Paracrine Wnt/β-catenin signalling is important during developmental processes, tissue regeneration and stem cell regulation. Wnt proteins are morphogens, which form concentration gradients across responsive tissues. Little is known about the transport mechanism for these lipid-modified signalling proteins in vertebrates. Here we show that Wnt8a is transported on actin-based filopodia to contact responding cells and activate signalling during neural plate formation in zebrafish. Cdc42/N-Wasp regulates the formation of these Wnt-positive filopodia. Enhanced formation of filopodia increases the effective signalling range of Wnt by facilitating spreading. Consistently, reduction in filopodia leads to a restricted distribution of the ligand and a limited signalling range. Using a simulation, we provide evidence that such a short-range transport system for Wnt has a long-range signalling function. Indeed, we show that a filopodia-based transport system for Wnt8a controls anteroposterior patterning of the neural plate during vertebrate gastrulation.

Activin and the Nodal-related proteins induce mesendodermal tissues during Xenopus development. These signals act through specific receptors to cause the phosphorylation, at their carboxyl termini, of Smad2 and Smad3. The phosphorylated Smad proteins form heteromeric complexes with Smad4 and translocate into the nucleus to activate the transcription, after the midblastula transition, of target genes such as Xbra and goosecoid (gsc). In this paper we use bimolecular fluorescence complementation (BiFC) to study complex formation between Smad proteins both in vivo and in response to exogenous proteins. The technique has allowed us to detect Smad2-Smad4 heteromeric interactions during normal Xenopus development and Smad2 and Smad4 homo- and heteromers in isolated Xenopus blastomeres. Smad2-Smad2 and Smad2-Smad4 complexes accumulate rapidly in the nuclei of responding cells following Activin treatment, whereas Smad4 homomeric complexes remain cytoplasmic. When cells divide, Smad2-Smad4 complexes associate with chromatin, even in the absence of ligand. Our observation that Smad2-Smad4 complexes accumulate in the nucleus only after the midblastula transition, irrespective of the stage at which cells were treated with Activin, may shed light on the mechanisms of developmental timing.

After activation by Wnt/β-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt–Fz–Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The μ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2μ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2μ2-mediated endocytosis is important to maintain Wnt/β-catenin signaling in vertebrates.

The thalamic complex is an essential part of the brain that requires a combination of specialized activities to attain its final complexity. In the following review we will describe the induction process of the mid-diencephalic organizer (MDO) where three different signaling pathways merge: Wnt, Shh, and Fgf. Here, we dissect the function of each signaling pathway in the thalamus in chronological order of their appearance. First we describe the Wnt mediated induction of the MDO and compartition of the caudal forebrain, then the Shh mediated determination of proneural gene expression before discussing recent progress in characterizing Fgf function during thalamus development. Then, we focus on transcription factors, which are regulated by these pathways and which play a pivotal role in neurogenesis in the thalamus. The three signaling pathways act together in a strictly regulated chronology to orchestrate the development of the entire thalamus.

Low‐density lipoprotein receptor‐related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt‐induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture‐based cDNA expression screen, we identified the non‐receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6‐Wnt signalling. Epistatically, they function upstream of β‐catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer‐induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de‐represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over‐activation of Wnt signalling at the level of the Wnt receptor, LRP6. Synopsis Tyr phosphorylation of LRP6 by Src and Fer is reported as a new mode of negative regulation of Wnt/β‐catenin signalling. LRP6 phosphorylation is stimulated by Wnt and leads to the disruption of LRP6 signalosome formation and removal of LRP6 from the cell surface. Tyr phosphorylation by Src and Fer inhibits LRP6. LRP6 phosphorylation disrupts signalosome formation. CK1γ counteracts inhibitory LRP6 phosphorylation by inactivating Fer.