David Kimelman

Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1.

Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3β (GSK), leading to the accumulation of cytosolic β‐catenin and activation of TCF/LEF‐1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF‐1, we investigated the roles of Axin and Frat1 in Wnt‐mediated activation of LEF‐1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl‐binding domain of either Frat1 or Axin was able to inhibit Wnt‐1‐induced LEF‐1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt‐1 appeared to promote the disintegration of the Frat1–Dvl–GSK–Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1‐mediated dissociation of GSK from Axin.

Crystal Structure of a β-Catenin/Tcf Complex

Abstract The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear β-catenin/Tcf complex, activating Wnt target genes. A crystal structure of β-catenin bound to the β-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to β-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in β-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.

GBP, an Inhibitor of GSK-3, Is Implicated in Xenopus Development and Oncogenesis

Dorsal accumulation of beta-catenin in early Xenopus embryos is required for body axis formation. Recent evidence indicates that beta-catenin is dorsally stabilized by the localized inhibition of the kinase Xgsk-3, utilizing a novel Wnt ligand-independent mechanism. Using a two-hybrid screen, we identified GBP, a maternal Xgsk-3-binding protein that is homologous to a T cell protooncogene in three well-conserved domains. GBP inhibits in vivo phosphorylation by Xgsk-3, and ectopic GBP expression induces an axis by stabilizing beta-catenin within Xenopus embryos. Importantly, antisense oligonucleotide depletion of the maternal GBP mRNA demonstrates that GBP is required for the establishment of the dorsal-ventral axis in Xenopus embryos. Our results define a family of GSK-3-binding proteins with roles in development and cell proliferation.

FROM CORTICAL ROTATION TO ORGANIZER GENE EXPRESSION : TOWARD A MOLECULAR EXPLANATION OF AXIS SPECIFICATION IN XENOPUS

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal‐determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt‐1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that β‐catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, β‐catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of β‐catenin and Tcf‐3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf‐3 likely works in the absence of β‐catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of β‐catenin establishes a range of subsequent dorsoventral asymmetries in the embryo. BioEssays 20:536–545, 1998.© 1998 John Wiley & Sons Inc.

ArticleCrystal Structure of a β-Catenin/Tcf Complex

Abstract The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear β-catenin/Tcf complex, activating Wnt target genes. A crystal structure of β-catenin bound to the β-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to β-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in β-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.

Activin-mediated mesoderm induction requires FGF.

The early patterning of mesoderm in the Xenopus embryo requires signals from several intercellular factors, including mesoderm-inducing agents that belong to the fibroblast growth factor (FGF) and TGF-beta families. In animal hemisphere explants (animal caps), basic FGF and the TGF-beta family member activin are capable of converting pre-ectodermal cells to a mesodermal fate, although activin is much more effective at inducing dorsal and anterior mesoderm than is basic FGF. Using a dominant-negative form of the Xenopus type 1 FGF receptor, we show that an FGF signal is required for the full induction of mesoderm by activin. Animal caps isolated from embryos that have been injected with the truncated FGF receptor and cultured with activin do not extend and the induction of some genes, including cardiac actin and Xbra, is greatly diminished, while the induction of other genes, including the head organizer-specific genes gsc and Xlim-1, is less sensitive. These results are consistent with the phenotype of the truncated FGF receptor-injected embryo and imply that the activin induction of mesoderm depends on FGF, with some genes requiring a higher level of FGF signaling than others.

A Dominant-Negative Form of p63 Is Required for Epidermal Proliferation in Zebrafish

Epidermal stem cells play a critical role in producing the multilayered vertebrate skin. Products of the p63 gene not only mark the epidermal stem cells, but also are absolutely required for the formation of mammalian epidermis. We find that early zebrafish embryos express a dominant-negative form of p63 (DeltaNp63), which accumulates in the nucleus just as epidermal growth begins. Using antisense morpholino oligonucleotides, we show that DeltaNp63 is needed for epidermal growth and limb development and is specifically required for the proliferation of epidermal cells by inhibiting p53 activity. While the structure of fish epidermis is very different from that of higher vertebrates, our study shows that DeltaNp63 has essential and ancient role in the development of skin.

Mesoderm induction: from caps to chips.

Vertebrate mesoderm induction is one of the classical problems in developmental biology. Various developmental biology approaches, particularly in Xenopus and zebrafish, have identified many of the key factors that are involved in this process and have provided major insights into how these factors interact as part of a signalling and transcription-factor network. These data are beginning to be refined by high-throughput approaches such as microarray assays. Future challenges include understanding how the prospective mesodermal cells integrate the various signals they receive and how they resolve this information to regulate their morphogenetic behaviours and cell-fate decisions.

Vertebrate mesendoderm induction and patterning.

Many of the key molecular events underlying the induction and patterning of the vertebrate mesoderm and endoderm have recently been elucidated. T-box transcription factors and TGF-beta and Wnt signaling pathways play crucial roles in the initial induction of the mesendoderm and the subdivision of the posterior mesoderm into rostral and caudal domains.