Using chemical tools to study Wnt signalling in neural development

通过在完整动物体内失活来研究特定蛋白质的功能存在几个挑战。基因删除或“敲除”技术完全消除了蛋白质，但由于蛋白质可能在不同组织或不同发育阶段发挥作用，敲除小鼠可能无法存活到所需的成熟阶段。药理学方法是有吸引力的替代方案，因为小分子可用于抑制遗传正常动物中的蛋白质功能，它们可以在特定时间施用和去除，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，小分子提出了自己的挑战。是否有一种小分子靶向感兴趣的蛋白质？它能被送到活的动物身上吗？最重要的是，小分子的脱靶效应能否最小化？为了研究信号蛋白在发育中的功能，我结合了基因靶向和小分子的优势，使用了一种称为诱导稳定的新方法，其中无毒药物调节任何特定蛋白质的稳定性。随着胚胎的发育和生长，每个细胞必须与相邻细胞精确协调，以便动物形成正确的模式。这些细胞必须与周围组织沟通，并随时决定细胞的命运。细胞是如何知道哪些刺激要回应，哪些刺激要忽略的？更深入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。研究发育过程的一个主要问题是，这些过程是随着时间的推移而发生的。例如，胚胎首先制造神经前体，然后分配其中一些细胞成为不同类型的神经组织。同时，由于胚胎正在生长和改变形状，所有这些组织都需要发育并在正确的时间转移到正确的位置。不知何故，这些细胞可以感知到一个“建筑计划”，并协调在头部制造大脑，在四肢发育的精确位置制造运动神经元。我目前的工作重点是Wnt信号中一种名为GSK-3的信号蛋白，这是一种已知对神经轴发育很重要的通路。在发育早期，Wnt信号被认为对胚胎后部的图案化很重要。过多的Wnt激活会导致前部头部结构截短，而抑制Wnt会导致头部结构扩大。GSK-3在Wnt通路中起着相反的作用，可能有助于维持动物不同部位接收到的信号量的平衡。我的工作使我们能够在胚胎发生过程中操纵GSK-3水平，比以前更精细的控制。此外，我还可以控制GSK-3在细胞内的定位，使用药物将其移入和移出细胞核。我正在使用的新化学工具可能也适用于其他蛋白质，这将为研究发育提供一套全新的工具。

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. Pharmacologic 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 I am 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 development 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. My work currently focuses on a signaling protein called GSK-3 in Wnt signalling, a pathway known to be important for the development of the neural axis. Early in development, Wnt signalling is thought be important for patterning the posterior part of the embryo. Too much Wnt activation results in a truncation of anterior head structures while inhibiting Wnts results in enlarged head structures. GSK-3 plays an opposing role in the Wnt pathway and presumably helps to maintain a balance in the amount of signals received in different parts of the animal. My work allows us to manipulate GSK-3 levels during embryogenesis with much finer control than previously available. In addtion, I can control the localization of GSK-3 within the cell, moving it into and out of the nucleus using a drug. The new chemical tools I am using may be applicable to other proteins as well which would provide a whole new set of tools to study development.