Repressor activity of Headless/Tcf3 is essential for vertebrate head formation

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.     

Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.     

Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation

Oriented cell division is an integral part of pattern development in processes ranging from asymmetric segregation of cell-fate determinants to the shaping of tissues. Despite proposals that it has an important function in tissue elongation, the mechanisms regulating division orientation have been little studied outside of the invertebrates Caenorhabditis elegans and Drosophila melanogaster. Here, we have analysed mitotic divisions during zebrafish gastrulation using in vivo confocal imaging and found that cells in dorsal tissues preferentially divide along the animal-vegetal axis of the embryo. Establishment of this animal-vegetal polarity requires the Wnt pathway components Silberblick/Wnt11, Dishevelled and Strabismus. Our findings demonstrate an important role for non-canonical Wnt signalling in oriented cell division during zebrafish gastrulation, and indicate that oriented cell division is a driving force for axis elongation. Furthermore, we propose that non-canonical Wnt signalling has a conserved role in vertebrate axis elongation, orienting both cell intercalation and mitotic division.     

The DIX domain targets dishevelled to actin stress fibres and vesicular membranes

Colorectal cancer results from mutations in components of the Wnt pathway that regulate beta-catenin levels. Dishevelled (Dvl or Dsh) signals downstream of Wnt receptors and stabilizes beta-catenin during cell proliferation and embryonic axis formation. Moreover, Dvl contributes to cytoskeletal reorganization during gastrulation and mitotic spindle orientation during asymmetric cell division. Dvl belongs to a family of eukaryotic signalling proteins that contain a conserved 85-residue module of unknown structure and biological function called the DIX domain. Here we show that the DIX domain mediates targeting to actin stress fibres and cytoplasmic vesicles in vivo. Neighbouring interaction sites for actin and phospholipid are identified between two helices by nuclear magnetic resonance spectroscopy (NMR). Mutation of the actin-binding motif abolishes the cytoskeletal localization of Dvl, but enhances Wnt/beta-catenin signalling and axis induction in Xenopus. By contrast, mutation of the phospholipid interaction site disrupts vesicular association of Dvl, Dvl phosphorylation, and Wnt/beta-catenin pathway activation. We propose that partitioning of Dvl into cytoskeletal and vesicular pools by the DIX domain represents a point of divergence in Wnt signalling.     

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.     

LDL-receptor-related protein 6 is a receptor for Dickkopf proteins

Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. Here we show that LRP6 (ref. 7) is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists.     

Frizzled-7 signalling controls tissue separation during Xenopus gastrulation.

Cell signalling through Frizzled receptors has evolved to considerable complexity within the metazoans. The Frizzled-dependent signalling cascade comprises several branches, whose differential activation depends on specific Wnt ligands, Frizzled receptor isoforms and the cellular context. In Xenopus laevis embryos, the canonical beta-catenin pathway contributes to the establishment of the dorsal-ventral axis. A different branch, referred to as the planar cell polarity pathway, is essential for cell polarization during elongation of the axial mesoderm by convergent extension. Here we demonstrate that a third branch of the cascade is independent of Dishevelled function and involves signalling through trimeric G proteins and protein kinase C (PKC). During gastrulation, Frizzled-7 (Fz7)-dependent PKC signalling controls cell-sorting behaviour in the mesoderm. Loss of zygotic Fz7 function results in the inability of involuted anterior mesoderm to separate from the ectoderm, which leads to severe gastrulation defects. This result provides a developmentally relevant in vivo function for the Fz/PKC pathway in vertebrates.     

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.     

Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.     

Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.