At vertebrate neuromuscular junctions (NMJs), Agrin plays pivotal roles in synapse development, but molecules that activate synapse formation at central synapses are largely unknown. Members of the Wnt family are well established as morphogens, yet recently they have also been implicated in synapse maturation. Here we demonstrate that the Drosophila Wnt, Wingless (Wg), is essential for synapse development. We show that Wg and its receptor are expressed at glutamatergic NMJs, and that Wg is secreted by synaptic boutons. Loss of Wg leads to dramatic reductions in target-dependent synapse formation, and new boutons either fail to develop active zones and postsynaptic specializations or these are strikingly aberrant. We suggest that Wg signals the coordinated development of pre- and postsynaptic compartments.

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wntbinding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields ϳ3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.

Wingless secretion provides pivotal signals during development by activating transcription of target genes. At Drosophila synapses, Wingless is secreted from presynaptic terminals and is required for synaptic growth and differentiation. Wingless binds the seven-pass transmembrane DFrizzled2 receptor, but the ensuing events at synapses are not known. We show that DFrizzled2 is endocytosed from the postsynaptic membrane and transported to the nucleus. The C terminus of DFrizzled2 is cleaved and translocated into the nucleus; the N-terminal region remains just outside the nucleus. Translocation of DFrizzled2-C into the nucleus, but not its cleavage and transport, depends on Wingless signaling. We conclude that, at synapses, Wingless signal transduction occurs through the nuclear localization of DFrizzled2-C for potential transcriptional regulation of synapse development.

Syndecan-1 is a cell-surface, heparan-sulphate proteoglycan (HSPG) predominantly expressed by epithelial cells. It binds specifically to many proteins, including oncoproteins. For example, it induces the assembly of a signalling complex between FGF ligands and their cognate receptors. But so far there has been no direct evidence that this proteoglycan contributes to tumorigenesis. Here we have examined the role of syndecan-1 (encoded by Sdc1) during mammary tumour formation in response to the ectopic expression of the proto-oncogene Wnt1. We crossed syndecan-1-deficient mice with transgenic mice that express Wnt1 in mammary gland (TgN(Wnt-1)1Hev; ref. 2). Ectopic Wnt-1 expression induces generalized mammary hyperplasia, followed by the development of solitary tumours (median time 22 weeks). We show that in Sdc1-/- mice, Wnt-1-induced hyperplasia in virgin mammary gland was reduced by 70%, indicating that the Wnt-1 signalling pathway was inhibited. Of the 39 tumours that developed in a test cohort of mice, only 1 evolved in the Sdc1-/- background. In addition, we show that soluble syndecan-1 ectodomain purified from mouse mammary epithelial cells stimulates the activity of a Wnt-1 homologue in a tissue culture assay. Our results provide both genetic and biochemical evidence that syndecan-1 can modulate Wnt signalling, and is critical for Wnt-1-induced tumorigenesis of the mouse mammary gland.