A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica

Regionalised activation of canonical Wnt signalling via beta-catenin stabilisation is a key early step in embryonic patterning in many metazoans, including the basally diverging cnidarians, but the upstream maternal cues appear surprisingly variable. In Clytia, regionalised beta-catenin stabilisation defining a presumptive 'oral' territory is determined by two maternally coded Frizzled family Wnt receptors of opposite localisation and function. We have identified a maternally coded ligand, CheWnt3, the RNA of which is localised to the animal cortex (future oral side) of the egg. Antisense morpholino oligonucleotide experiments showed that CheWnt3 is required maternally for regionalised oral beta-catenin stabilisation in the early embryo, being only the second clear example of a maternally required Wnt ligand after Xenopus Xwnt11. In line with the determinant role of the maternally localised Frizzleds, CheWnt3 overexpression by RNA injection initially had little effect on establishing the oral domain. Subsequently, however, overexpression had dramatic consequences for axis development, causing progressive expansion of beta-catenin stabilisation to yield spherical 'oralised' larvae. Upregulation of both CheFz1 and CheFz3 RNAs in CheWnt3 morpholino embryos indicated that CheWnt3 participates in an active axial patterning system involving reciprocal downregulation of the receptors to maintain oral and aboral territories. Localised introduction of CheWnt3 RNA induced ectopic oral poles in CheWnt3 morpholino embryos, demonstrating its importance in directing oral fate. These findings suggest that the complete ligand-dependent Wnt signalling cascade is involved in axial patterning in ancestral eumetazoans. In Clytia, two variant Frizzled receptors and one Wnt ligand produced from localised RNAs cooperate to initiate regionalised Wnt pathway activation.     

Three distinct RNA localization mechanisms contribute to oocyte polarity establishment in the cnidarian Clytia hemisphærica

Egg animal-vegetal polarity in cnidarians is less pronounced than in most bilaterian species, and its normal alignment with the future embryonic axis can be disturbed by low-speed centrifugation. We have analyzed the development of oocyte polarity within the transparent and autonomously functioning gonads of Clytia medusae, focusing on the localization of three recently identified maternal mRNAs coding for axis-directing Wnt pathway regulators. Animal-vegetal polarity was first detectable in oocytes committed to their final growth phase, as the oocyte nucleus (GV) became positioned at the future animal pole. In situ hybridization analyses showed that during this first, microtubule-dependent polarization event, CheFz1 RNA adopts a graded cytoplasmic distribution, most concentrated around the GV. CheFz3 and CheWnt3 RNAs adopt their polarized cortical localizations later, during meiotic maturation. Vegetal localization of CheFz3 RNA was found to require both microtubules and an intact gonad structure, while animal localization of CheWnt3 RNA was microtubule independent and oocyte autonomous. The cortical distribution of both these RNAs was sensitive to microfilament-disrupting drugs. Thus, three temporally and mechanistically distinct RNA localization pathways contribute to oocyte polarity in Clytia. Unlike the two cortical RNAs, CheFz1 RNA was displaced in fertilized eggs upon centrifugation, potentially explaining how this treatment re-specifies the embryonic axis.     

A critical role for endocytosis in Wnt signaling

Background  

The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.     

Wnt9a secreted from the walls of hepatic sinusoids is essential for morphogenesis, proliferation, and glycogen accumulation of chick hepatic epithelium

Hepatic epithelial morphogenesis, including hepatoblast migration and proliferation in the septum transversum, requires the interaction of hepatic epithelium with the embryonic sinusoidal wall. No factors that mediate this interaction have yet been identified. As the β-catenin pathway is active in hepatoblast proliferation, then Wnt ligands might activate the canonical Wnt pathway during liver development. Here, we investigated the role of Wnts in mediating epithelial vessel interactions in the developing chick liver. We found that Wnt9a was specifically expressed in both endothelial and stellate cells of the embryonic sinusoidal wall. Induced overexpression of Wnt9a resulted in hepatomegaly with hyperplasia of the hepatocellular cords, and in hyperproliferation of hepatocytes. Knockdown of Wnt9a caused a reduction in liver size, with hypoplasia of hepatocellular cord branching, and hypoproliferation of hepatoblasts, and also inhibited glycogen accumulation at later developmental stages. Wnt9a promoted in vivo stabilization of β-catenin through binding with Frizzled 4, 7, and 9, and activated TOPflash reporter expression in vitro via Frizzled 7 and 9. Our results demonstrate that Wnt9a from the embryonic sinusoidal wall is required for the proper morphogenesis of chick hepatocellular cords, proliferation of hepatoblasts/hepatocytes, and glycogen accumulation in hepatocytes. Wnt9a signaling appears to be mediated by an Fzd7/9-β-catenin pathway.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

An arrow for wingless to take-off

The Wnt family of secreted glycoproteins is involved in the regulation of diverse developmental processes. The classical Wnt/beta-catenin pathway has been thoroughly investigated resulting in the identification of a plethora of components involved in the activation of beta-catenin target genes. Moreover, two additional Wnt-triggered pathways have been identified. These various signalling cascades require at least one component that confers signalling specificity. This function is fulfilled at least in part by the Wnt receptor Frizzled. The recent identification of a potential Frizzled co-receptor, an LDL-receptor-related-protein (LRP), sheds more light on Wnt-signal transduction specificity and promises more exciting revelations.     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

The Frizzled family: receptors for multiple signal transduction pathways

Frizzled genes encode integral membrane proteins that function in multiple signal transduction pathways. They have been identified in diverse animals, from sponges to humans. The family is defined by conserved structural features, including seven hydrophobic domains and a cysteine-rich ligand-binding domain. Frizzled proteins are receptors for secreted Wnt proteins, as well as other ligands, and also play a critical role in the regulation of cell polarity. Frizzled genes are essential for embryonic development, tissue and cell polarity, formation of neural synapses, and the regulation of proliferation, and many other processes in developing and adult organisms; mutations in human frizzled-4 have been linked to familial exudative vitreoretinopathy. It is not yet clear how Frizzleds couple to downstream effectors, and this is a focus of intense study.     

Early embryonic death in mice lacking the beta-catenin-binding protein Duplin

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.     

You Wnt some,you lose some: oncogenes in the Wnt signaling pathway

The highly regulated Wnt signaling cascade plays a decisive role during embryonic patterning and cell-fate determination. The inappropriate expression of Wnt target genes, resulting from deregulation of this pathway, is also implicated in tumorigenesis. Thus, regulation of this pathway is of paramount importance. The Wnt signals are extracellularly regulated by a diverse group of antagonists, cofactors and coreceptors. In the cytoplasm, beta-catenin, a key effector of the Wnt signaling cascade, is highly regulated by a large and fascinating complex of proteins. In the nucleus, activation of target genes is regulated by a complex interplay of activators, repressors and other proteins. Recently, new factors in this pathway have been identified and the interplay and mechanisms of action of key players have been better characterized. Collectively, this represents an important step forward in our understanding of the role of Wnt signaling in development and oncogenesis.     

Dishevelled: The hub of Wnt signaling

Wnt signaling controls a variety of developmental and homeostatic events. As a key component of Wnt signaling, Dishevelled (Dvl/Dsh) protein relays Wnt signals from receptors to downstream effectors. In the canonical Wnt pathway that depends on the nuclear translocation of β-catenin, Dvl is recruited by the receptor Frizzled and prevents the constitutive destruction of cytosolic β-catenin. In the non-canonical Wnt pathways such as Wnt-Frizzled/PCP (planar cell polarity) signaling, Dvl signals via the Daam1-RhoA axis and the Rac1 axis. In addition, Dvl plays important roles in Wnt-GSK3β-microtubule signaling, Wnt-calcium signaling, Wnt-RYK signaling, Wnt-atypical PKC signaling, etc. Dvl also functions to mediate receptor endocytosis. To fulfill its multifaceted functions, it is not surprising that Dvl associates with various kinds of proteins. Its activity is also modulated dynamically by phosphorylation, ubiquitination and degradation. In this review, we summarize the current understanding of Dvl functions in Wnt signal transduction and its biological functions in mouse development, and also discuss the molecular mechanisms of its actions.