scholarly journals Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development.

1996 ◽  
Vol 133 (5) ◽  
pp. 1123-1137 ◽  
Author(s):  
M A Torres ◽  
J A Yang-Snyder ◽  
S M Purcell ◽  
A A DeMarais ◽  
L L McGrew ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Signaling Pathway ◽  
Ectopic Expression ◽  
Functional Class ◽  
Dominant Negative ◽  
Beta Catenin ◽  
Embryonic Cells ◽  
Xenopus Embryos ◽  
Secondary Axis ◽  
Functional Classes

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 755-765 ◽  
Author(s):  
S.B. Pierce ◽  
D. Kimelman
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Axis Formation ◽  
Serine Threonine Kinase ◽  
Spemann Organizer ◽  
Isolation And Characterization ◽  
Secondary Axis ◽  
Dorsal Axis ◽  
Negative Effect

Dorsal axis formation in the Xenopus embryo can be induced by the ectopic expression of several Wnt family members. In Drosophila, the protein encoded by the Wnt family gene, wingless, signals through a pathway that antagonizes the effects of the serine/threonine kinase zeste-white 3/shaggy. We describe the isolation and characterization of a Xenopus homolog of zeste-white 3/shaggy, Xgsk-3. A kinase-dead mutant of Xgsk-3, Xgsk-3K-->R, has a dominant negative effect and mimics the ability of Wnt to induce a secondary axis by induction of an ectopic Spemann organizer. Xgsk-3K-->R, like Wnt, induces dorsal axis formation when expressed in the deep vegetal cells, which do not contribute to the axis. These results indicate that the dorsal fate is actively repressed by Xgsk-3, which must be inactivated for dorsal axis formation to occur. Furthermore, our work suggests that the effects of Xgsk-3K-->R are mediated by an additional intercellular signal.

The role of F-cadherin in localizing cells during neural tube formation in Xenopus embryos

Development ◽  
1998 ◽  
Vol 125 (2) ◽  
pp. 301-312 ◽  
Author(s):  
A. Espeseth ◽  
G. Marnellos ◽  
C. Kintner
Keyword(s):  
Cell Adhesion ◽  
Neural Tube ◽  
Cell Adhesion Molecule ◽  
Ectopic Expression ◽  
Tube Formation ◽  
Cell Movements ◽  
Xenopus Embryos ◽  
Extensive Cell ◽  
Neural Tube Formation

The cell adhesion molecule F-cadherin is expressed in Xenopus embryos at boundaries that subdivide the neural tube into different regions, including one, the sulcus limitans, which partitions the caudal neural tube into a dorsal and ventral half (alar and basal plate, respectively). Here we examine the role of F-cadherin in positioning cells along the caudal neuraxis during neurulation. First, we show that ectopic expression of F-cadherin restricts passive cell mixing within the ectodermal epithelium. Second, we show that F-cadherin is first expressed at the sulcus limitans prior to the extensive cell movements that accompany neural tube formation, suggesting that it might serve to position cells at the sulcus limitans by counteracting their tendency to disperse during neurulation. We test this idea using an assay that measures changes in cell movements during neurulation in response to differential cell adhesion. Using this assay, we show that cells expressing F-cadherin localize preferentially to the sulcus limitans, but still disperse when located away from the sulcus limitans. In addition, inhibiting cadherin function prevents cells from localizing precisely at the sulcus limitans. These results indicate that positioning of cells at the sulcus limitans is mediated in part by the differential expression of F-cadherin.

scholarly journals Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.

1995 ◽  
Vol 15 (5) ◽  
pp. 2625-2634 ◽  
Author(s):  
S J Du ◽  
S M Purcell ◽  
J L Christian ◽  
L L McGrew ◽  
R T Moon
Keyword(s):  
Cell Fate ◽  
Functional Class ◽  
Chimeric Proteins ◽  
Axis Formation ◽  
Amino Terminal ◽  
Xenopus Embryos ◽  
Secondary Axis ◽  
Putative Transcription Factor ◽  
Carboxy Terminal ◽  
Xenopus Laevis Embryos

Wnts are secreted signaling factors which influence cell fate and cell behavior in developing embryos. Overexpression in Xenopus laevis embryos of a Xenopus Wnt, Xwnt-8, leads to a duplication of the embryonic axis. In embryos ventralized by UV irradiation, Xwnt-8 restores expression of the putative transcription factor goosecoid, and rescues normal axis formation. In contrast, overexpression of Xwnt-5A in normal embryos generates defects in dorsoanterior structures, without inducing goosecoid or a secondary axis. To determine whether Xwnt-4 and Xwnt-11 fall into one of these two previously described classes of activity, synthetic mRNAs were introduced into animal caps, normal embryos, and UV-treated embryos. The results indicate that Xwnt-4, Xwnt-5A, and Xwnt-11 are members of a single functional class with activities that are indistinguishable in these assays. To investigate whether distinct regions of Xwnt-8 and Xwnt-5A were sufficient for eliciting the observed effects of overexpression, we generated a series of chimeric Xwnts. RNAs encoding the chimeras were injected into normal and UV-irradiated Xenopus embryos. Analysis of the embryonic phenotypes and goosecoid levels reveals that chimeras composed of carboxy-terminal regions of Xwnt-8 and amino-terminal regions of Xwnt-5A are indistinguishable from the activities of native Xwnt-8 and that are the reciprocal chimeras elicit effects indistinguishable from overexpression of native Xwnt-5A. We conclude that the carboxy-terminal halves of these Xwnts are candidate domains for specifying responses to Xwnt signals.

scholarly journals EphA4 Signaling Regulates Blastomere Adhesion in the Xenopus Embryo by Recruiting Pak1 to Suppress Cdc42 Function

2007 ◽  
Vol 18 (3) ◽  
pp. 1030-1043 ◽  
Author(s):  
Nicolas Bisson ◽  
Luc Poitras ◽  
Alexander Mikryukov ◽  
Michel Tremblay ◽  
Tom Moss
Keyword(s):  
Catalytic Activity ◽  
Cell Adhesion ◽  
Cell Sorting ◽  
Ectopic Expression ◽  
Binding Domain ◽  
Dominant Negative ◽  
Membrane Targeting ◽  
Dependent Manner ◽  
Eph Receptors ◽  
Gtpase Binding Domain

The control of cell adhesion is an important mechanism by which Eph receptors regulate cell sorting during development. Activation of EphA4 in Xenopus blastulae induces a reversible, cell autonomous loss-of-adhesion and disruption of the blastocoel roof. We show this phenotype is rescued by Nckβ (Grb4) dependent on its interaction with EphA4. Xenopus p21Cdc42/Rac-activated kinase xPAK1 interacts with Nck, is activated in embryo by EphA4 in an Nck-dependent manner, and is required for EphA4-induced loss-of-adhesion. Ectopic expression of xPAK1 phenocopies EphA4 activation. This does not require the catalytic activity of xPAK1, but it does require its GTPase binding domain and is enhanced by membrane targeting. Indeed, membrane targeting of the GTPase binding domain (GBD) of xPAK1 alone is sufficient to phenocopy EphA4 loss-of-adhesion. Both EphA4 and the xPAK1-GBD down-regulate RhoA-GTP levels, and consistent with this, loss-of-adhesion can be rescued by activated Cdc42, Rac, and RhoA and can be epistatically induced by dominant-negative RhoA. Despite this, neither Cdc42 nor Rac activities are down-regulated by EphA4 activation or by the xPAK1-GBD. Together, the data suggest that EphA4 activation sequesters active Cdc42 and in this way down-regulates cell–cell adhesion. This novel signaling pathway suggests a mechanism for EphA4-guided migration.

scholarly journals Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes

2000 ◽  
Vol 113 (10) ◽  
pp. 1803-1811
Author(s):  
Y. Hanakawa ◽  
M. Amagai ◽  
Y. Shirakata ◽  
K. Sayama ◽  
K. Hashimoto
Keyword(s):  
Cell Adhesion ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Cell Contact ◽  
Beta Catenin ◽  
Hacat Cells ◽  
Subsequent Formation ◽  
Contact Sites ◽  
E Cadherin ◽  
Cell Cell

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

scholarly journals Induction of a secondary body axis in Xenopus by antibodies to beta-catenin.

1993 ◽  
Vol 123 (2) ◽  
pp. 477-484 ◽  
Author(s):  
P D McCrea ◽  
W M Brieher ◽  
B M Gumbiner
Keyword(s):  
Cell Adhesion ◽  
The Body ◽  
Body Axis ◽  
Beta Catenin ◽  
Gene Products ◽  
Embryonic Patterning ◽  
Xenopus Embryos ◽  
Segment Polarity ◽  
Segment Polarity Gene ◽  
Polarity Gene

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.

scholarly journals Antagonism of Cell Adhesion by an α-Catenin Mutant, and of the Wnt-signaling Pathway by α-Catenin in Xenopus Embryos

1997 ◽  
Vol 139 (4) ◽  
pp. 1033-1046 ◽  
Author(s):  
Ravinder N.M. Sehgal ◽  
Barry M. Gumbiner ◽  
Louis F. Reichardt
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Dominant Negative ◽  
Developmental Defects ◽  
Calcium Dependent ◽  
Adhesive Interactions

In Xenopus laevis development, β-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell–cell adhesion and can modulate β-catenin signaling. α-catenin links β-catenin to the actin-based cytoskeleton. To study the role of endogenous α-catenin in early development, we have made deletion mutants of αN-catenin. The binding domain of β-catenin has been mapped to the NH2-terminal 210 amino acids of αN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length αN-catenin or a mutant of β-catenin that lacks the internal armadillo repeats. We next show that coexpression of αN-catenin antagonizes the dorsalizing effects of β-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, α-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular β-catenin signaling pathway in vivo.

scholarly journals A beta-catenin/engrailed chimera selectively suppresses Wnt signaling

2000 ◽  
Vol 113 (10) ◽  
pp. 1759-1770 ◽  
Author(s):  
W.T. Montross ◽  
H. Ji ◽  
P.D. McCrea
Keyword(s):  
Cell Adhesion ◽  
Wnt Signaling ◽  
Transcriptional Repression ◽  
Organizer Region ◽  
Chimeric Protein ◽  
Homeobox Gene ◽  
Beta Catenin ◽  
Embryonic Axes ◽  
Xenopus Embryos ◽  
Cell Cell

beta-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, beta-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal ‘organizer’ region of early Xenopus embryos, beta-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that beta-catenin plays in Wnt signaling, we generated a chimeric protein, beta-Engrailed (beta-Eng), in which the C-terminal trans-activation domain of beta-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, beta-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress beta-catenin's signaling function apart from its role in cadherin-mediated cell-cell adhesion.

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2677-2685 ◽  
Author(s):  
E.M. Joseph ◽  
D.A. Melton
Keyword(s):  
Gene Function ◽  
Ectopic Expression ◽  
Embryonic Cells ◽  
Xenopus Embryos ◽  
Maternal Mrna ◽  
Dorsal Mesoderm ◽  
Mesoderm Formation

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

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