Many signaling cascades have been implicated in hematopoietic development. Among these is the wnt pathway, which has been proposed to be involved in the self-renewal of hematopoietic stem cells (HSCs) and in sustaining T-cell development. The downstream effectors of the canonical wnt pathway are the Tcf/Lef factors, which, prior to wnt-dependent activation, are bound to DNA in complex with the Groucho/TLE and act as sequence-specific transcriptional repressors. Binding of wnt to frizzled receptors leads to the stabilization and nuclear translocation of beta-catenin, which in turn binds Tcf/Lef factors, displacing the Groucho repressor complex to function as transcriptional activators of wnt-dependent genes. We postulate that Tcf-1 has a critical role in hematopoiesis. The objectives of this research proposal are to 1) map Tcf/Lef expression and activity in hematopoiesis and 2) further characterize the molecular role of Tcf-1 in cell survival, proliferation, and differentiation potential. The role of Tcf-1 in hematopoiesis and the molecular impact of its ablation will be further characterized. Tcf/Lef expression in hematopoietic progenitors will be analyzed by utilizing Real-Time PCR, while Tcf/Lef transcriptional activity will be mapped with the aid of transgenic reporter mice. Progenitor populations will be compared in Tcf-1 deficient and wildtype mice by staining for surface antigens and analyzing by flow cytometry. The progenitor subsets that are affected in Tcf-1-deficient mice will be analyzed with respect to their developmental potential both in vivo and in vitro. Survival and proliferation properties will be compared to the equivalent wildtype progenitors. The molecular basis of the defects will be examined by comparing their global expression profile to wildtype subsets. Retroviral transduction and reconstitution experiments will be used to further elucidate whether the function of Tcf-1 is redundant with homologous Tcf/Lef family proteins, whether this action is beta-catenin-dependent or -independent, and whether Tcf-1 is operating as a transcriptional activator or repressor. These studies support the NIH mission by contributing to our understanding of the normal molecular mechanisms involved in hematopoiesis. Defects in these signaling cascades are likely to contribute to developmental diseases and leukemic transformation. Understanding these molecular processes will help design diagnostic tests and targeted therapies for these diseases
