Functional conservation of Nematostella Wnts in canonical and noncanonical Wnt-signaling.

Cnidarians surprise by the completeness of Wnt gene subfamilies (11) expressed in an overlapping pattern along the anterior-posterior axis. While the functional conservation of canonical Wnt-signaling components in cnidarian gastrulation and organizer formation is evident, a role of Nematostella Wnts in noncanonical Wnt-signaling has not been shown so far. In Xenopus, noncanonical Wnt-5a/Ror2 and Wnt-11 (PCP) signaling are distinguishable by different morphant phenotypes. They differ in PAPC regulation, cell polarization, cell protrusion formation, and the so far not reported reorientation of the microtubules. Based on these readouts, we investigated the evolutionary conservation of Wnt-11 and Wnt-5a function in rescue experiments with Nematostella orthologs and Xenopus morphants. Our results revealed that NvWnt-5 and -11 exhibited distinct noncanonical Wnt activities by disturbing convergent extension movements. However, NvWnt-5 rescued XWnt-11 and NvWnt-11 specifically XWnt-5a depleted embryos. This unexpected 'inverse' activity suggests that specific structures in Wnt ligands are important for receptor complex recognition in Wnt-signaling. Although we can only speculate on the identity of the underlying recognition motifs, it is likely that these crucial structural features have already been established in the common ancestor of cnidarians and vertebrates and were conserved throughout metazoan evolution.

Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.

Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs.

One of the key end points for understanding the molecular basis of embryogenesis is the analysis of spatiotemporal patterns of gene expression. Methodical limitations due to low mRNA levels often prevent a tissue-specific resolution. In this study, we developed an improved laser microdissection technique and RT-PCR that allows marker gene detection in small tissue areas from sections of formalin-fixed paraffin-embedded Xenopus embryos. Tissue pieces were isolated by laser microbeam microdissection and captured by laser pressure catapulting. Neither laser treatment nor conventional histological or immunochemical staining impaired subsequent RNA analysis. Transcripts of tissue-specific marker genes such as endodermin (endoderm), epidermal cytokeratin (epidermal ectoderm), N-CAM (neural tube), myoD (somites), and sonic hedgehog (floor plate) were amplified by nested RT-PCR analysis from small areas of single sections.

Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+ signaling.

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/beta-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/beta-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/beta-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/beta-catenin pathway upstream of beta-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/beta-catenin signaling cascade downstream of beta-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.

Xenopus Eya1 demarcates all neurogenic placodes as well as migrating hypaxial muscle precursors.

We cloned two isoforms of the Xenopus Eya1 orthologue. They show identical patterns of expression that closely resemble the previously described expression of XSix1, but partly differ from the expression of Eya1 in other vertebrates. XEya1 is expressed in the somites and hypaxial muscle precursors, but not in the pronephros. Moreover, all ectodermal placodes except the lens placode strongly express XEya1. At neural plate stages, ectodermal XEya1 expression starts in two domains, the anterior neural folds and a domain lateral to the neural folds. At tailbud stages, XEya1 expression continues in the adenohypophysis, all neurogenic placodes and placodally-derived structures including cranial ganglia, the otic vesicle and lateral line primordia.

PCR-based RNA probes: a quick and sensitive method to improve whole mount embryo in situ hybridizations.

We have developed a PCR-based technique for the preparation of RNA probes that can be used for whole mount in situ hybridization on embryos. T3 or T7 RNA polymerase promoters were introduced at the 5' end of gene-specific oligonucleotide primers enabling direct in vitro transcription of purified PCR fragments. We show for various marker genes in Xenopus embryos that this method provides equivalent results as compared to conventional vector-based probe preparation, even when fluorescence detection (FISH) is applied. This method offers a rapid and useful means to prepare gene-specific in situ probes predominantly for expression screens or detection of splice variants that previously required time-consuming cloning steps.

Down-regulation of E-cadherin gene expression by collagen type I and type III in pancreatic cancer cell lines.

E-cadherin-mediated cell-cell adhesion is reduced in epithelial tumors, which is thought to be a prerequisite to acquire invasive properties. We observed that several pancreatic carcinoma cell lines with high metastatic potential expressed normal levels of E-cadherin and possessed functional E-cadherin/catenin adhesion complexes. When the cell lines PANC-1, BxPC-3, and PaTu8988s were cultured either on type I or type III collagen, E-cadherin gene expression was repressed, and E-cadherin and catenin protein concentrations were reduced. In contrast, growth on fibronectin and collagen type IV had no influence. Collagen type I- or type III-dependent reduction of E-cadherin expression led to decreased cell-cell adhesion, increased proliferation, and migratory activity as well as morphological transformation. Overexpression of activated c-Src in PANC-1 cells mimicked collagen-induced E-cadherin down-regulation and changed the elevated cell proliferation and migration. Conversely, treatment of cells with the Src-inhibitors PP1 or herbimycin A resulted in complete suppression of collagen type I-induced E-cadherin decrease. Our data demonstrate that specific collagens are able to promote metastatic behavior by down-regulation of E-cadherin gene expression in a Src-kinase-dependent manner. This points toward a novel mechanism for substrate-dependent signaling and underlines the significance of extracellular matrix environment for tumor growth and invasiveness.

Identification of two regulatory elements within the high mobility group box transcription factor XTCF-4.

Some members of the Wnt family of extracellular glycoproteins regulate target gene expression by inducing stabilization and nuclear accumulation of beta-catenin, which functions as a transcriptional activator after binding to transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Three different members of this family have been identified in Xenopus laevis thus far that differ in their ability to influence mesodermal differentiation and to activate expression of the Wnt target gene fibronectin. Here we report on the isolation and characterization of additional variants of XTCF-4. We show that the differential ability of these proteins and other members of the TCF family to activate target genes is neither due to preferential interaction with transcriptional cofactors of the groucho family or SMAD4 nor to different DNA binding affinities. Expression of these proteins in an epithelial cell line reveals differences in their ability to form a ternary complex with DNA and beta-catenin. Interestingly, formation of this ternary complex was not sufficient to activate target gene expression as previously thought. Our experiments identify two amino acid sequence motifs, LVPQ and SFLSS, in the central domain of XTCF-4 that regulate the formation of the DNA-TCF-beta-catenin complex or activation of target genes, respectively. Biochemical studies reveal that the phosphorylation state of these XTCF-4 variants correlates with their ability to form a ternary complex with beta-catenin and DNA but not to activate target gene expression. The described variants of XTFC-4 with their different properties in complex formation provide strong evidence that in addition to the regulation of beta-catenin stability the isoforms of TCF/LEF transcription factors and their posttranslational modifications define the cellular response of a Wnt/wingless signal.

Xenopus cadherin-6 is expressed in the central and peripheral nervous system and in neurogenic placodes.

We have cloned the cDNAs encoding the two Xenopus laevis cadherin-6 pseudoalleles (Xcad-6 and Xcad-6'), which belong to the classical type II cadherin subfamily. During embryogenesis, Xcad-6 expression starts at early neurula and increases dramatically until tadpole stages. Whole-mount in situ hybridization revealed that the transcripts are localized in the developing brain and eye. However, most prominent expression was found in the peripheral nervous system, including a major proportion of lateral line structures. Our results implicate an important contribution of cadherin-6 to the formation of placode derived nerves in Xenopus, an observation which has not been described for other species.

Altered subcellular distribution of cadherin-5 in endothelial cells caused by the serum of pre-eclamptic patients.

The main clinical features of pre-eclampsia are oedema and vascular leakage. Cadherin-5 mediates endothelial cell-cell contact in the vascular endothelium and may regulate permeability as a vascular function. Therefore, we addressed the question of whether pre-eclampsia alters cadherin-5 expression and intracellular distribution. Confluent human umbilical vein endothelial cells (HUVEC) were incubated with 20% serum from patients with pre-eclampsia (n = 18), haemolysis-elevated liver enzymes-low platelet syndrome (HELLP) (n = 12), pregnancy-induced hypertension (PIH) (n = 18) or normal pregnancy (n = 10). After incubation with sera from patients with pre-eclampsia, immunostaining analyses showed cadherin-5 accumulation in vesicular and tubular structures of the Golgi apparatus. Immunoblot analyses of HUVEC after pre-eclampsia serum incubation showed an increase of the stable form of cadherin-5 while degradation products decreased. Degradation of cadherin-5 takes place at the cell membrane, so this decrease may be due to a decrease of cadherin-5 in the cell membrane. The accumulation of cadherin-5 in the vesicular and tubular structures of the Golgi apparatus indicates that targeting of cadherin-5 to the plasma membrane could be disrupted. We suggest that intracellular retention of cadherin-5 caused by serum factors in patients with pre-eclampsia may decrease the number of adhesion complexes in the cell membrane, thereby contributing to endothelial dysfunction.

Expression of Xenopus homologs of the beta-catenin binding protein pontin52.

Identification of pontin52 as an interaction partner of the Wnt/Wg signal transducer beta-catenin implicated a role for this protein in Wnt signaling. Here we describe the isolation of two Xenopus homologs of pontin52, Xpontin and Xreptin, and report the first expression pattern of vertebrate pontin52 homologs. Whole-mount in situ hybridization studies reveal a strong expression of Xpontin in neural crest cells and in later stages in different gastrointestinal organs. Xreptin is also expressed in neural crest cells, in particular in a subpopulation that give raise to the adrenal medulla.

The HMG-box transcription factor XTcf-4 demarcates the forebrain-midbrain boundary.

A small subfamily of HMG-box transcription factors, the LEF/TCF group, serves as nuclear transducer of the Wnt-1/Wg signaling cascade. Upon Wnt-1/Wg signaling their members interact with beta-catenin and regulate the expression of Xenopus target genes siamois, twin, nodal related-3 or fibronectin. We have isolated a new HMG-box transcription factor in Xenopus that will be addressed XTcf-4 based on its homology to human and murine Tcf-4. Unlike XTcf-3, which is a maternal gene, and XLef-1 that is expressed after mid blastula transition (Molenaar et al., 1998. Mech. Dev. 75, 151-154), XTcf-4 expression starts at late neurula stage and is restricted to the anterior most midbrain demarcating the forebrain-midbrain boundary. The expression partially overlaps with a broad set of Xenopus Wnt family members in distinct patterns. XTcf-4 transcripts were also found partially co-localized with those of Xaxin, an intracellular antagonist of Wnt-1/Wg signaling.

An assay system to study migratory behavior of cranial neural crest cells in Xenopus.

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP.

The Wnt/Wg signal transducer beta-catenin controls fibronectin expression.

beta-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of beta-catenin following cadherin transfection in Xenopus fibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or beta-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position -368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-type Xenopus Tcf-3 (XTcf-3) was unable to activate FN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608-612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via beta-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, beta-catenin regulates the balance between cell-cell and cell-substrate adhesion.

Xenopus cadherin-11 (Xcadherin-11) expression requires the Wg/Wnt signal.

In this study we describe the isolation of Xcadherin-11, the Xenopus homologue to the mesenchymal cadherin-11. Similar to epithelial and neural cadherins, overexpression of Xcadherin-11 led to posteriorised phenotypes due to inhibition of convergent extension movement. Because zygotic expression of Xcadherin-11 starts with gastrulation, we analysed the ability of different growth factors involved in mesoderm differentiation to induce the expression of Xcadherin-11. Using the animal cap assay, we demonstrated that Xcadherin-11 is activated by Xwnt-8 or beta-catenin, but repressed by BMP-4. Activin did not induce Xcadherin-11 but its synergistic function was required for the Xwnt-8/beta-catenin-mediated activation of Xcadherin-11. Because Xcadherin-11 and Xenopus E- and N-cadherin are differentially regulated by growth factors in the Xenopus animal cap, our results also reveal that this assay provides a helpful model system to elucidate the molecular control mechanism of epithelial-mesenchymal conversion.

Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta.

Control of stability of beta-catenin is central in the wnt signaling pathway. Here, the protein conductin was found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induced beta-catenin degradation, whereas mutants of conductin that were deficient in complex formation stabilized beta-catenin. Fragments of APC that contained a conductin-binding domain also blocked beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfered with wnt-induced axis formation.

Expression of the Armadillo family member p120cas1B in Xenopus embryos affects head differentiation but not axis formation.

The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, beta-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120cas, the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120cas1B (mp120cas1B) in ventral blastomeres that this catenin cannot replace beta-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120cas1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120cas1B influences motility or orientation of migrating mesodermal cells.

Uncoupling of XB/U-cadherin-catenin complex formation from its function in cell-cell adhesion.

Xenopus XB/U-cadherin forms functional complexes with mouse alpha- and beta-catenins and p120(cas) when expressed in murine L-TK- fibroblasts. These cells were stably transfected with cDNAs encoding different cytoplasmic XB/U-cadherin mutants, each partially deleted in the different parts of the 38 most carboxyl-terminal amino acids. The binding of p120(cas) was not affected by carboxyl-terminal deletions, confirming its binding to a region more amino-terminal and distinct from the catenins. alpha- and beta-catenins associate with truncated XB/U-cadherins if either 19 amino acid half of the cadherin 38 amino acid tail is present, indicating that the site of catenin interaction is upstream of the deletions. However, for adhesive function of XB/U-cadherin constructs, the most carboxyl-terminal 19 amino acids are essential; if these amino acids are deleted, cadherin-catenin complexes unable to mediate cell-cell adhesion are formed. Nonadhesive complexes are solubilized by mild detergent, whereas functional complexes are stable. Provided that detergent stability of cadherin-catenin complexes is taken as a measure of their cytoskeletal association, our results give first evidence that cytoskeletal stabilization occurs independent of cadherin-catenin complex formation and requires the 19-amino acid cadherin carboxyl terminus.

Fritz: a secreted frizzled-related protein that inhibits Wnt activity.

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.