Understanding the molecular mechanisms of pattern formation during embryogenesis remains a challenge for biologists. One key family of signaling molecules that have been shown to play crucial roles in this process is the Wnt family. Wnt proteins are conserved secreted glycoproteins that govern major developmental processes including cell fate determination, cell proliferation, cell motility and establishment of the primay axis and head formation during vertebrate development [1-5]. In addition to regulating embryonic development and pattern formation, defects in Wnt signaling have also been implicated in tumorigenesis [2, 6-8]. Delineation of the molecular mechanisms of Wnt signaling can therefore illuminate the mechanisms of pattern formation during embryogenesis and contribute to our understanding of tumorigenesis. In our studies investigating the role of Wnt signaling in pattern formation, we have isolated a new effector molecule via a yeast two-hybrid screen, using Dishevelled (Dvl) as a bait. This protein termed Custos is required for canonical Wnt signaling, -catenin nuclear translocation and anterior development in both Xenopus and zebrafish embryos. Embryos depleted of Custos further lack pigmentation in the skin and eye suggesting a role for Custos in neural crest development. Interestingly, in a pilot yeast two-hybrid screen for binding partners for Custos, we isolated the nuclear membrane-associated protein nesprin1 as the major Custos-interacting protein. In building a model for how Custos functions, we propose Custos along with nesprin1 functions to regulate the cytoplasmic to nuclear trafficking of -catenin. Understanding the function of Custos and nesprin1 can provide key insights into the molecular mechanisms of canonical Wnt signaling during embryogenesis and the nuclear trafficking of -catenin, a key area of study that remains poorly resolved. Based on our studies, we hypothesize Custos and nesprin1 are an important factors required for canonical Wnt signaling during vertebrate embryogenesis. The primary objective of this proposal is to elucidate how Custos and nesprin1 regulates this process. We will build on our working model described above to test our hypothesis and delineate the role and function of Custos and nesprin1 in Wnt signal transduction. Our studies will utilize the distinct advantages of Xenopus laevis, zebrafish and mammalian cell culture-based systems. We have proposed two central aims in this application. The first will characterize the role of Custos in vertebrate embryogenesis in the zebrafish and Xenopus models and in role in mediating -catenin nuclear translocation using single molecule resolution imaging studies. The second will characterize the role of nesprin1 as a regulator of Custos function and its function during vertebrate development in both zebrafish and Xenopus models. Taken together our proposed studies will dissect the cytoplasmic to nuclear translocation of -catenin, an area of study that has remained exceptionally poorly resolved to date. Additionally, our studies will lead to a better understandin of how Wnt signaling regulates the process of embryogenesis as well as how deregulated Wnt signaling results in numerous and devastating human pathologies including cancers and birth defect disorders.
Understanding the development of human cancers and birth defect disorders remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key- signaling molecule that has been demonstrated to play causative roles in human cancer is the Wnt protein. Our studies are focused on functionally characterizing the role of two proteins termed Custos and nesprin1 that transduces a branch of Wnt signaling termed the canonical Wnt signaling pathway. These proteins further regulate the cytoplasmic to nuclear translocation of -catenin, which is a key hallmark of canonical Wnt signaling. We propose that these studies can provide important new insights into the mechanisms regulating embryogenesis as well as gaining insights into how deregulated Wnt signaling contributes to human pathologies including cancer formation and birth defect disorders.
