E-cadherin is required for cranial neural crest migration in Xenopus laevis.

The cranial neural crest (CNC) is a highly motile and multipotent embryonic cell population, which migrates directionally on defined routes throughout the embryo, contributing to facial structures including cartilage, bone and ganglia. Cadherin-mediated cell-cell adhesion is known to play a crucial role in the directional migration of CNC cells. However, migrating CNC co-express different cadherin subtypes, and their individual roles have yet to be fully explored. In previous studies, the expression of individual cadherin subtypes has been analysed using different methods with varying sensitivities, preventing the direct comparison of expression levels. Here, we provide the first comprehensive and comparative analysis of the expression of six cadherin superfamily members during different phases of CNC cell migration in Xenopus. By applying a quantitative RT-qPCR approach, we can determine the copy number and abundance of each expressed cadherin through different phases of CNC migration. Using this approach, we show for the first time expression of E-cadherin and XB/C-cadherin in CNC cells, adding them as two new members of cadherins co-expressed during CNC migration. Cadherin co-expression during CNC migration in Xenopus, in particular the constant expression of E-cadherin, contradicts the classical epithelial-mesenchymal transition (EMT) model postulating a switch in cadherin expression. Loss-of-function experiments further show that E-cadherin is required for proper CNC cell migration in vivo and also for cell protrusion formation in vitro. Knockdown of E-cadherin is not rescued by co-injection of other classical cadherins, pointing to a specific function of E-cadherin in mediating CNC cell migration. Finally, through reconstitution experiments with different E-cadherin deletion mutants in E-cadherin morphant embryos, we demonstrate that the extracellular domain, but not the cytoplasmic domain, of E-cadherin is sufficient to rescue CNC cell migration in vivo.

Regulation of distinct branches of the non-canonical Wnt-signaling network in Xenopus dorsal marginal zone explants.

BACKGROUND: A tight regulation of the Wnt-signaling network, activated by 19 Wnt molecules and numerous receptors and co-receptors, is required for the establishment of a complex organism. Different branches of this Wnt-signaling network, including the canonical Wnt/ß-catenin and the non-canonical Wnt/PCP, Wnt/Ror2 and Wnt/Ca(2+) pathways, are assigned to distinct developmental processes and are triggered by certain ligand/receptor complexes. The Wnt-signaling molecules are closely related and it is still on debate whether the information for activating a specific branch is encoded by specific sequence motifs within a particular Wnt protein. The model organism Xenopus offers tools to distinguish between Wnt-signaling molecules activating distinct branches of the network. RESULTS: We created chimeric Wnt8a/Wnt11 molecules and could demonstrate that the C-terminal part (containing the BS2) of Wnt8a is responsible for secondary axis formation. Chimeric Wnt11/Wnt5a molecules revealed that the N-terminus with the elements PS3-1 and PS3-2 defines Wnt11 specificity, while elements PS3-1, PS3-2 and PS3-3 are required for Wnt5a specificity. Furthermore, we used Xenopus dorsal marginal zone explants to identify non-canonical Wnt target genes regulated by the Wnt5a branch and the Wnt11 branch. We found that pbk was specifically regulated by Wnt5a and rab11fip5 by Wnt11. Overexpression of these target genes phenocopied the overexpression of their regulators, confirming the distinct roles of Wnt11 and Wnt5a triggered signaling pathways. Furthermore, knock-down of pbk was able to restore convergent extension movements in Wnt5a morphants. CONCLUSIONS: The N-terminal part of non-canonical Wnt proteins decides whether the Wnt5a or the Wnt11 branch of the Wnt-signaling network gets activated. The different non-canonical Wnt branches not only regulate cellular behavior, but, surprisingly, also regulate the expression of different target genes. One of these target genes, pbk, seems to be the relevant target gene executing Wnt5a-mediated regulation of convergent extension movements.

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

Neural crest specification by Prohibitin1 depends on transcriptional regulation of prl3 and vangl1.

A complex network of transcription factors regulates specification of neural crest cells at early neurula stage by stabilizing neural crest identity and activating neural crest effector genes so that distinct subpopulations evolve. In this network, c-myc acts on top of the gene hierarchy controlling snail2, AP2 and prohibitin1 (phb1) expression. While snail2 and AP2 are well studied neural crest specifier genes little is known about the role of phb1 in this process. To identify phb1 regulated genes we analyzed the transcriptome of neural crest explants of phb1 morphant Xenopus embryos. Among 147 phb1 regulated genes we identified the membrane-associated protein-tyrosine phosphatase PRP4A3 (prl3) and the atypical cadherin and Wnt-PCP component van gogh like1 (vangl1). Gain of function, loss of function and epistasis experiments allowed us to allocate both genes in the neural crest specification network between phb1 and twist. Interestingly, both, vangl1 and prl3 regulate only a small subset of neural crest marker genes. The identification of two membrane-associated proteins as novel neural crest specifiers indicates that in addition to gene regulation by combinatory effects of transcription factors also post-translational modifications (prl3) and cell-cell adhesion and/or regulation of cell-polarity (vangl1) specify the identity of neural crest cell populations. genesis 53:627-639, 2015. © 2015 Wiley Periodicals, Inc.

Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS.

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration.

Snail2 controls mesodermal BMP/Wnt induction of neural crest.

The neural crest is an induced tissue that is unique to vertebrates. In the clawed frog Xenopus laevis, neural crest induction depends on signals secreted from the prospective dorsolateral mesodermal zone during gastrulation. The transcription factors Snail2 (Slug), Snail1 and Twist1 are expressed in this region. It is known that Snail2 and Twist1 are required for both mesoderm formation and neural crest induction. Using targeted blastomere injection, morpholino-based loss of function and explant studies, we show that: (1) Snail1 is also required for mesoderm and neural crest formation; (2) loss of snail1, snail2 or twist1 function in the C2/C3 lineage of 32-cell embryos blocks mesoderm formation, but neural crest is lost only in the case of snail2 loss of function; (3) snail2 mutant loss of neural crest involves mesoderm-derived secreted factors and can be rescued synergistically by bmp4 and wnt8 RNAs; and (4) loss of snail2 activity leads to changes in the RNA levels of a number of BMP and Wnt agonists and antagonists. Taken together, these results identify Snail2 as a key regulator of the signals involved in mesodermal induction of neural crest.

Prohibitin1 acts as a neural crest specifier in Xenopus development by repressing the transcription factor E2F1.

Prohibitin 1 (phb1), which was initially described as an inhibitor of cell proliferation, is a highly conserved protein found in multiple cellular compartments. In the nucleus it interacts with the transcriptional regulators Rb and E2F1 and controls cell proliferation and apoptosis. Here we unravel an unexpected novel function for phb1 in Xenopus cranial neural crest (CNC) development. Xphb1 is maternally expressed; zygotically expressed neurula stage transcripts accumulate in the CNC and the neural tube. Knockdown of Xphb1 by antisense morpholino injection results in the loss of foxD3, snail2 and twist expression, whereas expression of c-myc, AP-2 and snail1 remains unaffected. Xphb2, its closest relative, cannot substitute for Xphb1, underlining the specificity of Xphb1 function. Epistatic analyses place Xphb1 downstream of c-myc and upstream of foxD3, snail2 and twist. To elucidate which subdomain in Xphb1 is required for neural crest gene regulation we generated deletion mutants and tested their rescue ability in Xphb1 morphants. The E2F1-binding domain was found to be necessary for Xphb1 function in neural crest development. Gain- and loss-of-function experiments reveal that Xphb1 represses E2F1 activity; suppression of E2F1 through Xphb1 is required for twist, snail2 and foxD3 expression in the CNC. With the Xphb1 dependency of a subset of CNC specifiers downstream of c-myc, we have identified a new branching point in the neural crest gene regulatory network.

Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway.

XWnt-5A, a member of the nontransforming Wnt-5A class of Wnt ligands, is required for convergent extension movements in Xenopus embryos. XWnt-5A knockdown phenocopies paraxial protocadherin (XPAPC) loss of function: involuted mesodermal cells fail to align mediolaterally, which results in aberrant movements and a selective inhibition of constriction. XWnt-5A depletion was rescued by coinjection of XPAPC RNA, indicating that XWnt-5A acts upstream of XPAPC. XWnt-5A, but not XWnt-11, stimulates XPAPC expression independent of the canonical Wnt/beta-catenin pathway. We show that transcriptional regulation of XPAPC by XWnt-5A requires the receptor tyrosine kinase Ror2. XWnt-5A/Xror2 signal through PI3 kinase and cdc42 to activate the JNK signaling cascade with the transcription factors ATF2 and c-jun. The Wnt-5A/Ror2 pathway represents an alternative, distinct branch of noncanonical Wnt signaling that controls gene expression and is required in the regulation of convergent extension movements in Xenopus gastrulation.

Giving the right tug for migration: cadherins in tissue movements.

Dynamically regulated cell-cell adhesion is crucial for morphogenesis during embryonic development and tumor progression. The cadherins as calcium-dependent cell-cell adhesion proteins represent key molecules in these tissue movements. How cadherins serve in maintaining tissue cohesion during migration, facilitate cell-cell communication and promote signaling will be summarized in this review.

En2, Pax2/5 and Tcf-4 transcription factors cooperate in patterning the Xenopus brain.

Among Xenopus Lef/Tcfs, XTcf-4 has an outstanding role. In early development it is located exclusively in the midbrain where it is essential for midbrain and isthmus development. In order to identify transcription factors responsible for the restriction of XTcf-4 expression we isolated a 3.8kb fragment of the XTcf-4 promoter. We found that this promoter fragment is sufficient to mimic endogenous XTcf-4 expression in the midbrain. Characterization of putative binding sites for en2 and pax2/5 revealed that en2, but not pax2/5 directly represses XTcf-4 promoter activity. Gain-of-function experiments in Xenopus embryos confirmed this en2-mediated repression. Loss-of-function experiments demonstrate that both en2 and pax2/5 are essential for endogenous XTcf-4 expression. The primary effect of pax2/5 depletion thereby appears to be a reduced en2 expression at neurula stages. Because en2 can compensate for the depletion of pax2/5, we assume a hierarchical regulation of gene expression in the midbrain/isthmus region with pax2/5 acting upstream of en2. Furthermore, since the XTcf-4 expression domain does not overlap with the expression domains of the isthmus marker genes en2 and pax2/5, we conclude that the knock-down of en2 and pax2/5 results in a downregulation of a paracrine growth factor regulating XTcf-4 expression. We found that the growth factor for this non-cell-autonomous effect of en2 and pax2/5 is wnt-1 acting on the -1437 Lef/Tcf binding site on the XTcf-4 promoter. We provide evidence that the main nuclear wnt transducer for the autoregulation of XTcf-4 is XTcf-1.

PAPC and the Wnt5a/Ror2 pathway control the invagination of the otic placode in Xenopus.

BACKGROUND: Paraxial protocadherin (PAPC) plays a crucial role in morphogenetic movements during gastrulation and somitogenesis in mouse, zebrafish and Xenopus. PAPC influences cell-cell adhesion mediated by C-Cadherin. A putative direct adhesion activity of PAPC is discussed. PAPC also promotes cell elongation, tissue separation and coordinates cell mass movements. In these processes the signaling function of PAPC in activating RhoA/JNK and supporting Wnt-11/PCP by binding to frizzled 7 (fz7) is important. RESULTS: Here we demonstrate by loss of function experiments in Xenopus embryos that PAPC regulates another type of morphogenetic movement, the invagination of the ear placode. Knockdown of PAPC by antisense morpholinos results in deformation of the otic vesicle without altering otocyst marker expression. Depletion of PAPC could be rescued by full-length PAPC, constitutive active RhoA and by the closely related PCNS but not by classical cadherins. Also the cytoplasmic deletion mutant M-PAPC, which influences cell adhesion, does not rescue the PAPC knockdown. Interestingly, depletion of Wnt5a or Ror2 which are also expressed in the otocyst phenocopies the PAPC morphant phenotype. CONCLUSIONS: PAPC signaling via RhoA and Wnt5a/Ror2 activity are required to keep cells aligned in apical-basal orientation during invagination of the ear placode. Since neither the cytoplasmic deletion mutant M-PAPC nor a classical cadherin is able to rescue loss of PAPC we suggest that the signaling function of the protocadherin rather than its role as modulator of cell-cell adhesion is required during invagination of the ear placode.

The polarising role of cell adhesion molecules in early development.

Polarising a cell or an embryo is a crucial and recurrent event during development, as it is important for cell differentiation and migration. Cells can become polarised along their apical-basal axis and also within the plane of the tissue layer to which they belong. The embryo develops three axes: the anteroposterior, the dorsoventral and the left-right axis. Recent work indicates instructive roles for cell adhesion molecules in establishing not only apical-basal polarity but also planar cell polarity and, surprisingly, in the generation of left-right asymmetry in vertebrates. Signalling cascades that regulate polarity formation seem to be conserved among different organisms, thereby raising the intriguing question of whether this also holds true for the cell adhesion molecules.

Benzylguanine thiol self-assembled monolayers for the immobilization of SNAP-tag proteins on microcontact-printed surface structures.

The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O(6)-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (microCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.

Design of chemically activated polymer microwells by one-step UV-lithography for stem cell adhesion.

A novel method to produce sub-microwalled chemically activated polymer microwells by one-step UV-lithography under ambient conditions which are selectively coated with gelatin is introduced. The dimensions as well as the shape of the resulting polystyrene structures are both tunable merely by the irradiation time through one and the same mask. It is shown that the UV-irradiation initiates three effects at those surface areas which are not covered by the mask: (i) oxidation, (ii) cross-linking, and (iii) degradation of polystyrene. The superposition of those effects results in the formation of microscaled, oxidized polymer wells separated by polymer walls, whereas the polymer walls are formed below the mask structures. Topographical changes induced by the UV-irradiation are investigated by atomic force microscopy after different irradiation times. It is shown by X-ray photoelectron spectroscopy and ellipsometric investigations that the chemical composition of the irradiated areas and the degradation of polystyrene reach an equilibrium state after an irradiation time of 10 min. The lateral distribution of the cross-linked and oxidized and of the nonmodified polystyrene after irradiation was determined by fluorescence microscopy and time-of-flight secondary ion mass spectrometry. After the irradiated samples were treated with gelatin solution, it was found that stem cells selectively attach to the irradiated areas. This is due to the selective immobilization of the gelatin on the irradiated polymer areas, which was proved by X-ray photoelectron spectroscopy experiments.

Polysialylated NCAM represses E-cadherin-mediated cell-cell adhesion in pancreatic tumor cells.

BACKGROUND & AIMS: Inhibition of cell-cell adhesion between epithelial cells represents an early step during tumor metastasis. Down-regulation or perturbation of E-cadherin-mediated adherens junctions is an essential requirement in this process. METHODS: The interaction between polysialylated neural cell adhesion molecule (PSA-NCAM) and the E-cadherin adhesion complex was studied by coimmunoprecipitation assays. The presence of PSA-NCAM was correlated with tumor invasion by using cell-cell aggregation and cell migration assays. The importance of polysialic acid (PSA) in the interaction of NCAM with E-cadherin and inhibition of cell-cell adhesion was confirmed by enzymatic removal of PSA from NCAM and down-regulation of PSA-transferases by siRNA. RESULTS: Expression of oncogenic K-Ras(V12) in pancreatic carcinoma cells resulted in induction of PSA-NCAM expression and reduced E-cadherin-mediated cellular adhesion. The association of PSA-NCAM with the E-cadherin adhesion complex correlated with decreased cell-cell aggregation and elevated cell migration of pancreatic carcinoma cells. Enzymatic removal of PSA from NCAM or reduction of polysialyltransferase expression led to reduced association between NCAM and E-cadherin and subsequently increased E-cadherin-mediated cell-cell aggregation and reduced cell migration. CONCLUSIONS: Our data suggest the induction of PSA-NCAM by oncogenic K-Ras as a novel molecular mechanism by which E-cadherin-mediated cellular adhesion is reduced and dissemination of tumor cells is facilitated.

Autoregulation of XTcf-4 depends on a Lef/Tcf site on the XTcf-4 promoter.

The restricted expression of XTcf-4 in the anterior midbrain is regulated via an active wnt/beta-catenin pathway (Kunz et al.,2004, Dev Biol 273:390-401). The molecular mechanism of this autoregulatory loop, however, remained elusive. Here we show that the activity of a 1,775 bp promoter fragment containing a consensus Lef/Tcf binding site at position -1,437 to -1,428 is upregulated by activating transcription factors of the Lef/Tcf family. Furthermore, chromatin immunoprecipitation revealed that endogenous beta-catenin is bound to the Lef/Tcf site on the promoter. Thus, regulation of XTcf-4 by canonical wnt-signaling is directly controlled by binding to and activating a consensus Lef/Tcf binding site within its own promoter.

Cold-inducible RNA binding protein (CIRP), a novel XTcf-3 specific target gene regulates neural development in Xenopus.

BACKGROUND: As nuclear mediators of wnt/beta-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/beta-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. RESULTS: We identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. CONCLUSION: Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns.

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (microCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide - even for the preparation of peptide-functionalized small surface areas.

Xenopus cadherin-6 regulates growth and epithelial development of the retina.

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.

Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1.

Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway.