XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Tcf/Lef transcription factors mediate signalling from Wingless/Wnt proteins by recruiting Armadillo/beta-catenin as a transcriptional co-activator. However, studies of Drosophila, Xenopus and Caenorhabditis elegans have indicated that Tcf factors may also be transcriptional repressors. Here we show that Tcf factors physically interact with members of the Groucho family of transcriptional repressors. In transient transfection assays, the Xenopus Groucho homologue XGrg-4 inhibited activation of transcription of synthetic Tcf reporter genes. In contrast, the naturally truncated Groucho-family member XGrg-5 enhanced transcriptional activation. Injection of XGrg-4 into Xenopus embryos repressed transcription of Siamois and Xnr-3, endogenous targets of beta-catenin-Tcf. Dorsal injection of XGrg-4 had a ventralizing effect on Xenopus embryos. Secondary-axis formation induced by a dominant-positive Armadillo-Tcf fusion protein was inhibited by XGrg-4 and enhanced by XGrg-5. These data indicate that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho.

At the heart of the canonical Wnt signaling cascade, adenomatous polyposis coli (APC), axin, and GSK3 constitute the so-called destruction complex, which controls the stability of ␤-catenin. It is generally believed that four conserved Ser/Thr residues in the N terminus of ␤-catenin are the pivotal targets for the constitutively active serine kinase GSK3. In cells that do not receive Wnt signals, glycogen synthase kinase (GSK) is presumed to phosphorylate ␤-catenin, thus marking the latter for proteasomal degradation. Wnt signaling inhibits GSK3 activity. As a consequence, ␤-catenin would no longer be phosphorylated and accumulate to form nuclear complexes with TCF/LEF factors. Although mutations in or near the N-terminal Ser/Thr residues stabilize ␤-catenin in several types of cancer, the hypothesis that Wnt signaling controls phosphorylation of these residues remains unproven. We have generated a monoclonal antibody that recognizes an epitope containing two of the four residues when both are not phosphorylated. The epitope is generated upon Wnt signaling as well as upon pharmacological inhibition of GSK3 by lithium, providing formal proof for the regulated phosphorylation of the Ser/Thr residues of ␤-catenin by Wnt signaling. Immunohistochemical analysis of mouse embryos utilizing the antibody visualizes sites that transduce Wnt signals through the canonical Wnt cascade.Wnt signal transduction controls multiple developmental events throughout the animal kingdom. Wnt/wingless proteins constitute a large family (Ͼ19 members in human) of secreted cysteine-rich glycoproteins. Wnts function as ligands for a receptor complex consisting of one of several members of the Frizzled (Fz) 1 family of serpentine receptors (1) and of LRP-5/6, homologues of the low density lipoprotein receptor (2-4).␤-catenin participates in a large cytoplasmic protein complex, containing the serine/threonine protein kinase glycogen synthase kinase-3 (GSK-3), the tumor suppressor gene product adenomatous polyposis coli (APC) and axin/conductin (5-8). In the absence of Wnt, GSK-3 (9, 10) is constitutively active and is believed to promote degradation of ␤-catenin by N-terminal phosphorylation and subsequent ubiquitination and proteasomal targeting (11,12). In response to Wnt, the activity of GSK-3 is inhibited by an incompletely understood mechanism. As a consequence, ␤-catenin breakdown is suppressed (13). Indeed, oncogenic mutations in ␤-catenin at putative GSK-3 phosphorylation sites stabilize ␤-catenin in colorectal cancer and melanoma (14), causing aberrant activation of TCF (14,15).Interspecies sequence comparison of ␤-catenin as well as the oncogenic point mutations in ␤-catenin have implied four Nterminal residues as the targets of upstream kinases: Ser-33, Ser-37, Thr-41, and Ser-45 (for an overview see ana.ed.ac.uk/ rnusse/pathway/bcatmut.html). To date however, no biochemical data exist to confirm the regulated phosphorylation of these residues. Such analyses are complicated by the fact that two separate pools of ␤-catenin co-exist ...

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Wnt proteins function as morphogens that can form long-range concentration gradients to pattern developing tissues. Here, we show that the retromer, a multiprotein complex involved in intracellular protein trafficking, is required for long-range signaling of the Caenorhabditis elegans Wnt ortholog EGL-20. The retromer functions in EGL-20-producing cells to allow the formation of an EGL-20 gradient along the anteroposterior axis. This function is evolutionarily conserved, because Wnt target gene expression is also impaired in the absence of the retromer complex in vertebrates. These results demonstrate that the ability of Wnt to regulate long-range patterning events is dependent on a critical and conserved function of the retromer complex within Wnt-producing cells.