👤 F Costantini

Axin is a negative regulator of canonical Wnt signaling, which promotes the degradation of beta-catenin, the major effector in this signaling cascade. While many protein-binding domains of Axin have been identified, their significance has not been evaluated in vivo. Here, we report the generation and analysis of mice carrying modified Axin alleles in which either the RGS domain or the six C-terminal amino acids (C6 motif) were deleted. The RGS domain is required for APC-binding, while the C6 motif has been implicated in the activation of c-Jun N-terminal kinase, but is not required for the effects of Axin on the Wnt/beta-catenin pathway, in vitro. Both mutant Axin alleles caused recessive embryonic lethality at E9.5-E10.5, with defects indistinguishable from those caused by a null allele. As Axin-DeltaRGS protein was produced at normal levels, its inability to support embryogenesis confirms the importance of interactions between Axin and APC. In contrast, Axin-DeltaC6 protein was expressed at only 25-30% of the normal level, which may account for the recessive lethality of this allele. Furthermore, many Axin(DeltaC6/DeltaC6) embryos that were heterozygous for a beta-catenin null mutation survived to term, demonstrating that early lethality was due to failure to negatively regulate beta-catenin. Show less

Ablations of the Axin family genes demonstrated that they modulate Wnt signaling in key processes of mammalian development. The ubiquitously expressed Axin1 plays an important role in formation of the embryonic neural axis, while Axin2 is essential for craniofacial skeletogenesis. Although Axin2 is also highly expressed during early neural development, including the neural tube and neural crest, it is not essential for these processes, apparently due to functional redundancy with Axin1. To further investigate the role of Wnt signaling during early neural development, and its potential regulation by Axins, we developed a mouse model for conditional gene activation in the Axin2-expressing domains. We show that gene expression can be successfully targeted to the Axin2-expressing cells in a spatially and temporally specific fashion. High levels of Axin in this domain induce a region-specific effect on the patterning of neural tube. In the mutant embryos, only the development of midbrain is severely impaired even though the transgene is expressed throughout the neural tube. Axin apparently regulates beta-catenin in coordinating cell cycle progression, cell adhesion and survival of neuroepithelial precursors during development of ventricles. Our data support the conclusion that the development of embryonic neural axis is highly sensitive to the level of Wnt signaling. Show less

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of beta-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of beta-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis. Show less

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling. Show less

Disheveled blocks the degradation of beta-catenin in response to Wnt signal by interacting with the scaffolding protein, Axin. To define this interaction in detail we undertook a mutational and binding analysis of the murine Axin and Disheveled proteins. The DIX domain of Axin was found to be important for association with Disheveled and two other regions of Axin (between residues 1-168 and 600-810) were identified that can promote the association of Axin and Disheveled. We found that the DIX domain of Disheveled is critical for association with Axin in vivo and for Disheveled activity. The Disheveled DIX domain controlled the ability of Disheveled to induce the accumulation of cytosolic beta-catenin whereas the PDZ domain was not essential to this function. Show less

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity. Show less

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians. Show less