Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.     

Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha

Wnt/beta-catenin signaling has been implicated in repressing adipogenesis. Several lines of evidence show that the possible mechanism is blockade of PPARgamma induction. However, the precise mechanisms remain to be elucidated. In this study, we demonstrated that Wnt3a conditioned medium suppresses C/EBPbeta/delta-induced adipogenesis of 3T3-L1 cells by inhibiting PPARgamma induction. In addition, the mutual activation of PPARgamma and C/EBPalpha was also repressed in the presence of Wnt3a. To further investigate the role of the canonical Wnt pathway in adipogenesis, we used mouse embryonic fibroblasts (MEFs) isolated from Lrp6-deficient embryos. Contrary to wild-type MEFs, Lrp6-deficient MEFs showed spontaneous adipogenesis and escaped the suppressive effect of exogenous Wnt3a. These findings suggest a critical role of Wnt/Lrp6/beta-catenin signaling in adipogenesis and cell fate decision of mesenchymal stem cells.     

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

非洲爪蟾早期胚胎发育中的Wnt信号

非洲爪蟾是脊椎动物胚胎发育研究中的几种重要模式生物之一,为揭示早期胚胎发育中的分子调控机制做出了显著的贡献.其中一个重要的发现就是细胞信号通路在胚胎发育中起到非常关键的调控作用.本文简单介绍Wnt信号在爪蟾早期胚胎发育不同时期的几种调控作用.     

Twist plays an essential role in FGF and SHH signal transduction during mouse limb development

Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist(-/-) forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signalling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist(-/-) embryos is also retarded. The overall activity of genes involved in SHH signalling is reduced.Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signalling are expressed in normal patterns. Twist(+/-);Gli3(+/XtJ) mice display more severe polydactyly than that seen in either Twist(+/-) or Gli3(+/XtJ) mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction.     

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.     

Sonic hedgehog expression during early tooth development in Suncus murinus

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.     

Fate of HERS during tooth root development

Tooth root development begins after the completion of crown formation in mammals. Previous studies have shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of the root throughout root formation and do not disappear. Most of the HERS cells are attached to the surface of the cementum, and others separate to become the epithelial rest of Malassez. HERS cells secrete extracellular matrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network to contact dentin. HERS cells also participate in the cementum development and may differentiate into cementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue to communicate with each other through the network structure. Furthermore, HERS cells interact with cranial neural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells is crucial for tooth root development.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Downregulation of adenomatous polyposis coli by microRNA-663 promotes odontogenic differentiation through activation of Wnt/beta-catenin signaling

MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/β-catenin signaling pathway, has a miR-663 binding site in its 3′-untranslated region (3′UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/β-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.     

Nodal signaling in Xenopus gastrulae is cell-autonomous and patterned by beta-catenin

The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, we have previously shown that the activin-response element (DE) of the goosecoid promoter is uniformly activated across the vegetal half of midgastrula-stage embryos. Here, we further examine the nature of this activity by measuring DE activation by endogenous signals over time. We find that the spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated and also conclude that DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, we demonstrate that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal-deficient cells, suggesting that nodals function at short range in this context. Finally, we show that DE activation by endogenous signals occurs in the absence of dorsal beta-catenin-mediated signaling, but that the timing of dorsal initiation is altered. We conclude that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling.     

Wnt11-R signaling regulates a calcium sensitive EMT event essential for dorsal fin development of Xenopus

In the frog embryo, a sub-population of trunk neural crest (NC) cells undergoes a dorsal route of migration to contribute to the mesenchyme in the core of the dorsal fin. Here we show that a second population of cells, originally located in the dorsomedial region of the somite, also contributes to the fin mesenchyme. We find that the frog orthologue of Wnt11 (Wnt11-R) is expressed in both the NC and somite cell populations that migrate into the fin matrix. Wnt11-R is expressed prior to migration and persists in the mesenchymal cells after they have distributed throughout the fin. Loss of function studies demonstrate that Wnt11-R activity is required for an epithelial to mesenchymal transformation (EMT) event that precedes migration of cells into the fin matrix. In Wnt11-R depleted embryos, the absence of fin core cells leads to defective dorsal fin development and to collapse of the fin structure. Experiments using small molecule inhibitors indicate that dorsal migration of fin core cells depends on calcium signaling through calcium/calmodulin-dependent kinase II (CaMKII). In Wnt11-R depleted embryos, normal migration of NC cells and dorsal somite cells into the fin and normal fin development can be rescued by stimulation of calcium release. These studies are consistent with a model in which Wnt11-R signaling, via a downstream calcium pathway, regulates fin cell migration and, more generally, indicates a role for non-canonical Wnt signaling in regulation of EMT.