Wnt genes belong to a multigene family encoding secreted proteins that activate receptor-mediated signal transduction pathways involved in both development and disease. The best-understood Wnt pathway is the Wnt/β-catenin pathway. In this pathway, the Wnt signal leads to activation of the nuclear functions of β-catenin, which in turn activates gene expression leading to cell survival, proliferation, or differentiation. A second vertebrate Wnt pathway, the Wnt/Ca²⁺ pathway, promotes intracellular Ca²⁺ release and regulates cell movements in development and in some cancers. We have three goals in studying Wnts. Our first goal is to understand the normal functions of Wnt pathways in vertebrates, focusing on regeneration and response to injury. Our second goal is to understand the biochemistry of a Wnt signal. Our third goal is to leverage this understanding of the normal biology of Wnts to determine whether Wnt signaling is involved in various injuries and diseases and, if it is, to make contributions to developing therapies.

Wnt Signaling in Vertebrates

One of our short-term goals is to complete the identification of the majority of proteins that regulate both Wnt pathways. We are employing RNA interference (RNAi) screens in Drosophila cultured cells (in collaboration with Ramanuj Das Gupta and Norbert Perrimon (HHMI, Harvard Medical School), RNAi screens in human cultured cells (in collaboration with Sheng Ding, Scripps Research Institute), and RNAi screens with Rosetta-Merck. The RNAi screens have led to numerous candidate genes that might function in the Wnt pathways.

We are also identifying new components of Wnt pathways by proteomic approaches based on immunoprecipitation of Wnt pathway complexes from human cultured cells followed by mass spectrometry (with Michael MacCoss, University of Washington). Immunoprecipitation of Dishevelled protein complexes coupled with mass spectrometry revealed the presence of a complex of proteins that regulate Dishevelled stability. This is a different complex of proteins than is used by cells to regulate β-catenin in the same signaling pathway. We have recently expanded our use of mass spectrometry and are quickly learning a great deal that was heretofore unsuspected with regard to how the Wnt pathways work.

In higher resolution studies, we are continuing to work with Wenqing Xu (University of Washington) on the x-ray crystallographic structure of selected key regulators of Wnt signaling, and we have finished the structure of full-length β-catenin. These studies will contribute to identifying the networks of protein interactions that constitute the Wnt pathways, and to determining how these interactions change during signaling.

Functions of Wnt Signaling Pathways in Regeneration

Besides advancing basic science, a better understanding of normal Wnt functions will likely be useful in designing safe and effective therapies for a range of clinical conditions. One set of projects asks whether Wnt signaling is involved in regeneration. Because only some body structures in vertebrates regenerate, there is considerable interest in identifying the basis for this ability. Various signaling pathways have previously been shown to be involved in regeneration, but surprisingly little is known about the involvement of Wnts.

In the adult zebrafish, the tail fin regenerates quickly after injury, and we have recently found (using both genetic and nongenetic methods) that Wnt/β-catenin signaling is required for this to occur. We have also found that Wnts are required for the normal outgrowth of the embryonic zebrafish tail, suggesting that Wnts play roles in embryonic development that are recapitulated following injury in the adult. Activating Wnt/β-catenin signaling is sufficient to enhance proliferation during regeneration, suggesting that activating β-catenin function may serve a positive role in regenerative therapies.

Conversely, we show that a β-catenin-independent Wnt pathway serves a negative role in regulating regeneration of the tail fin. Activation of this pathway blocks regeneration, while genetic reduction in this pathway enhances regeneration. Wnt signals thus serve to regulate regeneration in a complex manner.

The Development of Therapies Based on Wnt Pathways

A general challenge for basic research is to identify areas in human medicine where insights gleaned from research can be translated into advances in understanding diseases and in developing potential therapies. For example, we have shown that a mutation linked to the retinal disease familial exudative vitreoretinopathy (FEVR) produces a truncated Frizzled-4, which oligomerizes with wild-type Frizzled, trapping it in the endoplasmic reticulum and preventing it from signaling. This result may explain why some FEVR mutations are genetically dominant.

Recent work has suggested that Wnt/β-catenin signaling may be attenuated in Alzheimer's disease. To determine whether polymorphisms in Wnt pathway components contribute to a susceptibility to Alzheimer's disease, we are examining single-nucleotide polymorphisms (SNPs) in Wnt pathway components in both affected and control individuals (with John Hardy, National Institute on Aging). Results to date do suggest that some SNPs in the Wnt coreceptor LRP6 are more common in affected individuals, though whether this contributes to the disease is not yet clear. The SNPs in LRP6 result in the protein functioning less efficiently than wild-type protein, which supports the likelihood of a functional link. If this can be validated, it will open the door to possible genetic testing and prophylactic treatment of affected individuals.

In the area of stem cell research, we are studying effects and roles of Wnts in human embryonic stem cells (with Charles Murry, University of Washington), human hematopoietic stem cells (with Mickie Bhatia, McMaster University) and mouse adult neural stem cells (with Philip Horner, University of Washington). Recent exciting progress with all three types of stem cells may lead to improved cell therapies for a range of clinical conditions. For example, systemic activation of β-catenin signaling with glycogen synthase kinase 3 (GSK-3) inhibitors in mouse models of hematopoietic stem cell transplantation enhances the success of the stem cells. With human embryonic stem cells cultured in vitro, we have shown that we can enhance differentiation into cardiomyocytes by controlling Wnt signaling. These studies suggest that Wnt signaling may modulate progenitor and stem cells in vitro and in vivo and in a manner that will promote regenerative therapies.

Finally, we are collaborating with Sheng Ding and others to identify small-molecule modulators of Wnt signaling. We have identified several new compounds that show promise for regulating the Wnt signaling pathway and which we hope will contribute to new therapeutic approaches for myriad diseases.

Grants from the National Institutes of Health and an award from the Alzheimer's Association provided partial support for some of these projects.