Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

Frizzled-4 is required for normal bone acquisition despite compensation by Frizzled-8

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.     

Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5

Wnt7b is a Wnt ligand that has been demonstrated to play critical roles in several developmental processes, including lung airway and vascular development and chorion-allantois fusion during placental development. Wnt signaling involves the binding of Wnt ligands to cell surface receptors of the frizzled family and coreceptors of the LRP5/6 family. However, little is known of the ligand-receptor specificity exhibited by different Wnts, Fzds, and LRPs in Wnt signaling. Expression analysis of Fzds and LRP5/6 in the developing lung and vasculature showed that Fzd1, -4, -7, and -10 and LRP5/6 are expressed in tissue-specific patterns during lung development. Fzd1, -4, and -7 are expressed primarily in the developing lung mesenchyme, and Fzd10 is expressed in airway epithelium. LRP5 and LRP6 are expressed in airway epithelium during lung development, whereas LRP5 but not LRP6 expression is observed in the muscular component of large blood vessels, including the aorta. Cell transfection studies demonstrate that Wnt7b can activate the canonical Wnt pathway but not the noncanonical Wnt pathway in a cell-specific manner. Biochemical analysis demonstrates that Wnt7b can bind to Fzd1 and -10 on the cell surface and cooperatively activate canonical Wnt signaling with these receptors in the presence of LRP5. Together, these data demonstrate that Wnt7b signals through Fzd1 and -10 and LRP5 and implicate these Wnt coreceptors in the regulation of lung airway and vascular development.     

Wnt5b–Ryk pathway provides directional signals to regulate gastrulation movement

Noncanonical Wnts are largely believed to act as permissive cues for vertebrate cell movement via Frizzled (Fz). In addition to Fz, Wnt ligands are known to regulate neurite outgrowth through an alternative receptor related to tyrosine kinase (Ryk). However, Wnt–Ryk signaling during embryogenesis is less well characterized. In this study, we report a role for Wnt5b as an instructive cue to regulate gastrulation movements through Ryk. In zebrafish, Ryk deficiency impairs Wnt5b-induced Ca²⁺ activity and directional cell movement. Wnt5b–Ryk signaling promotes polarized cell protrusions. Upon Wnt5b stimulation, Fz2 but not Ryk recruits Dishevelled to the cell membrane, suggesting that Fz2 and Ryk mediate separate pathways. Using co-culture assays to generate directional Wnt5b cues, we demonstrate that Ryk-expressing cells migrate away from the Wnt5b source. We conclude that full-length Ryk conveys Wnt5b signals in a directional manner during gastrulation.     

A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities

Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.     

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.     

Secreted Frizzled-related protein potentiation versus inhibition of Wnt3a/β-catenin signaling

Wnt signaling regulates a variety of cellular processes during embryonic development and in the adult. Many of these activities are mediated by the Frizzled family of seven-pass transmembrane receptors, which bind Wnts via a conserved cysteine-rich domain (CRD). Secreted Frizzled-related proteins (sFRPs) contain an amino-terminal, Frizzled-like CRD and a carboxyl-terminal, heparin-binding netrin-like domain. Previous studies identified sFRPs as soluble Wnt antagonists that bind directly to Wnts and prevent their interaction with Frizzleds. However, subsequent observations suggested that sFRPs and Frizzleds form homodimers and heterodimers via their respective CRDs, and that sFRPs can stimulate signal transduction. Here, we present evidence that sFRP1 either inhibits or enhances signaling in the Wnt3a/β-catenin pathway, depending on its concentration and the cellular context. Nanomolar concentrations of sFRP1 increased Wnt3a signaling, while higher concentrations blocked it in HEK293 cells expressing a SuperTopFlash reporter. sFRP1 primarily augmented Wnt3a/β-catenin signaling in C57MG cells, but it behaved as an antagonist in L929 fibroblasts. sFRP1 enhanced reporter activity in L cells that were engineered to stably express Frizzled 5, though not Frizzled 2. This implied that the Frizzled expression pattern could determine the response to sFRP1. Similar results were obtained with sFRP2 in HEK293, C57MG and L cell reporter assays. CRD_sFRP1 mimicked the potentiating effect of sFRP1 in multiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRD_sFRP1 showed little avidity for Wnt3a compared to sFRP1, implying that the mechanism for potentiation by CRD_sFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/β-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors.     

Expression of Wnt signaling components during Xenopus pronephros development

Background

The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth.

Methodology/Principal Findings

Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development.

Conclusion/Significance

Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney.     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.     

Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin

Although Wnt signaling is considered a key regulatory pathway for bone formation, inactivation of β-catenin in osteoblasts does not affect their activity but rather causes increased osteoclastogenesis due to insufficient production of osteoprotegerin (Opg). By monitoring the expression pattern of all known genes encoding Wnt receptors in mouse tissues and bone cells we identified Frizzled 8 (Fzd8) as a candidate regulator of bone remodeling. Fzd8-deficient mice displayed osteopenia with normal bone formation and increased osteoclastogenesis, but this phenotype was not associated with impaired Wnt signaling or Opg production by osteoblasts. The deduced direct negative influence of canonical Wnt signaling on osteoclastogenesis was confirmed in vitro and through the generation of mice lacking β-catenin in the osteoclast lineage. Here, we observed increased bone resorption despite normal Opg production and a resistance to the anti-osteoclastogenic effect of Wnt3a. These results demonstrate that Fzd8 and β-catenin negatively regulate osteoclast differentiation independent of osteoblasts and that canonical Wnt signaling controls bone resorption by two different mechanisms.     

LGR5 interacts and cointernalizes with Wnt receptors to modulate Wnt/β-catenin signaling

LGR5, a seven-transmembrane domain receptor of the rhodopsin family, is a Wnt target gene and a bona fide marker of adult stem cells in the gastrointestinal tract and hair follicle bulge. Recently, we and others demonstrated that LGR5 and its homologues function as receptors of the R-spondin family of stem cell factors to potentiate Wnt/β-catenin signaling. However, the mechanism of how LGR5 enhances the signaling output remains unclear. Here we report that following costimulation with the ligands R-spondin1 and Wnt3a, LGR5 interacts and forms a supercomplex with the Wnt coreceptors LRP6 and Fzd5 which is rapidly internalized and then degraded. Internalization of LGR5 is mediated through a dynamin- and clathrin-dependent pathway. Inhibition of this endocytic process has no effect on LGR5 signaling. Deletion of the C-terminal tail of LGR5 maintains its ability to interact with LRP6, yet this LGR5 mutant exhibits increased signaling activity and a decreased rate of endocytosis in response to R-spondin1 compared to the wild-type receptor. This study provides direct evidence that LGR5 becomes part of the Wnt signaling complex at the membrane level to enhance Wnt/β-catenin signaling. However, internalization of LGR5 does not appear to be essential for potentiating the canonical Wnt signaling pathway.     

Selective Modulation of Wnt Ligands and Their Receptors in Adipose Tissue by Chronic Hyperadiponectinemia

Background

Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue.

Materials and Methods

We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells.

Results

The Wnt5b, Wnt6, Frizzled 6 (Fzd6), and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6) were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII) and phosphorylated Jun N-terminal kinase (p-JNK) were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin.

Conclusion

Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors accompanied by the inhibition of the Wnt/Ca²⁺ and JNK signaling pathways, which may be involved in the altered adipocyte cellularity, endogenous adiponectin production, and anti-inflammatory action induced by hyperadiponectinemia.