Neucrin, a novel secreted antagonist of canonical Wnt signaling, plays roles in developing neural tissues in zebrafish

Wnt signaling plays crucial roles in neural development. We previously identified Neucrin, a neural-specific secreted antagonist of canonical Wnt/β-catenin signaling, in humans and mice. Neucrin has one cysteine-rich domain, in which the positions of 10 cysteine residues are similar to those in the second cysteine-rich domain of Dickkopfs, secreted Wnt antagonists. Here, we have identified zebrafish neucrin to understand its roles in vivo. Zebrafish Neucrin also has one cysteine-rich domain, which is significantly similar to that of mouse Neucrin. Zebrafish neucrin was also predominantly expressed in developing neural tissues. To examine roles of neucrin in neural development, we analyzed neucrin knockdown embryos. Neural development in zebrafish embryos was impaired by the knockdown of neucrin. The knockdown of neucrin caused increased expression of the Wnt/β-catenin target genes. In contrast, overexpression of neucrin reduced the expression of the Wnt/β-catenin target genes. The knockdown of neucrin affected specification of dorsal region in the midbrain and hindbrain. The knockdown of neucrin also suppressed neuronal differentiation and caused increased cell proliferation and apoptosis in developing neural tissues. Neucrin is a unique secreted Wnt antagonist that is predominantly expressed in developing neural tissues and plays roles in neural development in zebrafish.     

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.     

Gtpbp2 is a positive regulator of Wnt signaling and maintains low levels of the Wnt negative regulator Axin

Background

Canonical Wnt signals, transduced by stabilized β-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/β-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here.

Results

Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3β but upstream of β-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b.

Conclusions

We conclude that Gtpbp2 is required for canonical Wnt/β-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the β-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant β-catenin signaling in cancer and other Wnt-related diseases.

     

Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

The Amotl2 gene inhibits Wnt/β-catenin signaling and regulates embryonic development in zebrafish

The Motin family proteins can regulate cell polarity, cell mobility, and proliferation during embryonic development by controlling distinct signaling pathways. In this study, we demonstrate that amotl2 knockdown in zebrafish wild-type embryos results in embryonic dorsalization, and this effect can be antagonized by co-knockdown of the dorsal inducer β-catenin2. Overexpression of amotl2 in masterblind (mbl) homozygous embryos, in which canonical Wnt signaling is up-regulated due to an axin1 mutation, transforms eyeless phenotype into smaller eyes, whereas co-knockdown of amot, amotl1, and amotl2 leads to development of smaller eyes in mbl heterozygotes. In cultured mammalian cells, Motin family members all possess the ability to attenuate Wnt/β-catenin signaling. Focusing on Amotl2, we show that Amotl2 can associate with and trap β-catenin in the Rab11-positive recycling endosomes, and as a result, the amount of β-catenin in the cytosol and nucleus is reduced. Thus, our findings provide novel insights into functions of Motin family members and regulation of Wnt/β-catenin signaling.     

Caprin-2 enhances canonical Wnt signaling through regulating LRP5/6 phosphorylation

The low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic β-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor–dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/β-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt,TGFbeta, Hedgehog and JAK/STAT signaling pathways

Cell-cell interactions play important roles in a variety of developmental processes, and therefore molecules involved in the signaling pathways have been studied extensively. Recently, the draft genome sequence of the basal chordate, Ciona intestinalis, was determined. Here we annotated genes for the signaling pathways of Wnt, transforming growth factor beta (TGFbeta), Hedgehog, and JAK/STAT in the genome of Ciona intestinalis. The Ciona genome contains ten wnt genes, six frizzled genes, four sFRP genes, ten TGFbeta family member genes, five TGFbeta-receptor genes, and five Smad genes; most of the genes were found with less redundancy than in vertebrate genomes. The other genes in the signaling pathways are present as a single copy in the Ciona genome. In addition, all of the identified genes for the signaling pathway, except for a few genes, have EST evidence, and their cDNAs are available from the Ciona intestinalis gene collection. Therefore, Ciona intestinalis may provide an experimental system for exploring the basic genetic cascade associated with the signaling pathways in chordates.     

Accumulation of β-catenin by lithium chloride in porcine myoblast cultures accelerates cell differentiation

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation to determine cell fate during embryogenesis. Lithium chloride (LiCl) is known to activate canonical Wnt signaling by inhibiting glycogen synthetase kinase-3β and consequently stabilizing free cytosolic β-catenin. To understand the role of the Wnt/β-catenin pathway in the regulation of porcine myoblast differentiation, we studied the effects of LiCl on cultured porcine myoblasts and β-catenin expression. A supplementation of 25 mM LiCl induced myoblast differentiation into myotubes over 3 days of culture. By semi-quantitative RT-PCR analyses, levels of mRNA encoding MyoD, Myogenin, Myf5 and several Wnt-responsive genes in the cultured myoblast cells were significantly increased after LiCl treatment. Using Western blotting and immunofluorescence analysis, we found that the protein levels of β-catenin were consistently increased by LiCl. Meanwhile, phosphorylated GSK-3β at Ser9 levels were also increased as an indicator of GSK-3β inactivation. Additionally, the nuclear staining of endogenous β-catenin was also significantly increased in porcine myoblasts 48 h after LiCl treatment. These results provided additional evidence that Wnt/β-catenin is a significant pathway that regulates myogenic differentiation. An enhanced level of β-catenin plays a positive role in porcine myoblast differentiation.     

The Ubiquitin Ligase RNF220 Enhances Canonical Wnt Signaling through USP7-Mediated Deubiquitination of β-Catenin

Wnt/β-catenin signaling plays critical roles in embryonic development and disease. Here, we identify RNF220, a RING domain E3 ubiquitin ligase, as a new regulator of β-catenin. RNF220 physically interacts with β-catenin, but instead of promoting its ubiquitination and proteasomal degradation, it stabilizes β-catenin and promotes canonical Wnt signaling. Our analysis showed that RNF220 interacts with USP7, a ubiquitin-specific peptidase, which is required for RNF220 to stabilize β-catenin. The RNF220/USP7 complex deubiquitinates β-catenin and enhances canonical Wnt signaling. Interestingly, the stability of RNF220 itself is negatively regulated by Gsk3β, which is a key component of the β-catenin destruction complex and is inhibited upon Wnt stimulation. Accordingly, the RNF220/USP7 complex works as a positive feedback regulator of β-catenin signaling. In colon cancer cells with stimulated Wnt signaling, knockdown of RNF220 or USP7 impairs Wnt signaling and expression of Wnt target genes, suggesting a potentially novel role of RNF220 in Wnt-related tumorigenesis.     

SCFβ-TRCP E3 ubiquitin ligase targets the tumor suppressor ZNRF3 for ubiquitination and degradation

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation-and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a contextdependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.