Chika Yokota

Maternal Wnt11 Activates the Canonical Wnt Signaling Pathway Required for Axis Formation in Xenopus Embryos

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.

Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions.

Canonical Wnt/β-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/β-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/β-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate β-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/β-catenin signaling.

R-spondin1 Is a High Affinity Ligand for LRP6 and Induces LRP6 Phosphorylation and β-Catenin Signaling

R-spondin proteins are newly identified secreted molecules that activate β-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (Kd = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.

Conversion of ectoderm into a neural fate by ATH‐3, a vertebrate basic helix–loop–helix gene homologous to Drosophila proneural gene atonal

We have isolated a novel basic helix–loop–helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH‐3, from Xenopus and mouse. ATH‐3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH‐3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non‐neural development. This ATH‐3‐induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non‐neural types adopt a neural fate. In a Xenopus animal cap assay, ATH‐3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker‐inducing ability without affecting general neurogenic activities. These results provide evidence that ATH‐3 can directly convert non‐neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH‐3 activity by phosphorylation.

Wnt Signal Amplification via Activity, Cooperativity, and Regulation of Multiple Intracellular PPPSP Motifs in the Wnt Co-receptor LRP6

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/β-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of β-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal βcatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.

In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid

In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas‐like structures accompanied by an intestinal epithelium‐like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus‐like structure, including secretory granules. The pancreas‐specific molecular markers insulin and XlHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well‐differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3–5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15 h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.

Expression cloning of Xantivin, a Xenopus lefty/antivin-related gene, involved in the regulation of activin signaling during mesoderm induction.

In a screening for activin-responsive genes, we isolated a Xenopus lefty/antivin-related gene, called Xantivin (Xatv). In the animal cap assay, the expression of Xatv was induced by activin signaling, and in the embryo, by nodal-related genes. Overexpression of Xatv in the marginal zone caused suppression of mesoderm formation and gastrulation defects, and inhibited the secondary axis formation induced by Xnr1 and Xactivin, suggesting that Xatv acted as a feedback inhibitor of activin signaling. However, in the animal cap, Xatv failed to antagonize Xnr1 and Xactivin. This result suggested that Xatv has different responses in the marginal zone and in the animal region, and antagonizes to a higher degree activin signaling in the marginal zone.

ENDODERM DIFFERENTIATION AND INDUCTIVE EFFECT OF ACTIVIN-TREATED ECTODERM IN XENOPUS

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk‐rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin‐treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.

Xnr2 and Xnr5 Unprocessed Proteins Inhibit Wnt Signaling Upstream of Dishevelled

Nodal and Nodal‐related proteins activate the Activin‐like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal‐related proteins apart from the canonical Activin‐like signal pathway. Here we report a novel function of Nodal‐related proteins using cleavage mutants of Xenopus nodal‐related genes (cmXnr2 and cmXnr5), which are known to be dominant‐negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin‐like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin‐Xnr2 and cmActivin‐Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced. Developmental Dynamics 234:900–910, 2005.