力的先导化合物。然而，小分子也有自己的挑战。有没有一种小分子可以靶向目标蛋白？它能被送到活体动物身上吗？最重要的是，小分子的脱靶效应能被最小化吗？为了研究发育中的信号蛋白的功能，我们结合了基因靶向和小分子的优势，使用一种称为诱导稳定的新方法，即无毒药物调节任何感兴趣的特定蛋白质的稳定性。在胚胎发育和成长的过程中，每个细胞都必须与邻近的细胞精确协调，以使动物形成正确的图案。这些细胞必须随时与周围组织沟通并决定细胞的命运。细胞如何知道对哪些刺激作出反应，对哪些刺激不予理睬？更彻底地了解关键信号分子在特定类型细胞中的作用，将使我们更好地了解动物是如何构建的，以及当发育出错时会发生什么。我的工作旨在通过采用新的化学工具来解决这些问题，以帮助我们更好地了解胚胎发育。研究发展过程的一个主要问题是这些过程是随时间发生的。例如，胚胎首先产生神经前体，然后将其中一些细胞分配成不同类型的神经组织。与此同时，由于胚胎正在生长和形状变化，所有这些组织都需要发育并在正确的时间移到正确的位置。不知怎的，这些细胞可以感知“建筑计划”，并协调形成头部的大脑和肢体发育的运动神经元。具体来说，这项工作将侧重于制造新的工具来研究β -连环蛋白，这是一种在发育和癌症等疾病中很重要的分子。由于这种分子的重要性，我们将其作为这些新技术的“测试案例”。通过这种方式，我们的“测试案例”将教会我们很多关于这些新方法的知识，也可能对未来的生物学研究普遍有用。第二个目标是利用我们现有的糖原合成酶激酶3 （GSK-3）的药物依赖等位基因来研究神经嵴迁移。这种蛋白质也是发育和疾病的重要调节因子。我们以前使用类似的方法表明，GSK-3在腭发育的关键时期是必要的。使用这些小鼠，我们发现在妊娠中期添加这种蛋白质足以挽救突变小鼠的腭裂。我们希望利用这些方法来了解不同发育过程中所需的基因活动的时间和数量。综上所述，该项目将为研究界提供重要的新化学生物学工具，并深入了解神经嵴迁移的分子机制。更好地了解神经嵴迁移可能有助于我们更好地理解人类发育和疾病过程，如癌症转移。

Studying the functions of specific proteins by inactivation within an intact animal presents several challenges. Genetic deletion, or 'knockout,' technology completely eliminates a protein, but since the protein may have roles in different tissues or at different stages of development, a knockout mouse may not survive to the desired stage of maturity. Drug-based approaches are attractive alternatives because small molecules can be used to inhibit protein function in a genetically normal animal, they can be administered and removed at specific times and are thus reversible, and they often provide attractive lead compounds for drug development. However, small molecules present their own challenges. Is there a small molecule that targets the protein of interest? Can it be delivered to a live animal? Most importantly, can off-target effects of the small molecule be minimized? To study the function of signaling proteins in development we are combining the advantages of gene targeting and small molecules, using a novel approach called inducible stabilization in which a non-toxic drug regulates the stability of any specific protein of interest. As an embryo develops and grows, each cell must be precisely coordinated with its neighbors in order for the animal to be properly patterned. These cells must be communicating with surrounding tissues and making cell fate decisions at all times. How do cells know which stimuli to respond to and which stimuli to ignore? A more thorough understanding of what key signaling molecules are doing in specific types of cells will give us a better understanding of how an animal is built, as well as what happens when development goes awry. My work aims to address these questions by adapting novel chemical tools to help us better understand embryonic development. A major problem when studying developmental processes is that these processes occur over time. For example, first the embryo makes neural precursors, then it allocates some of these cells to become different types of neural tissue. Meanwhile, because the embryo is growing and changing in shape, all these tissues need to develop and be moved to the right place at the right time. Somehow the cells can sense an 'architectural plan' and coordinate to make brains in the head and motor neurons precisely where the limbs are developing. Specifically, this work will focus on making new tools to study beta-catenin, a molecule that is important in development and in diseases such as cancer. Because of the importance of this molecule, we are using it as a 'test case' for these new technologies. In this way, our 'test case' will teach us a great deal about these new methods and will also likely be generally useful for future biological studies. A second aim makes use of our existing drug-dependent allele of glycogen synthase kinase-3 (GSK-3) to study neural crest migration. This protein is also an important regulator of development and disease. We have previously shown, using similar methods, that GSK-3 is necessary during a critical period in palate development. Using these mice, we found that adding back this protein during mid-gestation was sufficient to rescue cleft palate in mutant mice. We hope to use these kinds of approaches to understand the timing and amounts of gene activity required in different developmental processes. Taken together, this project will provide important new chemical biology tools for the research community as well as gaining insight into molecular mechanisms underlying neural crest migration. A better understanding of neural crest migration will likely help us better understand human development and diseases processes such as cancer metastasis.