Small molecule control of Wnt signal transduction

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.

The Wnt/beta-catenin signalling pathway is evolutionarily conserved and plays important roles in proliferation and patterning. While this pathway has been well studied genetically and developmentally, many of the biochemical interactions remain unclear. Building new molecular tools to study Wnt signalling will provide important insight into a variety of biological processes. This proposal has two aims: 1) to develop new chemical methods to study Wnt/beta-catenin signalling, and 2) to better understand the roles of GSK-3 and beta-catenin during neural crest delamination and migration. To increase our 'tool kit', my lab is engineering conditional drug-dependent proteins that can be rapidly, specifically and reversibly regulated. Our approach is to fuse destabilizing domains (DD) to our protein of interest. In the absence of drug, the fusion protein is unstable and rapidly degraded. The addition of drug restores protein stability and function. We have previously used this approach to regulate GSK-3b in vivo (Nature 2007). In current BBSRC funded studies, we have designed a beta-catenin fusion protein which is drug-sensitive, allowing us to switch function on and off. There are now several 'next generation' peptide tags available that are reported to have better stabilization dynamics and improved pharmacology. This project aims to 1) test the efficacy and limitations of the new destabilizing domains, using b-catenin as a new 'druggable' target; and, 2) combine this approach with our GSK-3b allele, allowing drug-dependent control at two distinct points in the Wnt pathway. Furthermore, we will test our new b-catenin constructs in mouse mutants lacking b-catenin. Together, this project will allow us to test the requirements for these molecules during delamination and migration of the neural crest.

Because of the pivotal roles of Wnt/b-catenin signalling in biological processes including embryonic development, diabetes, neurodegeneration, cancer and stem cell differentiation, this proposal will impact a number of areas of basic biology. Beta-catenin is already a very interesting therapeutic target in human disease; however, designing drugs that target beta-catenin activity is challenging. Thus, understanding and controlling beta-catenin function will be informative and medically relevant. Our work is likely to have the greatest direct impact for following academic beneficiaries as outlined in the previous section. From a 'tool-making' point of view, development of drug-dependent conditional systems will be very useful for the study of any biological system. For example, the ability to control quantity and duration of a biological signal will be an important tool when testing predictions of systems biologists. It is increasingly clear that the ability to predict and test large-scale biological systems will be critical for our understanding of human biology. Our experiments will provide additional tools for testing protein interactions and will also help us refine our in vivo experiments. Our work is likely to have great impact for society: These benefits are indirect; however, by increasing the tools available to biological scientists, we will accelerate our understanding of human development and disease. For example, our tools will be immediately available for use in studying important disease processes such as metastasis, diabetes or neurodegeneration. Therefore, these studies may have long term global health and economic impacts. Our work may also have direct medical implications: Neural crest cells contribute to multiple tissue types in the body. These cells display incredible plasticity, giving rise to diverse tissues ranging from bone and cartilage to adipocytes and neurons. As a result, abnormalities in neural crest development lead to diverse congenital anomalies such as cleft palate, and Hirschsprung's Disease, where enteric innervation of the bowel is missing. Moreover, neural crest derivatives often reactivate during post-natal life, leading to highly invasive tumours such as melanomas. Even so, comparatively little is known about the genes necessary for later steps in neural crest development. In particular, very little is known about the genetic requirements for maintenance of neural crest stem cell, neural crest delamination or migration, or how these affect ultimate differentiation of the neural crest. The biochemical and genetic evidence suggests that GSK-3 and b-catenin are important players in all of these processes. Our project will pinpoint the role(s) of GSK-3/b-catenin during neural crest migration and development. In addition, because GSK-3 affects multiple target genes, this project will also provide a wealth of data on intersecting pathways, including those that regulate cell cycle, cytoskeleton and signalling. These data may be useful for understanding neurocristopathies, cancer metastasis and regenerative medicine. The data from this project will be immediately useful. We will present our data at international conferences and we will rapidly publish, in peer-reviewed journals, any data generated. Any tools generated will be available to the academic community for dissemination as well as adaptation for broader uses.