PAWS1 controls Wnt signalling through association with casein kinase 1α

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.     

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/β-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.     

Adhesive subdivisions intrinsic to the epithelial somites.

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(-/-) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4. 1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.     

FGF signalling controls the timing of Pax6 activation in the neural tube

We have recently demonstrated that Pax6 activation occurs in phase with somitogenesis in the spinal cord. Here we show that the presomitic mesoderm exerts an inhibitory activity on Pax6 expression. This repressive effect is mediated by the FGF signalling pathway. The presomitic mesoderm displays a decreasing caudorostral gradient of FGF8, and grafting FGF8-soaked beads at the level of the neural tube abolishes Pax6 activation. Conversely, when FGF signalling is disrupted, Pax6 is prematurely activated in the neural plate. We propose that the progression of Pax6 activation in the neural tube is controlled by the caudal regression of the anterior limit of FGF activity. Hence, as part of its posteriorising activity, FGF8 downregulation acts as a switch from early (posterior) to a later (anterior) state of neural epithelial development.     

Ginkgolide B promotes osteoblast differentiation via activation of canonical Wnt signalling and alleviates osteoporosis through a bone anabolic way

Osteoporosis has become a worldwide problem as the population ages. Although many advances have been made in the treatment of osteoporosis in the past few years, the outcome are sometimes disturbing because of the adverse effects of these treatments. Further studies are still needed to identify novel alternate agents to improve the therapeutic effect. Ginkgolide B (GB), a derivative of Ginkgo biloba leaves, has numerous pharmacological effects, including anticancer and anti‐inflammation activities. However, the effect of GB on the regulation of osteoblast activity and bone formation effect has not yet been investigated. In this study, we showed the in vitro and in vivo effects of GB on osteoblast differentiation and bone formation. We found that GB promotes osteoblast differentiation of Bone Mesenchymal Stem Cells (BMSCs) and MC3T3‐E1 cells in vitro in a Wnt/β‐catenin‐dependent manner. In an in vivo study, we constructed a cranial defect model in rats and treated with GB. Histomorphometric and histological analyses confirmed that the usage of GB significantly promotes bone formation. Further study on ovariectomy (OVX) rats demonstrated that GB is capable of alleviating ovariectomy‐induced bone loss by enhancing osteoblast activity. Our findings indicate that GB is a potential therapeutic agent of osteoporosis through an anabolic way in bone.     

LRP5 negatively regulates differentiation of monocytes through abrogation of Wnt signalling

Molecular changes involved in cell differentiation are only partially known. Circulating inflammatory cells need to differentiate to perform specialized functions in target tissues. Here, we hypothesized that low‐density lipoprotein receptor–related protein 5 (LRP5) is involved, through its participation in the canonical Wnt/β‐catenin signalling, in the differentiation process of monocytic cells. To this aim, we characterized differentiation mechanisms of HL60 cells and primary human monocytes. We show that silencing the LRP5 gene increased differentiation of HL60 cells and human monocytes, suggesting that LRP5 signalling abrogates differentiation. We demonstrate that the mechanisms behind this blockade include sequestration of β‐catenin at the cellular membrane, inhibition of the Wnt signalling and increase of apoptosis. We further demonstrate the involvement of LRP5 and the Wnt/β‐catenin signalling in the process because cellular differentiation can be rescued by the addition of downstream Wnt target genes to the monocytic cells.     

Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.     

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.