François Fagotto

Nucleo-cytoplasmic Shuttling of Axin, a Negative Regulator of the Wnt-β-Catenin Pathway

Axin is a negative regulator of the Wnt pathway essential for down-regulation of β-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.

EphrinB/EphB Signaling Controls Embryonic Germ Layer Separation by Contact-Induced Cell Detachment

Tissue boundary formation in the early vertebrate embryo involves cycles of cell attachment and detachment at the boundary, and cell contact-dependent signaling by membrane-bound EphB receptors and ephrinB ligands.

Selective Pharmacological Targeting of a DEAD Box RNA Helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

RanBP3 enhances nuclear export of active β-catenin independently of CRM1

β-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of β-catenin is regulated is not well defined. Therefore, we used the nuclear marker RanGTP to screen for novel nuclear β-catenin binding proteins. We identified a cofactor of chromosome region maintenance 1 (CRM1)–mediated nuclear export, Ran binding protein 3 (RanBP3), as a novel β-catenin–interacting protein that binds directly to β-catenin in a RanGTP-stimulated manner. RanBP3 inhibits β-catenin–mediated transcriptional activation in both Wnt1- and β-catenin–stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with β-catenin–induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated β-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active β-catenin toward the cytoplasm. Modulation of β-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. We conclude that RanBP3 is a direct export enhancer for β-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

Plasma membrane recruitment of dephosphorylated β-catenin upon activation of the Wnt pathway

The standard model of Wnt signaling specifies that after receipt of a Wnt ligand at the membranous receptor complex, downstream mediators inhibit a cytoplasmic destruction complex, allowing β-catenin to accumulate in the cytosol and nucleus and co-activate Wnt target genes. Unexpectedly, shortly after Wnt treatment, we detected the dephosphorylated form of β-catenin at the plasma membrane, where it displayed a discontinuous punctate labeling. This pool of β-catenin could only be detected in E-cadherin–/– cells, because in E-cadherin+/+ cells Wnt-induced, membranous β-catenin was concealed by a constitutive junctional pool. Wnt-signaling-dependent dephosphorylated β-catenin colocalized at the plasma membrane with two members of the destruction complex – APC and axin – and the activated Wnt co-receptor LRP6. β-catenin induced through the Wnt receptor complex was significantly more competent transcriptionally than overexpressed β-catenin, both in cultured cells and in early Xenopus embryos. Our data reveal a new step in the processing of the Wnt signal and suggest regulation of signaling output beyond the level of protein accumulation.

A role for maternal β-catenin in early mesoderm induction in Xenopus

Mesoderm formation results from an inducing process that requires maternal and zygotic FGF/MAPK and TGFβ activities, while maternal activation of the Wnt/β‐catenin pathway determines the anterior–dorsal axis. Here, we show a new role of Wnt/β‐catenin signaling in mesoderm induction. We find that maternal β‐catenin signaling is not only active dorsally but also all around the equatorial region, coinciding with the prospective mesoderm. Maternal β‐catenin function is required both for expression of dorsal genes and for activation of MAPK and the mesodermal markers Xbra and eomesodermin. β‐catenin acts in a non‐ cell‐autonomous manner upstream of zygotic FGF and nodal signals. The Wnt/β‐catenin activity in the equatorial region of the early embryo is the first example of a maternally provided mesoderm inducer restricted to the prospective mesoderm.

Looking beyond the Wnt pathway for the deep nature of β‐catenin

After two decades of stardom, one would think that β‐catenin has revealed all of its most intimate details. Yet the essence of its duality has remained mysterious—how can a single protein both be the core link between cadherins and the cytoskeleton, and the nuclear messenger for Wnt signalling? On the basis of the available evidence and on molecular and evolutionary considerations, I propose that β‐catenin was a born nuclear transport receptor, which by interacting with adhesion molecules acquired the property to coordinate nuclear functions with cell–cell adhesion. While Wnt signalling diverted this activity, the original pathway might still function in modern eukaryotes.

Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro, but not in the embryo

Adhesion differences between cell populations are in principle a source of strong morphogenetic forces promoting cell sorting, boundary formation and tissue positioning, and cadherins are main mediators of cell adhesion. However, a direct link between cadherin expression, differential adhesion and morphogenesis has not yet been determined for a specific process in vivo. To identify such a connection, we modulated the expression of C-cadherin in the Xenopus laevis gastrula, and combined this with direct measurements of cell adhesion-related parameters. Our results show that gastrulation is surprisingly tolerant of overall changes in adhesion. Also, as expected, experimentally generated, cadherin-based adhesion differences promote cell sorting in vitro. Importantly, however, such differences do not lead to the sorting of cells in the embryo, showing that differential adhesion is not sufficient to drive morphogenesis in this system. Compensatory recruitment of cadherin protein to contacts between cadherin-deprived and -overexpressing cells could contribute to the prevention of sorting in vivo.

A Molecular Base for Cell Sorting at Embryonic Boundaries: Contact Inhibition of Cadherin Adhesion by Ephrin/Eph-Dependent Contractility

The mechanism responsible for subdividing the embryo into individual tissues is a fundamental, yet still poorly understood, question in developmental biology. Various general hypotheses have been proposed, involving differences in cell adhesion, contractility, or contact-mediated repulsion. However, the key parameter in tissue separation, i.e., the regulation of cadherin-based adhesion at the boundary, has not yet been investigated. We show that cadherin clustering is specifically inhibited at the vertebrate notochord-presomitic mesoderm boundary, preventing formation of adhesive bonds between cells of the two different types. This local regulation depends on differentially expressed ephrins and Eph receptors, which increase cell contractility and generate a membrane blebbing-like behavior along the boundary. Inhibiting myosin activity is sufficient to induce cadherin clustering and formation of stable contacts across the boundary, causing notochord and presomitic tissues to fuse. Local inhibition of cadherin adhesion explains how sharp separation can be achieved in response to cell-cell contact signals.

The cellular basis of tissue separation.

The subdivision of the embryo into physically distinct regions is one of the most fundamental processes in development. General hypotheses for tissue separation based on differential adhesion or tension have been proposed in the past, but with little experimental support. During the last decade, the field has experienced a strong revival, largely driven by renewed interest in biophysical modeling of development. Here, I will discuss the various models of boundary formation and summarize recent studies that have shifted our understanding of the process from the simple juxtaposition of global tissue properties to the characterization of local cellular reactions. Current evidence favors a model whereby separation is controlled by cell surface cues, which, upon cell-cell contact, generate acute changes in cytoskeletal and adhesive properties to inhibit cell mixing, and whereby the integration of multiple local cues may dictate both the global morphogenetic properties of a tissue and its separation from adjacent cell populations.