The vertebrate transcription factors Tcf/Lef (T cell factor/lymphocyte enhancer binding protein) were initially identified by their differential expression and DNA binding during T cell and B cell differentiation. We collaborated with the Clevers (Utrecht) and Peifer (UNC) labs in cloning the Drosophila Tcf homolog dTcf. We mapped the gene to the base of the fourth chromosome and identified mutations that partially or fully inactivate dTcf. We demonstrated that dTcf interacts with Armadillo/B-catenin to transduce Wingless/WNT signaling. We demonstrated that the genomic structure of dTcf is very similar to that of human Tcf-1 suggesting they share a common ancestor. We are now focusing on the regulation and functioning of dTcf. We are generating antibodies to the Drosophila protein. We have also identified a target of dTcf in the visceral mesoderm as the Drosophila Transforming Growth Factor-Beta ortholog, dpp. Reporter constructs under the control of an enhancer sequence from dpp, which mimic dpp expression in the visceral mesoderm, have two putative dTcf binding sites. We show that dTcf protein shifts the mobility of oligomers containing these sites. The reporter constructs can be activated ectopically by stimulating the wingless pathway in the visceral mesoderm but not by overexpressing dTCF itself. However, ectopic expression of a dominant negative form of dTcf only blocks expression in a subset of cells. We are investigating if this interaction is direct by changing the sequences of these dTcf binding sites and transforming the resulting constructs back into flies to test their function. We show that mutation of either site results in a partial expansion of reporter gene expression in the visceral mesoderm. Mutation of both sites results in complete expansion of reporter gene expression throughout the visceral mesoderm. These results suggest that the primary function of dTcf is direct repression of dpp in the visceral mesoderm. Activation by the Wingless signaling pathway is primarily indirect.This project has been discontinued as a separate project and has now been incorporated into that covered under Z01 BC 05272-08 LB. - Drosophila, T cell factor/lymphoid enhancer, binding protein, armadillo/B-catenin, Wingless/WNT signaling,
