Protein interactions of Axin family members: Dishevelled proteins

Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, ß-catenin, leading to the nuclear accumulation of ß-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. A novel zebrafish gene encoding Ccd1 has been identified that possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 forms homomeric and heteromeric complexes with Dvl and Axin and activates TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos leads to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) causes the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 is inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype is rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning (Shiomi, 2003).

Wnt proteins transduce their signals through Dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3beta (GSK), leading to the accumulation of cytosolic beta-catenin and activation of TCF/LEF-1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF-1, the roles of Axin and Frat1 (a novel proto-oncogene) in Wnt-mediated activation of LEF-1 were examined in mammalian cells. 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. Dvl, Axin and GSK can form a ternary complex bridged by Axin, and Frat1 can be recruited into this complex, probably by Dvl. The observation that the Dvl-binding domain of either Frat1 or Axin is 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 (Li, 1999).

Axin has been shown to interact with the Wnt signaling components GSK, APC and beta-catenin. It has been proposed that Axin functions as a scaffold protein and that the binding of GSK and beta-catenin to Axin may facilitate the phosphorylation of beta-catenin by GSK. The phosphorylation is thought to contribute to the destabilization of beta-catenin. To investigate the effects of Axin and its various domains on Wnt-1-induced activation of LEF-1, Axin or its mutants was co-expressed with Wnt-1 and LEF-1 reporter plasmids in NIH 3T3 cells. Wnt-1, when co-expressed with LEF-1 in NIH 3T3 cells, leads to marked increases in transcription of the luciferase reporter gene under the control of a LEF-1 regulatory sequence. These results suggest that Wnt-1 is able to activate LEF-1 through NIH 3T3 cell endogenous signaling components. The expression of Axin inhibits Wnt-1-stimulated luciferase activity, which is consistent with previous findings that Axin antagonizes Wnt-1's effects. Two Axin mutants were generated to assess the roles of specific Axin domains in inhibiting Wnt-1 action. The mutants are AxinC, containing the C-terminal DIX domain; AxinN, a deletion of the DIX domain retaining the APC, GSK and beta-catenin-binding domains. AxinN inhibits Wnt-1-mediated LEF-1 activation. However, AxinC is also able to inhibit Wnt-1-induced increases in luciferase activity in co-transfected 3T3 cells. Similar inhibitory effects of AxinC on Wnt-1-induced LEF-1 activation have also been observed in COS-7 and HEK cells (Li, 1999).

To confirm that AxinC blocks the activation of the reporter system by secreted Wnt proteins, a paracrine paradigm was used where the Wnt-1 cDNA and LEF-1 reporter gene were transfected into two separate groups of COS-7 cells. These two groups of cells were combined after transfection, and luciferase activities were determined after the cells were co-cultured for 24 h. Since Wnt-1 and the reporter gene are produced in different cells, the effect of Wnt-1 can be attributed only to the interaction of secreted Wnt-1 proteins with cell surface receptors on cells containing the reporter gene. The effects of Axin and its two mutants were tested in the paracrine paradigm. When either Axin or its mutants are co-transfected with the LEF reporter plasmid, they are able to inhibit the effect of Wnt-1 produced by separate cells. This result confirms that AxinC as well as Axin and AxinN can inhibit the effect of secreted Wnt (Li, 1999).

The inhibitory effect of AxinC suggests that it may interact with proteins that are involved in Wnt-1 signaling. Since the binding sites for beta-catenin, GSK and APC are not included in AxinC, a test was performed to see whether AxinC interacts with Dvl using an immunoprecipitation approach. COS-7 cells were co-transfected with cDNAs encoding hemagglutinin (HA)-tagged AxinC and Flag-tagged Dvl. The next day, Dvl-Flag was immunoprecipitated with an anti-Flag antibody, and the immunocomplexes were detected by Western blot analysis using an anti-HA antibody. AxinC is detected in the immunocomplexes, demonstrating that AxinC binds to Dvl. To determine to which portions of the Dvl molecule AxinC binds, AxinC-HA was co-transfected with a number of Dvl mutants: either DvlN-Flag, DvlPDZ-Flag or DvlC-Flag. Only DvlN-Flag was co-immunoprecipitated with AxinC-HA, indicating that DvlN, which contains a DIX domain, interacts with AxinC. The interaction between AxinC and DvlN was also confirmed using purified recombinant proteins from Escherichia coli. Since there is no Wnt signaling pathway in E.coli, the interaction between AxinC and DvlN should be direct (Li, 1999).

The interaction between AxinC and Dvl suggests that full-length Axin may also bind to Dvl. Dvl has been shown to interact with Axin in a co-immunoprecipitation experiment. The interactions between endogenous Dvl and Axin proteins were also detected; Axin is detected in immunocomplexes pulled down by the anti-Dvl-3 monoclonal antibody. To test whether Axin binds only to DvlN, Axin was co-expressed with DvlPDZ or DvlC. Interestingly, Axin binds not only to DvlN but also to DvlPDZ. Axin does not bind to DvlC. The Axin mutants, AxinN and AxinN1, can be co-immunoprecipitated with DvlPDZ but not with DvlC or DvlN. All of these results indicate that the binding of Axin to Dvl is probably mediated by two interactions; one between DvlN and AxinC and the other between DvlPDZ and an N-terminal sequence of Axin (Li, 1999).

Axin has been shown to bind to GSK. Because Axin sequences involved in interactions with GSK and Dvl do not overlap, it is possible that Axin, Dvl and GSK form a ternary complex. When Myc-tagged GSK is co-expressed with Dvl-HA, GSK and Dvl are not co-immunoprecipitated, suggesting that GSK does not interact directly with Dvl. However, co-expression of Axin allows GSK and Dvl to be co-immunoprecipitated. Thus, GSK, Dvl and Axin can form a complex, which is probably bridged by Axin. In addition, endogenous beta-catenin was detected in the immunocomplexes pulled down via Dvl. This is probably due to the interaction between Axin and beta-catenin (Li, 1999).

A GSK inhibitor, GBP, was identified recently in Xenopus based on its ability to bind to GSK and inhibit GSK-mediated phosphorylation of a protein substrate (Yost, 1998). The mammalian homolog of GBP, named Frat1, was cloned independently for its tumor-promoting activity in lymphocytes (Jonkers, 1997). Ectopic overexpression of GBP or the C-terminal GSK-binding domain of GBP or human FRAT2 can mimic Wnt's effects in Xenopus (Yost, 1998). It was confirmed that endogenous GSK is bound to endogenous Frat in mammalian cells. In addition, expression of Frat1 stimulates LEF-1-dependent transcriptional activity in COS-7 cells, indicating that Frat1 can also mimic Wnt's effect in mammalian cells. The observation that the GSK-interacting site deletion mutant of Frat1 (FratN) could inhibit Wnt-1-mediated LEF-1 activation suggests that Frat1 may be a mediator of Wnt-1 signaling (Li, 1999).

The dominant-negative effect of FratN implies that FratN may interact with intracellular factors in the Wnt signaling pathway. Whether Frat1 interacts with Dvl and Axin was investigated. Frat1 and FratN can be co-immunoprecipitated with Dvl, but not with Axin. Co-immunoprecipitation of Frat1 and Dvl mutants was performed to delineate the Dvl sequences involved in Frat1 binding. DvlPDZ, but not DvlN, binds to Frat1 and FratN. Thus, the N-terminal half of Frat1 interacts with Dvl via binding to the PDZ domain of Dvl. The interaction between FratN and DvlPDZ was also confirmed using recombinant proteins purified from E.coli. Importantly, the endogenous GSK and Dvl proteins are bound to the endogenous Frat1, demonstrating that these interactions occur even without overexpression (Li, 1999).

Dsh (Xdsh) was examined in embryos using epitope-tagged constructs in immunoprecipitation assays to confirm that Frat1 and Dvl interact directly; to investigate whether their interaction might play a role in Wnt signaling in an intact vertebrate system, the ability of Xenopus Frat1 ortholog, GBP could bind to Xenopus Dsh (Xdsh). GBP-Flag immunoprecipitates Xdsh-Myc only in the presence of Xgsk-3-Myc. To determine if the binding of GBP to Xgsk-3 is required for the GBP-Xdsh interaction, a GBP mutant that does not bind Xgsk-3 (Yost, 1998) was immunoprecipitated after co-expression with Xdsh-Myc and Xgsk-3-Myc. Xdsh-Myc is precipitated by the mutant GBP-Flag in a manner dependent upon Xgsk-3-Myc, even though no binding of the mutant GBP-Flag and Xgsk-3-Myc is observed. It has been concluded that the direct interaction of Dvl and Frat is conserved in Xenopus and that this interaction may require a GSK-dependent phosphorylation event (Li, 1999).

The inhibition of Wnt-1-mediated LEF-1 activation by Axin C and FratN implies that the interactions between Dvl and Axin and between Dvl and Frat1 may be required for Wnt-1 signaling. A possible model to explain the involvement of these interactions in this signaling pathway is that Dvl, Axin, Frat1 and GSK form a complex. Although Axin may compete with Frat1 for the Dvl PDZ domain, Axin and Frat1 may still be able simultaneously to bind to Dvl, because Axin can still bind to Dvl via the DIX domain interaction. Additionally, Dvl and GSK bind to different portions of Frat1, and Dvl, Axin and GSK have been shown to form a complex. Therefore, it is theoretically possible that Frat1, Dvl, GSK and Axin form a bigger complex. To test this possibility, combinations of Axin-Myc, GSK-Myc and Dvl-HA were coexpressed along with Frat1-Flag. When Frat1-Flag is co-expressed with Axin-HA even in the presence of GSK or Dvl, little Axin is detected in the immunocomplexes precipitated with the anti-Flag antibody. This indicates that Frat1 has little affinity for Axin and that Frat1 and Axin may not form a ternary complex with Dvl or GSK. Frat1 is co-expressed with Dvl and GSK, and it was found that Dvl could not precipitate GSK in the presence of Frat1, or vice versa. Thus, unlike Axin, Frat1 could not bridge the formation of a stable complex of GSK-Frat1-Dvl in the assay system used. However, when all four proteins are expressed together, Axin is able to be immunoprecipitated by Frat1-Flag, and increased levels of Dvl and GSK proteins are also detected in the immunocomplexes. These results indicate that Frat1 may form a quaternary complex with GSK, Dvl and Axin. However, when Dvl is used to pull down the complexes, co-expression of Frat1 reduces the levels of the Axin and GSK in the immunocomplexes. These results are interpreted to suggest that GSK, Dvl and Axin may form a more stable complex than the quaternary complex that contains Frat1 (Li, 1999).

Having established that Frat1 forms a complex with Dvl-Axin-GSK, it was then of interest know the effect of Wnt on the formation of the complex. Wnt-1 was co-expressed with Frat1, Dvl, Axin and GSK in COS-7 cells. The expression of Wnt-1 significantly decreases the levels of Axin and Dvl in the immunocomplexes pulled down via Frat1, while the levels of GSK in the immunocomplexes do not seem to change when compared with the absence of Wnt-1. The simplest explanation of this observation is that Wnt may promote the disintegration of the quaternary complex. Because the level of GSK associated with Frat1 remains the same in the absence and presence of Wnt-1, GSK maintains its association with Frat1 after the disintegration of the complex (Li, 1999).

The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 has been found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bind one another. Furthermore, Dvl-1 forms a homo-oligomer. Axin also forms a homo-oligomer, and its DIX domain is necessary. The N-terminal region of Dvl-1, including its DIX domain, bind to Axin directly. Dvl-1 inhibits Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin; the DIX domain of Dvl-1 is required for this inhibitory activity. Expression of Dvl-1 in L cells induces the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolishes this activity. Although expression of Axin in SW480 cells causes the degradation of beta-catenin and reduces the cell growth rate, expression of an Axin mutant that lacks the DIX domain does not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin (Kishida, 1999).

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. An exploration was carried out of the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that functions upstream of GSK-3beta. Expression of Dvl-2 activates TCF-dependent transcription. This is blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activates transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevents GSK-3beta binding also generates a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localize within expressing cells. When Dvl-2 localization is altered using a C-terminal CAAX motif, Axin is also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggests that Dvl-association determinants within Axin are contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta (Smalley, 1999).

The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bind one another. Furthermore, Dvl-1 forms a homo-oligomer. Axin also forms a homo-oligomer, and its DIX domain is necessary for homo-oligomerization. The N-terminal region of Dvl-1, including its DIX domain, bind to Axin directly. Dvl-1 inhibits Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 is required for this inhibitory activity. Expression of Dvl-1 in L cells induces the nuclear accumulation of beta-catenin; deletion of the DIX domain abolishes this activity. Although expression of Axin in SW480 cells causes the degradation of beta-catenin and reduces the cell growth rate, expression of an Axin mutant that lacks the DIX domain does not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin (Kishida, 1999).

Protein interactions of Axin family members: Casein kinase Iepsilon

Wnt and its intracellular effector beta-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, casein kinase Iepsilon (CKIepsilon) has been identified. CKIepsilon mimics Wnt in inducing a secondary axis in Xenopus, stabilizing beta-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIepsilon by kinase-defective CKIepsilon or CKIepsilon antisense-oligonucleotides attenuates Wnt signaling. CKIepsilon is in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIepsilon appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate beta-catenin (Sakanaka, 1999).

Some of the downstream molecules in the Wnt pathway have been shown to form complexes containing negative regulators (GSK-3beta, axin, and adenomatous polyposis coli tumor suppressor protein) and a positive regulator (beta-catenin) in vivo. The possibility that CKIepsilon is also in a complex with these molecules was examined. Axin, a negative regulator of the Wnt pathway, binds to CKIepsilon. Endogenous CKIepsilon coimmunoprecipitates with overexpressed axin. Because the C-terminal domain is unique for CKIepsilon/delta isoforms, a test was performed to see whether deletion of this domain alters activity. Binding of axin to DeltaC-CKIepsilon and DeltaC-KN-CKIepsilon (KN stands for Kinase Negative) is much reduced compared to wild-type CKIepsilon and KN-CKIepsilon. These results suggest that the C-terminal domain of CKIepsilon is important for its interaction with axin, which may be the reason that DeltaC-CKIepsilon and CKIalpha did not activate the Wnt pathway in either Xenopus or mammalian cells. To further study the complex of CKIepsilon with axin and GSK-3beta, it was shown that GSK-3beta coimmunoprecipitates with CKIepsilon, but much less with DeltaC-CKIepsilon, and only in the presence of axin. This finding suggests that CKIepsilon is a positive regulatory molecule in the Wnt pathway and its interaction through its C terminus with the axin-GSK-3beta complex is likely to be important for its activity. Furthermore, endogenous CKIepsilon has been detected in the complex with overexpressed Dvl3, an upstream molecule of the axin-GSK-3beta complex (Sakanaka, 1999).

Protein interactions of Axin family members: Regulation of JNK pathway

Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and the structural requirement for this activation have been determined. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that is termed MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, that is, binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. The results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade (Zhang, 1999).

Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of ß-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. This study describes the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the ß-catenin degradation complex that consists of Axin/Conductin and GSK3ß and allows efficient phosphorylation of ß-catenin, thereby inhibiting ß-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on ß-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. These data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical ß-catenin pathway and stimulates signaling via JNK (Schwarz-Romond, 2002).

Compared with Diversin, Diego contains six ankyrin repeats instead of eight, and has 35% amino acid sequence identity within the ankyrin repeats, but little identity (18%) in the residual domains. Diego acts downstream of Frizzled and controls planar polarization of epithelial cells in the eye and wing; such polarization depends upon JNK activity. Coimmunoprecipitation experiments show that Diego interacts with mammalian CKIepsilon but not with Drosophila Axin. Further, Diversin stimulates JNK-dependent transcription in 293 cells. Diversin also promotes JNK activation that is induced by Dishevelled or Wnt11. In zebrafish embryos, injection of Diversin mRNA results in abnormal gastrulation movements, that is, the convergence and extension of injected embryos are defective. Low amounts of Diversin mRNA induce failure of gastrulation movements, but little ventralization, whereas at higher dosages, ventralization is predominant. Similarly, injection of low amounts of Diversin MOs also interfers with gastrulation movements and induces a general undulation of the embryo along its anterior-posterior axis, as revealed by in situ hybridization for myoD at the 5-10 somite stage. Thus, both activation and inhibition of the Wnt/JNK pathway perturb gastrulation movements. Injection of Diego mRNA also induces deficits in convergence and extension movements, but does not affect axis formation at any concentration tested. Diego could not rescue the Diversin MO-induced dorsalization. Taken together, these data indicate that Diversin functions both in the Wnt/ß-catenin and the Wnt/JNK pathway, and that Diego acts only in the Wnt/JNK pathway. Diego and Diversin are therefore structurally and functionally not entirely homologous (Schwarz-Romond, 2002).

Biochemical analysis allows the molecular mechanism by which Diversin functions in the canonical Wnt pathway to be assigned. Efficient ß-catenin degradation requires a two-step mechanism, a priming phosporylation at Ser 45 catalyzed by CKIepsilon or CKIalpha, and subsequent phosphorylation on three equally spaced serine/threonine residues by GSK3ß. Diversin recruits the priming kinase CKIepsilon to the Axin/Conductin-GSK3ß complex. Separate domains of Diversin, the central and C-terminal regions, mediate these two interactions. Both Diversin and GSK3ß bind simultaneously to dimeric Axin/Conductin, and they use identical binding sites. Diversin-mediated recruitment of CKIepsilon allows phosporylation of Ser 45 of ß-catenin, thus creating a classical GSK3ß recognition motif and initiating the subsequent phosphorylation cascade. A minimal fusion molecule that contains the catalytic domain of CKIepsilon and the Axin/Conductin-binding domain of Diversin is fully functional in ß-catenin signaling, showing the role of Diversin as a molecular linker. Diversin is inactive in the presence of Frat-1/GBP, which displaces GSK3ß, showing the importance of GSK3ß in the complex. Taken together, these data demonstrate that Diversin functions in the canonical Wnt pathway by engaging CKIepsilon to the ß-catenin degradation complex, and allows priming phosphorylation and degradation of ß-catenin (Schwarz-Romond, 2002).

Diversin activates the JNK branch of the Wnt-signaling pathway, that controls the establishment of planar cell polarity in Drosophila and gastrulation movements in vertebrates. In zebrafish, inhibition and overexpression of Diversin cause defects in gastrulation movements, that is, a reduction in body length and undulation of the body axis -- these defects are similar to those observed in pipetail (Wnt5a) mutants. Thus, Diversin controls gastrulation movements, as does the Drosophila protein Diego. However, Diego is only in part a functional homolog of Diversin, because it does not interact with Drosophila Axin and has not been implicated in Wnt/ß-catenin signaling. Specific to Diversin in vertebrates is its role at a branchpoint of intracellular Wnt signaling, where it represses the canonical Wnt/ß-catenin pathway and simultaneously activates the JNK pathway (Schwarz-Romond, 2002).

Axin is a scaffold protein that controls multiple important pathways, including the canonical Wnt pathway and JNK signaling. An Axin-interacting protein, Aida, blocks Axin-mediated JNK activation by disrupting Axin homodimerization. During investigation of in vivo functions of Axin/JNK signaling and aida in development, it was found that Axin, besides ventralizing activity by facilitating β-catenin degradation, possesses a dorsalizing activity that is mediated by Axin-induced JNK activation. This dorsalizing activity is repressed when aida is overexpressed in zebrafish embryos. Whereas Aida-MO injection leads to dorsalized embryos, JNK-MO and MKK4-MO can ventralize embryos. The anti-dorsalization activity of aida is conferred by its ability to block Axin-mediated JNK activity. It is further demonstrated that dorsoventral patterning regulated by Axin/JNK signaling is independent of maternal or zygotic Wnt signaling. Thus a dorsalization pathway has been identified that is exerted by Axin/JNK signaling and its inhibitor Aida during vertebrate embryogenesis (Rui, 2007).

Protein interactions of Axin family members: Disabled-2

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, the N-terminal domain of Dab2 has been shown to interact with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse fibroblasts attenuates canonical Wnt/ß-catenin-mediated signaling, including accumulation of ß-catenin, activation of ß-catenin/T-cell-specific factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a time-dependent dissociation of endogenous Dab2-Dvl-3 and Dvl-3-axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2-Dvl-3 association and subsequent loss of Dvl-3-axin interactions. In addition, Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo fibroblasts that lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear ß-catenin and cyclin D1 protein levels. Based on these results, it is proposed that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the ß-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor (Hocevar, 2003).

Axin and GSK3-β control Smad3 protein stability and modulate TGF-β signaling

The broad range of biological responses elicited by transforming growth factor-β (TGF-β) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. This stud shows that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-β (GSK3-β). Smad3 physically interacts with Axin and GSK3-β only in the absence of TGF-β. Reduction in the expression or activity of Axin/GSK3-β leads to increased Smad3 stability and transcriptional activity without affecting TGF-β receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-β. Mechanistically, Axin facilitates GSK3-β-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β, and suggest a new function of the Axin/GSK3-β complex in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression (Guo, 2008).

Axin mutation

During Caenorhabditis elegans vulval development, activation of receptor tyrosine kinase/Ras and Notch signaling pathways causes three vulval precursor cells (VPCs) to adopt induced cell fates. A Wnt signaling pathway is also activated in cell fate specification within the VPCs, via regulation of the Hox gene lin-39. Either mutation of pry-1 or expression of an activated BAR-1 ß-catenin protein causes an Overinduced phenotype, in which greater than three VPCs adopt induced cell fates. This indicates that pry-1, which encodes a C. elegans axin homolog, acts as a negative regulator of Wnt signaling in the VPCs. Loss of activity of the APC homolog apr-1 increases the penetrance of this Overinduced phenotype, suggesting that APR-1 may play a negative role in Wnt signaling in this process in C. elegans, similar to APC proteins in other systems. The Overinduced phenotype is suppressed by reduction of function of the genes pop-1 TCF and lin-39 Hox. Surprisingly, the Overinduced phenotype caused by hyperactivated Wnt signaling is not dependent on signaling through the Ras pathway. These data suggest that hyperactivation of Wnt signaling is sufficient to cause VPCs to adopt induced fates and that a canonical Wnt pathway may play an important role during C. elegans vulval induction (Gleason, 2002).

How does overactivation of the Wnt pathway lead to extra vulval induction, even when activation of the Ras pathway has been compromised? It is believed that overexpression of one or more Wnt target genes causes these extra inductions. Currently, the only known target of the Wnt pathway in the VPCs is lin-39. It is also known that LIN-39 protein levels increase in P6.p in a Ras pathway-dependent manner. This suggests that LIN-39 may function in the adoption of induced vulval fates when expressed at higher levels. Therefore, it is proposed that overactivation of the Wnt pathway may cause levels of LIN-39 to exceed some threshold in P3.p, P4.p, and P8.p, causing those cells to sometimes adopt induced cell fates. In this model, the Wnt pathway normally plays a permissive role in the maintenance of LIN-39 expression in all VPCs to prevent these cells from adopting the Fused fate, but overactivation of the Wnt pathway can phenocopy a cell in which both the Wnt and Ras pathways are active. This would be consistent with the dependence of the Overinduced phenotype on lin-39 activity in these experiments, and the independence of that phenotype with regard to Ras signaling. Preliminary experiments have suggested that expression of high amounts of LIN-39 alone in L2/L3 larvae is not sufficient to phenocopy the Overinduced phenotype described here. This suggests that there may be additional targets of the Wnt pathway that contribute to the adoption of induced fates when Wnt signaling is overactivated. Future experiments will attempt to identify target genes regulated by overactivation of the Wnt pathway, as well as to determine the functional relationship between factors acting downstream of Wnt signaling, such as LIN-39, and transcription factors known to act downstream of Ras signaling during vulval induction, such as the winged helix protein LIN-31 and the Ets domain protein LIN-1 (Gleason, 2002).

masterblind (mbl) is a zebrafish mutation characterized by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. Inhibition of Gsk3ß in zebrafish embryos either by overexpression of dominant negative dn gsk3ß mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos show increased sensitivity to inhibition of GSK3ß by lithium or dn Xgsk3ß that leads to the loss of eyes. Overexpression of gsk3ß mRNA rescues eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1, which delineates the telencephalon, is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain is not restored upon overexpression of gsk3ß mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3ß that binds and phosphorylates ß-catenin, thereby destabilizing it. mbl maps on LG 3 close to a candidate gene axin1. In mbl a point mutation was detected in the conserved minimal Gsk3ß-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescues mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos results in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. It is suggested that the function of Axin1/Mbl protein is to antagonize the Wnt signal and in doing so to establish and maintain the most anterior CNS. These findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate (van de Water, 2001).

Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, the role of the Wnt/Axin/ß-catenin pathway in this process was explored. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and data is presented suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Smad2 homolog Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. It is suggested that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information (Kapsimali, 2004).

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. 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, intramembranous ossification was examined 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, these 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 (Yu, 2005).

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 beta-catenin. Axin has been considered so far as a cytoplasmic protein. This study shows 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. The sequence requirement for export have been characterized and two export domains, which do not contain classical nuclear export consensus sequences, were identified; Axin binds directly to the export factor CRM1 in the presence of RanGTP (Wiechens, 2004).

Developmental roles of vertebrate axins

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. Mouse Fused is not to be confused with Drosophila fused, which mediates the Hedgehog signal to activate decapentaplegic. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling: see Drosophila Loco) and Dishevelled proteins. Axin blocks the stimulation of the Wnt signaling pathway, regulating an early step in axis formation downstream of GSK-3, the mammalian homolog of Drosophila Shaggy. The Axin sequence homologous to the RGS domain is found between amino acids 213 and 338. A C-terminal 51 amino acid segment is 40% identical to a conserved sequence near the N terminus of Drosophila Dsh and its vertebrate homologs. 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. Ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Based on its ability to block ectopic axis formation in Xenopus embryos by Xwnt8, Dishevelled, or dominant negative GSK-3, Axin appears to negatively regulate signaling through the Wnt pathway, either at the level of GSK-3 or further downstream. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in both mammals and amphibians (Zeng, 1997).

Axin has been identified as a regulator of embryonic axis induction in vertebrates; it inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos have indicated that Axin functions downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin; subsequent studies have shown that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). The roles of different Axin domains have been examined with respect to the effects on axis formation and beta-catenin levels. 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 are required for targeting to either of these sites, whereas COOH-terminal sequences increase localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) reveals strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity (Fagotto, 1999).

In Xenopus, the dorsal factor in the vegetal cortical cytoplasm (VCC) of the egg is responsible for axis formation of the embryo. Previous studies have shown that VCC dorsal factor has properties similar to activators of the Wnt/beta-catenin-signaling pathway. In this study, the relationship of components of the pathway to the VCC dorsal factor was examined. Initially examined was whether beta-catenin protein, which is known to be localized on the dorsal side of early embryos, accounts for the dorsal axis activity of VCC. Reduction of beta-catenin mRNA and protein in oocytes does not diminish the activity of VCC to induce a secondary axis in recipient embryos. The amount of beta-catenin protein is not enriched in VCC compared to animal cortical cytoplasm, which has no dorsal axis activity. These results indicate that beta-catenin is unlikely to be the VCC dorsal axis factor. The next series of studies looked at the effects of four Wnt-pathway-interfering constructs (dominant-negative Xdsh, XGSK3, Axin, and dominant-negative XTcf3) on the ability of VCC to induce expression of the early Wnt target genes, Siamois and Xnr3. The activity of VCC is inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. VCC decreases neither the amount nor the activity of exogenous XGSK3, suggesting that the VCC dorsal factor does not act by affecting XGSK3 directly. Finally, six Wnt-pathway activating constructs (Xwnt8, Xdsh, dominant negative XGSK3, dominant negative Axin, XAPC and beta-catenin) were tested for their responses to the four Wnt-pathway-interfering constructs. Only XAPC exhibits the same responses as VCC; it is inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. Although the connection between XAPC and the VCC dorsal factor is not yet clear, the fact that APC binds Axin suggests that the VCC dorsal factor might act on Axin rather than XGSK3 (Marikawa, 1999).

Regulation of the stability of ß-catenin protein is a critical role of Wnt signaling cascades. In early Xenopus development, dorsal axis specification depends on regulation of ß-catenin by both cytoplasmic and nuclear mechanisms. While the cytoplasmic protein axin is known as a key component of the cytoplasmic ß-catenin degradation complex, loss-of-function studies are needed to establish whether it is required for dorso-ventral patterning in the embryo, and to test where in the embryo it carries out its function. Embryos lacking maternal axin protein have increased levels of soluble ß-catenin protein and increased nuclear localization of ß-catenin in ventral nuclei at the blastula stage. These embryos gastrulate abnormally and develop with excessive notochord and head structures, and reduced tail and ventral components. They show increased expression of dorsal markers, including siamois, Xnr3, chordin, gsc, Xhex, and Otx2, decreased expression of Xwnt 8 and Xbra, and little alteration of BMP4 and Xvent1 and -2 mRNA levels. The ventral halves of axin-depleted embryos at the gastrula stage have dramatically increased levels of chordin expression, and severely decreased levels of Xwnt 8 mRNA expression, while BMP4 transcript levels are only slightly reduced. This dorso-anterior phenotype is rescued by axin mRNA injected into the vegetal pole of axin-depleted oocytes before fertilization. Interestingly, the phenotype was rescued by ventral but not dorsal injection of axin mRNA, at the 4-cell stage, although dorsal injection into wild-type embryos does cause ventralization. These results show directly that the localized ventral activity of maternal axin is critical for the correct patterning of the early Xenopus embryo (Kofron, 2001).

In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/ß-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, it has been shown that Wnt/ß-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/ß-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/ß-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/ß-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/ß-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/ß-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration (Stoick-Cooper, 2007).

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, a mouse model was developed for conditional gene activation in the Axin2-expressing domains. 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 β-catenin in coordinating cell cycle progression, cell adhesion and survival of neuroepithelial precursors during development of ventricles. These data support the conclusion that the development of embryonic neural axis is highly sensitive to the level of Wnt signaling (Yu, 2007).

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. Overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. It is suggested that Wnt signaling regulates the establishment of the bilateral repression. A second role was identified for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, it was shown that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries (Carl, 2007).

Structural and functional asymmetries are common features of the nervous systems of both invertebrates and vertebrates. The best described neuroanatomical asymmetries in vertebrates are found in the diencephalic epithalamus, where both the habenulae and the dorsally adjacent pineal complex are lateralized in many species. The epithalamus is part of a conserved output pathway of the limbic system, connecting telencephalic nuclei to the interpeduncular nucleus (IPN) in the ventral midbrain (Carl, 2007).

During early development in zebrafish, bilaterally located parapineal cells migrate leftward from the pineal complex to form a left-sided nucleus that sends ipsilateral axonal projections to the left habenula. The paired habenular nuclei themselves show various asymmetries, including differences in gene expression, subnuclear regionalization, timing of neuronal differentiation, and neuropil organization. Left-right asymmetries in habenular neuronal organization are converted into a dorsal-ventral asymmetry in the targeting of the habenular axons in the midbrain IPN, with left-sided habenular axons predominantly innervating the dorsal IPN and right-sided axons projecting to the ventral IPN (Carl, 2007).

The parapineal influences the elaboration of habenular asymmetries. For instance, the parapineal modulates gene expression in the left habenula, and ablation of parapineal cells results in the left habenula adopting some right-sided character. In contrast, ablation of left-sided habenula precursors can influence the orientation of parapineal migration. Taken together, these results suggest that there is communication between the various structures in the epithalamus to ensure coordinated and consistent elaboration of lateralized neuroanatomical asymmetries (Carl, 2007).

The earliest known indication of brain asymmetry in zebrafish is the expression of Nodal pathway genes within the left epithalamus from about 18 hpf. Epithalamic Nodal signaling influences the laterality of the habenulae and parapineal, but asymmetry per se appears to be established independently of this pathway. As the Nodal pathway is activated unilaterally in the epitahalmus, other mechanisms must act upstream to initiate this asymmetry. Within the lateral plate mesoderm (LPM), Nodal signaling has evolutionarily conserved roles in the development of asymmetries, and in zebrafish, it appears that activation of Nodal pathway genes in the left epithalamus is dependent upon the activity of the Nodal ligand Southpaw (Spw) emanating from the left LPM. Whether this activity of Spw is direct or indirect is unknown. It has been proposed that the role of left-sided LPM Nodal signaling may be indirect, through removal of repression of Nodal pathway gene expression in the left epithalamus (Carl, 2007).

This study addresses the role of the Wnt/Axin1/β-catenin signaling pathway in the regulation of asymmetric Nodal pathway gene expression and in the elaboration of brain asymmetries. The role of this pathway in the development of brain asymmetries has not previously been assessed, but some studies suggest that Wnt signaling can influence visceral asymmetries. For instance, overexpression of Xwnt8 in Xenopus can lead to cardiac left-right reversals as can overactivation of the Wnt/β-catenin pathway in medaka. In chick, Wnt/β-catenin signaling is suggested to be a left determinant of Nodal pathway gene expression in the LPM, as early upregulation of the pathway results in bilateral Nodal gene expression. Furthermore, mice lacking Wnt3a exhibit asymmetry defects that are likely due to a requirement for Wnt3a acting in and around the node during the period when asymmetries first become evident (Carl, 2007).

This study used a variety of approaches to establish roles for Wnt/β-catenin signaling and the Wnt pathway scaffolding protein Axin1 in both the regulation of Nodal pathway activation and in the differentiation of lateralized brain nuclei. masterblind (mbl) embryos carry a mutation in Axin1 that disrupts the binding of GSK3β, reducing the ability of GSK3β to degrade β-catenin and consequently leading to overactivation of Wnt/β-catenin signaling in the anterior neural plate. mbl mutant embryos show bilateral activation of Nodal pathway genes in the epithalamus but not the viscera. This activation can occur independently of the activity of Spw, suggesting that overactivation of Wnt signaling bilaterally removes repression of epithalamic Nodal pathway gene expression. Evidence is provided that this likely reflects a role for Wnt signaling during late gastrulation. Later overactivation of Wnt signaling during somitogenesis stages can disrupt lateralized Nodal pathway gene expression concordantly in the LPM and brain in both zebrafish and medaka. This is consistent with a role for Spw in the ipsilateral removal of repression of epithalamic Nodal pathway gene expression. Finally, Axin1 is shown to be required downstream of Nodal signaling during the elaboration of epithalamic asymmetries. These results provide evidence that the Wnt/Axin1/β-catenin signaling pathway plays several critical roles during the establishment and elaboration of asymmetries in the forming CNS (Carl, 2007).

Axin directs the amplification and differentiation of intermediate progenitors in the developing cerebral cortex

The expansion of the mammalian cerebral cortex is safeguarded by a concerted balance between amplification and neuronal differentiation of intermediate progenitors (IPs). Nonetheless, the molecular controls governing these processes remain unclear. This study found that the scaffold protein Axin is a critical regulator that determines the IP population size and ultimately the number of neurons during neurogenesis in the developing cerebral cortex. The increase of the IP pool is mediated by the interaction between Axin and GSK-3 in the cytoplasmic compartments of the progenitors. Importantly, as development proceeds, Axin becomes enriched in the nucleus to trigger neuronal differentiation via beta-catenin activation. The nuclear localization of Axin and hence the switch of IPs from proliferative to differentiative status are strictly controlled by the Cdk5-dependent phosphorylation of Axin at Thr485. The results demonstrate an important Axin-dependent regulatory mechanism in neurogenesis, providing potential insights into the evolutionary expansion of the cerebral cortex (Fang, 2013).

The fate decision of NPCs between amplification and differentiation controls the number of neurons produced during brain development and ultimately determines brain size. However, it is unclear how the NPCs make this fundamental choice. This study shows that the subcellular localization of a signaling scaffold protein, Axin, defines the activation of specific signaling networks in NPCs, thereby determining the amplification or neuronal differentiation of NPCs during embryonic development. Cytoplasmic Axin in NPCs enhances IP generation, which ultimately leads to increased neuron production, whereas nuclear Axin in IPs promotes neuronal differentiation. Intriguingly, the Cdk5-dependent phosphorylation of Axin facilitates the nuclear accumulation of the protein, thereby functioning as a 'brake' to prevent the overproduction of IPs and induce neuronal differentiation (Fang, 2013).

The expansion of cortical surface may result from increased numbers of neuroepithelial (NE) cells and radial glial cells (RGs) or from an amplified IP pool. NE/RG augmentation evidently controls the global enlargement of cortical surface. The amplification of a subset of RGs expressing the transcription factor Cux2 was recently suggested to facilitate upper-layer neuron expansion (Franco, 2012). However, there is a lack of experimental evidence indicating whether IP amplification also substantially contributes to the expansion of upper-layer cortical neurons and the cerebral cortex. Nonetheless, upper-layer neurons are generated during mid- and late neurogenesis, at which time IPs play the primary role in neuron production. Moreover, the enlargement of IP-residing SVZ is temporally correlated with the increased number of upper-layer neurons and expanded cortical surface. Therefore, it is tempting to speculate that the amplification of IPs during mid- and late corticogenesis has facilitated the evolutionary expansion of the cerebral cortex. The present findings demonstrate that increasing Axin levels during midcorticogenesis, which leads to the transient amplification of IPs without affecting the RG pool, is sufficient to expand the surface of the neocortex. Previous studies show that Axin expression is tightly regulated by different posttranslational modifications including deubiquitination, SUMOylation, methylation, and phosphorylation, which increase the stability of Axin; meanwhile, polyubiquitination and poly-ADP-ribosylation lead to its degradation. Thus, the adaptive evolution of the Axin gene that regulates its posttranslational modifications and hence its expression level might be involved in the evolutionary expansion of the cerebral cortex (Fang, 2013).

To ensure the development of a cerebral cortex of the proper size, the amplification and neuronal differentiation of IPs need to be precisely controlled. A reduced number of IPs due to precocious depletion of NEs/RGs or inhibition of IP generation/proliferation ultimately lead to the generation of fewer cortical neurons, resulting in a smaller cortex - a characteristic feature of human microcephalic syndromes (Fang, 2013).

In contrast, the overexpansion of IPs generates an excessive number of neurons, which is associated with macrocephaly and autism. The current findings demonstrate that Axin strictly controls the process of indirect neurogenesis to ensure the production of a proper number of neurons. Although cytoplasmic Axin simultaneously maintains the RG pool and promotes IP amplification to sustain rapid and long-lasting neuron production, subsequent enrichment of Axin in the nuclei of IP daughter cells triggers neuronal differentiation and prevents the overexpansion of IPs. In addition, the results demonstrate that Cdk5-mediated phosphorylation regulates the nucleocytoplasmic shuttling of Axin, thereby controlling the switching of NPCs from proliferative to differentiation status (Fang, 2013).

The findings show that Axin phosphorylation in IPs triggers neuronal differentiation in a rostrolateral-high to caudo-medial-low gradient correlated with the spatial gradient of neurogenesis. Thus, the gradient of Axin phosphorylation may provide a quantitative tool for evaluating the temporal and spatial gradient of IP differentiation into neurons. Importantly, nuclear Axin phosphorylation is rapidly induced in IP daughter cells in the G1 phase, which is the stage when progenitor cells actively respond to neurogenic signals; this suggests that the timing of Axin phosphorylation-dependent IP differentiation is regulated by diffusible extracellular signals. Therefore, understanding how Axin phosphorylation is regulated in IPs by extracellular cues and niches should shed new light on the molecular basis underlying the gradient-specific differentiation of IPs (Fang, 2013).

The findings also highlight the importance of Cdk5 in embryonic neurogenesis. Although Cdk5 plays critical roles in neuronal development and is implicated in the neurogenesis of cultured neural stem cells, it remains unclear whether Cdk5 regulates embryonic neurogenesis. The current findings provide in vivo evidence that Cdk5 is required for the neuronal differentiation of IPs, at least in part through phosphorylating Axin. Intriguingly, although cdk5/ cortices exhibited an accumulation of IPs and reduced neuron production during early-mid neurogenesis, the brain size of these mutant mice remained unchanged by the end of neurogenesis. This may be due to the compensatory increase of neuron production from the expanded pool of IPs during the mid-to-late neurogenesis stages. Therefore, elucidating how Cdk5 is involved in different stages of neurogenesis may provide insights into the molecular control of neuronal number and subtypes (Fang, 2013).

Several factors that regulate the generation and amplification of IPs have been identified. Nonetheless, key questions remain open: how RGs determine to differentiate into IPs instead of neurons, how RG-to-IP transition and IP differentiation are coordinated, and how IP amplification and differentiation are balanced. The present results show that the interaction between cytoplasmic Axin and GSK-3β maintains the RG pool and promotes IP production. The signaling mechanisms underlying the action of Axin-GSK-3β interaction require further investigation. It is hypothesized that Axin regulates IP differentiation from RGs via various molecular mechanisms. First, the Axin-GSK-3β complex may reduce the level of Notch receptor or β-catenin, leading to the suppression of Notch- and Wnt-mediated signaling, respectively. Given that Axin and GSK-3β can associate with the centrosome and mitotic spindle, Axin-GSK-3β interaction may also modulate cleavage plane orientation. Furthermore, Axin-GSK-3β can interact with and affect the microtubule-binding activity of adenomatous polyposis coli (APC), which is required for establishing the apical-basal polarity and asymmetric division of RGs. Finally, interaction with Axin can cause GSK-3β inhibition, which may enhance IP amplification through the activation of Shh signaling (Fang, 2013).

The timing of IPs to undergo cell-cycle exit balances the proliferative and neurogenic divisions of IPs and switches the RG-to- IP transition to the neuronal differentiation of IPs. This study shows that the interaction between Axin and β-catenin in the nucleus switches the division of IPs from proliferative to neurogenic by enhancing the neurogenic transcriptional activity of β-catenin. Indeed, Axin and β-catenin are required for the signal transduction of Wnt, RA, and TGF-β, which triggers and promotes neuronal differentiation. Thus, Axin in the nucleus may serve to transduce and converge multiple neurogenic signaling pathways to β-catenin during neurogenesis. However, the mechanism by which nuclear Axin enhances the transcriptional activity of β-catenin requires further investigation. Given that β-catenin exerts its transcriptional regulation of target genes through association with T cell factor/lymphoid enhancer factor (Tcf/Lef), it is hypothesized that nuclear Axin facilitates β-catenin/Tcf/Lef complex formation to enhance transcription (Fang, 2013).

Although Axin was previously recognized as a negative regulator of canonical Wnt signaling, suppressing cell division by recruiting GSK-3β and β-catenin into the β-catenin destruction complex for β-catenin degradation, the present results show that cytoplasmic Axin and nuclear Axin act distinctly from canonical Wnt signaling through specific binding to GSK-3β and β-catenin, respectively. Therefore, the current findings corroborate the notion that Wnt signaling components play multifaceted roles in NPCs during neurogenesis, independent of canonical Wnt signaling as demonstrated in previous studies (Fang, 2013).

In conclusion, the present study identified distinct roles of Axin in IP amplification and neuron production. The results demonstrate that the modulation of Axin levels, subcellular localization, phosphorylation, and its interaction with key signaling regulators (e.g., GSK-3β and β-catenin) in NPCs ultimately control neuron production and expansion of the cerebral cortex. Given that Axin is a key regulator of the switch from IP amplification to differentiation, the characterization of the signals that control this switch will not only advance current understanding of how the cerebral cortex expands during evolution but also provide important insights into neurodevelopmental disorders such as microcephaly (Fang, 2013).

Axin Evolutionary homologs part 1/2 |

Axin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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