Maternal Wnt/β-Catenin Signaling Coactivates Transcription through NF-κB Binding Sites during Xenopus Axis Formation

Maternal Wnt/β-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/β-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between β-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/β-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual β-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus β-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous β-Catenin levels in an NF-κB motif dependent manner. These results suggest that β-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells

SNW1 Is a Critical Regulator of Spatial BMP Activity, Neural Plate Border Formation, and Neural Crest Specification in Vertebrate Embryos

In frog and fish embryos, SNW1 is a protein required for the spatio-temporal activity of BMP signaling necessary for neural plate border formation and specification of neural crest tissue

Modulation of the β-Catenin Signaling Pathway by the Dishevelled-Associated Protein Hipk1

BACKGROUND:Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY:We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS:These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation

Current perspectives of the signaling pathways directing neural crest induction

The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse

Beta-catenin, MAPK and Smad signaling during early Xenopus development

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. In this study, we have systematically analyzed and compared the signaling pattern of four major pathways by localization of the activated key components beta-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). We have determined semi-quantitatively the distribution of these components at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages, by immunofluorescence on serial cryosections. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, we have manipulated maternal beta-catenin signaling and determined the effect on the other pathways in the blastula embryo. We found that the patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on beta-catenin at this stage

Nucleo-cytoplasmic shuttling of Axin, a negative regulator of the Wnt-beta-catenin Pathway

Axin is a negative regulator of the Wnt pathway essential for down-regulation of beta-catenin. Axin has been considered so far as a cytoplasmic protein. Here we show that, although cytoplasmic at steady state, Axin shuttles in fact in and out of the nucleus; Axin accumulates in the nucleus of cells treated with leptomycin B, a specific inhibitor of the CRM1-mediated nuclear export pathway and is efficiently exported from Xenopus oocyte nuclei in a RanGTP- and CRM1-dependent manner. We have characterized the sequence requirement for export and identified two export domains, which do not contain classical nuclear export consensus sequences, and we show that Axin binds directly to the export factor CRM1 in the presence of RanGTP