dishevelled


EVOLUTIONARY HOMOLOGS

Besides Dishevelled and Discs large, a third Drosophila protein, Canoe, contains the GLGF/DHR motif. canoe interacts genetically with Notch and scabrous in eye, bristle and wing development, suggesting that cno has a common role with sca and N in the morphogenesis of these tissues. As there appears to be a direct physical interaction between Notch receptor and Dishevelled (Axelrod, 1996), providing a link between Notch and wingless signaling, perhaps Canoe plays a role in modifying this interaction (Miyamoto, 1995).

Dishevelled domain structure and signaling pathways

Dishevelled (Dsh/Dvl) proteins are known to mediate Wnt signaling by up-regulating beta-catenin (Drosophila homolog: Armadillo) levels and stimulating T cell factor (TCF)/LEF-1-dependent transcription (see Drosophila Pangolin). A new, TCF independent, Dvl-mediated signaling pathway has been identified in the mouse. Dvl proteins, when expressed in COS-7 cells, stimulate c-Jun-dependent transcription activity and the kinase activity of the c-Jun N-terminal kinase (JNK: see Drosophila Basket). The DEP domain of Dvl1 is essential for JNK activation. By contrast, all three conserved domains of Dvl, including DIX, PDZ, and DEP, are required for up-regulation of beta-catenin and for stimulation of LEF-1-mediated transcription in mammalian cells. Thus, Dvl can lead to two different signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1, which are involved in JNK activation by many stimuli, do not appear to play a major role in Dvl-mediated JNK activation, because the dominant negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK activation. This suggests that Dvl may activate JNK via novel pathways (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 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. 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, 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 (Drosophila homolog: Shaggy), 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).

Recent studies have shown that Drosophila Dishevelled (Dsh), an essential component of the wingless signal transduction pathway, is also involved in planar polarity signaling through the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway in Drosophila. Expression of a mouse homolog of Dsh (mDvl-1) in NIH3T3 cells activates JNK/SAPK, and its activator MKK7. A C-terminal half of mDvl-1, which contains the DEP domain, is sufficient for the activation of JNK/SAPK, whereas an N-terminal half of mDvl-1 as well as the DEP domain is required for stimulation of TCF/LEF-1-dependent transcriptional activation; this is a beta-catenin-dependent process. A single amino acid substitution (Met for Lys) within the DEP domain [mDvl-1 (KM)] abolishes the JNK/SAPK-activation of mDvl-1, but does not affect the activation of LEF-1-dependent transcription. Ectopic expression of mDvl-1 (KM) or an N-terminal half of mDvl-1, but not the C-terminal half, is able to induce a secondary axis in Xenopus embryos. Because the secondary axis formation is dependent on the Wnt/beta-catenin signaling pathway, these results suggest that distinct domains of mDvl-1 are responsible for the two downstream signaling pathways: the beta-catenin pathway and the JNK/SAPK pathway in vertebrates (Moriguchi, 1999).

The presence of multiple domains in Dsh suggests that it may interact with different signaling pathways via different domains. Several structural motifs are conserved in Dsh. The N-terminal DIX domain (DIX named after Dishevelled and axin) can interact physically with and has homologies to the C-terminal region of axin, a negative regulator of Wnt signaling. The medial PDZ domain of Dsh represents a globular protein-protein interaction domain contained in many adaptor molecules found in cellular junctional complexes. PDZ domains bind C-terminal ends of membrane receptors and/or interact with other PDZ domains. Finally, the C-terminal DEP domain (named after Dishevelled, Egl-10 and plekstrin) is found in several molecules that regulate G-protein functions. Xenopus Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant-negative approaches have not supported a role for Dsh in primary axis formation. The Dsh protein is post-translationally modified at the dorsal side of the embryo: timing and position of this regulation suggest a role for Dsh in dorsal-ventral patterning. To create functional links between these properties of Dsh the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics were examined. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis has revealed that axis inducing abilities do not segregate with Xdsh membrane association. The DIX region and a short stretch at the N-terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos, suggesting that multimerization is important for its proper function (Rothbacker, 2000).

Only one Dsh molecule has been found so far in Xenopus, and Xdsh may represent the obligatory component through which Frizzled molecules mediate their function(s) in Xenopus. During embryogenesis, distinct Frizzled molecules may instruct Xdsh to activate either a beta-catenin-dependent or beta-independent pathway, or both. So far, three maternally expressed Xenopus Frizzled proteins (Xfz-3, -7 and -8) have been identified in Xenopus. They all can translocate Xdsh to the membrane; in contrast, only Xfz-8 can induce a secondary axis. Xfz-7 is expressed most abundantly in early cleavage stages and thus may function as a primary anchor to recruit endogenous Xdsh to the membrane. Initial phosphorylation of Xdsh soon after fertilization may reflect such membrane association events. At present, it is unclear how differential Xdsh phosphorylation is achieved in the embryo One possibility is that as yet unidentified Wnt-like growth factors activate the Frizzled receptor system. A recent study in zebrafish supports this hypothesis, since a dominant-negative Fz blocks endogenous D/V signaling. Alternatively, Xdsh in early embryos might be activated de novo by a dorsally activated kinase in a Frizzled-independent manner. While deletion analysis shows that different domains are involved in phosphorylation and membrane association versus axis formation, the exact mechanisms by which the activity of Xdsh is regulated in the embryo remain to be determined. In particular, the way in which phosphorylation may influence axis formation or other early events, including polarity of cells in the developing embryo, remains unclear. Two independent pieces of evidence clearly support the idea that endogenous Xdsh and possibly its phosphorylation are actually important in dorso-ventral patterning. (1) CKIepsilon causes Xdsh phosphorylation while being involved in Wnt signaling and is thus a good candidate for the endogenous Xdsh phosphorylation. Interactions of Dsh and CKIepsilon with axin and the GBP-GSK-3 complex suggest that the regulation of Dsh via phosphorylation or segregation to the membrane may be important factors in the activation of axis formation. Thus, while membrane association and phosphorylation normally may be required to regulate endogenous Dsh activities, in overexpression, a membrane association-and phosphorylation-deficient Dsh may cause axis formation by influencing the balance of this multiprotein complex. (2) The demonstration of active transport of Xdsh to the dorsal side additionally supports the hypothesis that Xdsh is involved in dorsal-ventral patterning (Rothbacker, 2000 and references therein).

For Dsh to be clearly implicated in activating the downstream Wnt signaling pathway in the embryo, it has to meet several criteria: Dsh protein should be present and active at the correct time and place in the embryo. In addition, activation of Dsh function should induce secondary axes, while blocking its activity should block primary axis formation. New evidence is presented that maternal Dsh RNA is translated into a protein product, which is present throughout early cleavage stage embryos. Furthermore, Dsh protein becomes phosphorylated in a dorsal location at the appropriate time and place, correlating well with the initiation of dorso-ventral axis specification and the translocation of beta-catenin to nuclei. Such activation of members of the Wnt signaling pathway was shown to lead to axis formation, and ectopic expression of Dsh itself induces secondary axis formation. These results suggest that endogenous Xdsh is present in multimeric complexes. Because such pre-arranged multimers may not be destabilized and inactivated by dominant-negative forms, the evidence previously offered for lack of need for Xdsh is inconclusive. The evidence presented here therefore revives the possibility that Xdsh is indeed involved in endogenous axial patterning (Rothbacker, 2000 and references therein).

Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. These results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation (Salic, 2000).

The central mechanistic features of beta-catenin phosphorylation and ubiquitination have been well described in several systems by genetic approaches and by overexpression in cell lines and embryos. These studies have identified dsh, axin, GSK3, APC, and GBP/FRAT1 as important regulators of beta-catenin levels. However, there is disagreement as to whether APC acts in a positive or negative fashion; whether GBP plays an important role, or whether it just buffers GSK3; how the components interact; what the precise function of axin and APC is in regulating beta-catenin phosphorylation, and finally, the role of dsh in this process. To address these issues, cytoplasmic extracts from Xenopus eggs were used to reconstitute the regulated degradation of beta-catenin. By measuring beta-catenin stability directly rather than steady-state levels of the protein, the complicating effects of transcription and translation present in other systems were avoided. By adding known amounts of purified proteins to extracts and measuring degradation kinetics, a quantitative analysis of wnt signaling can be performed. This system also allows an examination of mutant proteins that might not show phenotypes if expressed in vivo, due to rapid turnover. In extracts, beta-catenin was degraded with a half-life of 1 hr, dropping to less than 15 min in the presence of added axin, which suggestes that axin is rate limiting for degradation. Addition of dsh, a positive regulator epistatically upstream of GSK3, stabilizes beta-catenin, suggesting that the wnt pathway from dsh to beta-catenin had been recapitulated in extracts (Salic, 2000).

This in vitro system allowed for a test of the biochemical requirement for several different components of the wnt pathway, ruling out the necessary involvement of any machinery upstream of protein translation. beta-catenin degradation depends on axin, GSK3, and APC; extracts depleted of any of these proteins cannot degrade beta-catenin. Of the four defined domains in axin, only the C-terminal DIX domain is dispensable for activity. The DIX domain of axin binds a related domain in dsh, and this interaction is required for dsh function; hence, binding of axin and dsh through the DIX domains confers signal-dependent regulation of beta-catenin. Another domain of dsh required for activity is PDZ, which binds GBP. Dsh and GBP synergize to inhibit beta-catenin degradation in extracts and beta-catenin phosphorylation in vitro, which are both axin-dependent processes. The inhibitory effect of GBP and dsh on beta-catenin degradation in extracts is reversed by high concentrations of GSK3 due to titration of dsh/GBP on axin. These observations further support a role for dsh, as an adaptor protein recruiting GBP to the axin/GSK3/APC/beta-catenin complex and inhibiting locally the enzymatic activity of GSK3. These recent results suggest that the axin-GSK3 interaction is highly dynamic and that increasing the local GBP concentration at the level of the complex efficiently promotes the dissociation of GSK3 from its site on axin (Salic, 2000).

The simplest model would have beta-catenin and GSK3 binding to their sites on axin and GBP binding to its site on dsh. When dsh and axin bind via their DIX domains, GSK3 would be inhibited. How does APC fit into this model? In addition to the mechanistic confusions about the role of APC, there are suggestions that APC can be an inhibitor of beta-catenin degradation in some circumstances or an activator in others. Specifically in Xenopus, injection of APC can cause secondary axis formation, the opposite of the ventralizing effects of axin injections. In an in vitro system, there is an absolute requirement for APC in beta-catenin degradation, consistent with genetic experiments in Drosophila and experiments in cultured cells. The confusion is likely to be simply due to the tendency of overexpressed APC to act as a dominant inhibitor. In Xenopus extracts, overexpressed APC is a potent inhibitor of beta-catenin degradation, although these experiments yield a mixture of truncated and full-length APC proteins. The role of APC in beta-catenin degradation is clear from the in vitro experiments. An interaction of APC with the RGS domain of axin is essential for beta-catenin degradation, demonstrating that endogenous APC is in fact a negative regulator of beta-catenin levels in Xenopus. Although beta-catenin binds to axin beads in a purified system, the two proteins show little interaction in the nanomolar range. Extracts contain an activity that promotes the binding of beta-catenin to axin at low concentrations. The activity is inhibited by the RGS domain of axin, suggesting that it is either APC or APC complexed to other proteins. Also, axinDeltaRGS (which cannot bind APC) does not bind beta-catenin in extracts. With purified components in vitro, APC accelerates the binding of beta-catenin to axin, recapitulating the stimulating effect of extracts on binding between the two proteins. Taken together, these results identify APC as the activity stimulating the axin-beta-catenin interaction in extracts (Salic, 2000).

beta-catenin interacts with numerous cellular proteins, some not directly involved in wnt signaling. The requirement for an APC-like molecule makes sense if one realizes that not only the stability of beta-catenin is important but also its availability for other interactions, both transcriptional and cytoskeletal. Aside from participating in beta-catenin degradation, APC also prevents the interaction of beta-catenin with Tcf3, thus inhibiting the transcriptional activation function of beta-catenin. Additionally, APC maintains a pool of beta-catenin in a state that can either lead to its degradation or release in response to a wnt signal. The phosphorylation state of APC is maintained by rapid futile cycles of opposing phosphorylation and dephosphorylation reactions. Changes in the phosphorylation of APC by GSK3 acts as a switch to trigger either beta-catenin degradation or its discharge in the cytoplasm. Although it has been suggested that phosphorylation of axin modulates binding to beta-catenin, no evidence could be found for such a regulation in extracts because (1) APC is the principal mediator of the axin-beta-catenin interaction and (2) using purified axin and beta-catenin, an increase in the affinity of beta-catenin for axin due to phosphorylation of the latter by GSK3 could not be detected. These data suggests that the APC/axin/GSK3/beta-catenin complex exists in two states. In one state, beta-catenin is bound tightly to axin via APC; beta-catenin and APC are both phosphorylated by GSK3. In this situation, phosphorylated beta-catenin can only be removed through SCF-dependent degradation while phosphorylated APC continues to bind beta-catenin molecules avidly. Upon binding of active dsh to axin, GBP interacts with GSK3, removing it from axin competitively. This inhibits phosphorylation on both beta-catenin and APC. The former stabilizes beta-catenin to ubiquitination, and the latter releases intact beta-catenin so that it can interact with other partners. The beta-catenin-binding site on axin is also important because its deletion impairs the function of axin. Although this scenario is written as a set of irreversible steps, it is likely that all binding events are dynamic. The fact that dominant-negative GSK3 blocks degradation of beta-catenin in extracts suggests that the interaction between endogenous axin and GSK3 must be dynamic. The dissociation rate for GSK3 bound to axin must be fast enough to allow its displacement by the dominant-negative mutant on a time scale of perhaps less than 1 hr. The same must be true for the axin-APC interaction, since the RGS domain of axin (which interacts with APC) blocks beta-catenin degradation (Salic, 2000).

The stability of beta-catenin is subject to a system of weak protein interactions and posttranslational modifications. The ability to dissect this pathway in vitro without altering appreciably its kinetics and without simplifying essential steps should aid an understanding of these weak interactions and their physiological consequences. Several remaining questions should be clarified by studies in partially purified systems and ultimately by reconstitution from purified components. These include the mechanism of signal transmission from frizzled to dsh and the mechanism of dsh activation. In particular, does the wnt signal regulate the GBP-dsh or the dsh-axin interaction? It will also be of interest to know whether APC suffices by itself to mediate beta-catenin degradation or whether associated proteins are also required. Finally, it will be of interest to know whether signaling pathways cross-regulate the wnt pathway and how that might work. In vitro reconstitution should be useful in deciphering the molecular events involved in transducing the wnt signal (Salic, 2000).

Gastrulation in the amphibian embryo is driven by cells of the mesoderm. One of the genes that confers mesodermal identity in Xenopus is Brachyury (Xbra), which is required for normal gastrulation movements and ultimately for posterior mesoderm and notochord differentiation in the development of all vertebrates. Xbra is a transcription activator, and interference with transcription activation leads to an inhibition of morphogenetic movements during gastrulation. To understand this process, a screen was carried out for downstream target genes of Brachyury. This approach results in the isolation of Xwnt11, whose expression pattern is almost identical to that of Xbra at gastrula and early neurula stages. Activation of Xwnt11 is induced in an immediate-early fashion by Xbra and its expression in vivo is abolished by a dominant-interfering form of Xbra, Xbra-EnR. Overexpression of a dominant-negative form of Xwnt11 (a C-terminally truncated form of the protein), like overexpression of Xbra-EnR, inhibits convergent extension movements. This inhibition can be rescued by Dsh, a component of the Wnt signaling pathway and also by a truncated form of Dsh that cannot signal through the canonical Wnt pathway involving GSK-3 and beta-catenin. Together, these results suggest that the regulation of morphogenetic movements by Xwnt11 occurs through a pathway similar to that involved in planar polarity signaling in Drosophila (Tada, 2000).

The morphological effects of different Dishevelled constructs reveal similarities in the signaling pathways required for convergent extension in Xenopus and the establishment of planar polarity in Drosophila. In Drosophila, mutations in dsh cause defects in the orientation of cells within epithelia of the wing, thorax and eye. For example, hairs in the wing usually point distally; the dsh1 allele causes these hairs to become oriented in a highly abnormal fashion. Genetic and biochemical studies show that the 'planar polarity' signaling required to establish correct cellular orientation does not involve components usually placed downstream of Dsh, including Shaggy (GSK-3), Armadillo (beta-catenin) and Pangolin (Tcf-3). Rather, it consists of small GTPases such as RhoA and Rac followed by the activation of JNK/SAPK-like kinases. The Dsh genes have three conserved domains. The N-terminal DIX (Dishevelled-Axin) domain is involved in protein-protein interactions and is necessary for the stabilization of beta-catenin. The PDZ domain is also involved in protein-protein interactions, and may be involved in recruiting signaling proteins into larger, membrane-associated complexes. Finally, the DEP domain (Dishevelled-EGL10-Pleckstrin) is thought to be involved in G protein signaling and membrane localization and also plays a role, perhaps independent of G proteins, in activation of JNK/SAPK-like kinases (Tada, 2000 and references therein).

In Drosophila, use of transgenic embryos expressing different domain deletions reveals that the DEP domain is essential for planar polarity signaling, whereas the DIX domain, which is essential for signaling through the canonical Wnt pathway, is not involved. Similarly, in these Xenopus experiments the DEP domain (as well as the PDZ domain) but not the DIX domain is required to restore activin-induced elongation in animal caps expressing dn-wnt11. The similarities in the signaling pathways required for morphogenetic movements in Xenopus and the establishment of planar polarity in Drosophila raises the intriguing possibility that Xwnt11 may function to control cell polarity during gastrulation in Xenopus (Tada, 2000).

Colorectal cancer results from mutations in components of the Wnt pathway that regulate ß-catenin levels. Dishevelled (Dvl or Dsh) signals downstream of Wnt receptors and stabilizes ß-catenin during cell proliferation and embryonic axis formation. Moreover, Dvl contributes to cytoskeletal reorganization during gastrulation and mitotic spindle orientation during asymmetric cell division. Dvl belongs to a family of eukaryotic signaling proteins that contain a conserved 85-residue module of called the DIX domain. The DIX domain is shown to mediate targeting to actin stress fibers and cytoplasmic vesicles in vivo. Neighboring interaction sites for actin and phospholipid are identified between two helices by nuclear magnetic resonance spectroscopy (NMR). Mutation of the actin-binding motif abolishes the cytoskeletal localization of Dvl, but enhances Wnt/ß-catenin signaling and axis induction in Xenopus. By contrast, mutation of the phospholipid interaction site disrupts vesicular association of Dvl, Dvl phosphorylation, and Wnt/ß-catenin pathway activation. It is proposed that partitioning of Dvl into cytoskeletal and vesicular pools by the DIX domain represents a point of divergence in Wnt signaling (Capelluto, 2002).

Cell proliferation and morphogenesis involve subcellular redistributions of signaling proteins such as Dvl. On Wnt stimulation of embryonic kidney cells, endogenous Dvl migrates to the perinuclear region where the signaling machinery resides, and also localizes to actin stress fibers. These contractile bundles of actin filaments are anchored to the substratum by focal adhesion plaques, and their dynamics underlie cell motility. Consistent with these observations, endogenous dishevelled-2 (Dvl2) was found to co-localize with sets of actin stress fibers that span Chinese hamster ovary K1 (CHO) cells. Dvl2 also exhibits a punctate pattern, suggesting some distribution to cytoplasmic vesicles. The identity of these Dvl-associated vesicles in a variety of cell types has remained unclear since their discovery. They may be endosomal, Golgi-derived, or transport vesicles as indicated by double labelling with GAP-43, a protein that also co-localizes with another ß-catenin regulator, glycogen synthase kinase-3ß. However, RhoB, a marker for early endosomes and prelysosomal compartments, does not co-stain with Dvl2. In Xenopus embryos, Dvl associates with 0.3-0.5-µm diameter vesicles that appear to transport Dvl towards the prospective dorsal side of the embryo during cortical rotation (Capelluto, 2002).

To establish whether the DIX domain is responsible for Dvl targeting, the localization of Dvl2 constructs was investigated in CHO cells, which exhibit well-characterized Wnt responses. Similar to endogenous Dvl2, the overexpressed full-length protein co-localizes with actin stress fibers, as does its isolated DIX domain. Similarly, axin, a protein that destabilizes ß-catenin, possesses a DIX domain that also co-localizes with stress fibers. Furthermore, endogenous actin immunoprecipitates with full-length Dvl2 and the isolated DIX domains of Dvl2 and axin, but not the highly expressed Dvl2DeltaDIX protein. Thus the Dvl2 DIX domain is necessary and sufficient for actin stress fiber association, and this DIX function seems to be conserved (Capelluto, 2002).

DIX domains are known to mediate oligomerization of Dvl and axin. These homo- and hetero-meric complexes are also formed in the presence of dodecylphosphocholine (DPC). This detergent mimics the predominant phospholipid in mammalian membranes and forms small micelles suitable for NMR studies of membrane-associated proteins. The DIX domain, which has a relative molecular mass of 9,800 (Mr 9.8K), exists as a homodimer of approximately 21.2K (Capelluto, 2002).

The Dvl2 DIX domain forms a predominantly helical structure. NMR studies predict two alpha-helices spanning residues Ala 35 to Gln 48 (alpha1) and Ala 51 to Tyr 55 (alpha2), and helical and extended elements from Asn 82 to Leu 89 (alpha3) and Val 29 to Ile 31, respectively (Capelluto, 2002).

An actin-binding element was identified between the alpha2 and alpha3 helices. Lys 58, Ser 59 and Met 60 appear to be critically involved in the actin interaction. These residues lie in the conserved YFFKSM-60 sequence, which shares hydrophobic and basic character with an established actin-binding motif represented by FSFKKS and V(T/H)VKKV in the MARCKS and actobindin proteins, respectively. The most conserved residue of this motif, Lys 58, was mutated to alanine. The K58A mutation renders the DIX domain unable to bind actin by immunoprecipitation, results in a diffuse distribution, and does not compromise the structural integrity of the domain. Consequently, the YFFKSM-60 motif is functionally required for Dvl2 binding to actin stress fibers (Capelluto, 2002).

In addition to actin association, the Dvl2 DIX domain also appears to localize to cytoplasmic vesicles. The latter interaction was further investigated with concanavalin A (Con A), a lectin that binds glycoproteins in many cytoplasmic membranes. Similar to full-length Dvl2, some DIX domain localizes to these cytoplasmic vesicles. By contrast, the Dvl2DeltaDIX construct, which retains the plasma-membrane-targeting DEP domain, was more diffusely distributed, suggesting that the DIX domain contributes to the vesicular association of Dvl2 (Capelluto, 2002).

A phospholipid interaction loop is apparent. Micelle interactions primarily involve the conserved VKEEIS sequence between the principal actin-binding motif and alpha3. Notably, actin and phospholipid binding may compete. To examine these phospholipid interactions in vivo, the Lys 68 and Glu 69 residues of the Dvl2 DIX domain were mutated to alanines. The K68A/E69A mutant does not associate with vesicles, although actin stress fiber co-localization and structural integrity are retained. Thus the VKEEIS motif of the DIX domain mediates phospholipid interactions that recruit Dvl2 to cytoplasmic vesicles (Capelluto, 2002).

Stabilization of ß-catenin through canonical Wnt signaling involves membrane targeting of Dvl. Therefore mutation of the DIX domain's functional sites could differentially affect intracellular ß-catenin stability. Elevated levels of cytosolic ß-catenin were found in CHO cells expressing full-length Dvl2, consistent with the inhibition of ß-catenin destruction by Dvl. The critical role of the DIX domain in Wnt regulation was confirmed by the inability of Dvl2DeltaDIX to stabilize ß-catenin. Moreover stabilization of ß-catenin is lost when the vesicle-targeting motif of Dvl2 is mutated (Dvl2(K68A/E69A). However, actin binding seems to be dispensable, since the ß-catenin in cells expressing the Dvl2 K58A mutant [Dvl2(K58A)] mirrors the elevated level seen with wild-type Dvl2. Thus the DIX domain mediates formation of a vesicular pool of Dvl that stabilizes ß-catenin and ensures commitment to canonical Wnt signaling (Capelluto, 2002).

The localization of Dvl to vesicles correlates with its regulated phosphorylation in Wnt and planar cell polarity pathways. Dvl phosphorylation can be catalysed by casein kinase Iepsilon and PAR1, and can be inhibited by PP2A, a serine/threonine phosphatase. Accordingly, phosphorylated and dephosphorylated Dvl2 are found in CHO cells, with PP2A converting the former to the latter state. Similarly Dvl2(K58A), which is disabled for actin binding, is heavily phosphorylated in vivo. However Dvl2(K68A/E69A), which is impaired in vesicular localization, is not phosphorylated. Consequently Dvl2 phosphorylation and DIX-mediated vesicle association are linked, although a causal relationship remains to be established (Capelluto, 2002).

The biological implications of targeting Dvl to actin or vesicles was further examined by Xenopus axis induction. Endogenous ß-catenin signaling activates genes that are essential for dorsal axis formation including Xnr3 and siamois2. Furthermore ectopic Wnt/ß-catenin signaling, such as Dvl2 overexpression, induces Xnr3 and siamois expression and dorsal axis duplication. The Dvl2(K58A) mutant, which does not bind actin but expresses comparably to wild-type Dvl2, also induces Xnr3/siamois expression and boosts the axis duplication frequency. Thus the actin pool of Dvl is sequestered from downstream Wnt/ß-catenin signaling. By contrast, the Dvl2(K68A/E69A) mutant, which is defective in vesicle targeting, does not induce Xnr3/siamois expression and axis duplication, suggesting that vesicular Dvl2 is needed for ß-catenin-dependent transcriptional programmes. Axis duplication was also not induced by a Dvl2(K58A/K68A/E69A) triple mutant that is compromised in both vesicle and actin interactions, emphasizing the fact that vesicular Dvl is required for Wnt signaling. Finally, axin associates comparably with each of the Dvl2 mutants when expressed in Xenopus embryos. Consequently these mutations specifically block vesicle and actin targeting and not axin association, which relies on several Dvl regions (Capelluto, 2002).

The DIX domain represents a novel signaling module that mediates actin stress fiber and vesicular targeting. Dvl thus partitions between these two pools, and commits to distinct pathways. This segregation is not only critical for proliferation and morphogenesis, but may also contribute to oncogenesis, with the most frequent mutations of the axin tumor suppressor found in colorectal, medullablastoma and hepatocellular tumors centered on its DIX domain (Capelluto, 2002).

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).

Canonical Wnt/beta-catenin signaling is crucial during embryonic development. Upon Wnt stimulation, Dishevelled proteins relay the signal from upstream Frizzled receptors to downstream effectors. By using affinity purification followed by ion-trap mass spectrometry, K-homology splicing regulator protein (KSRP) was identified as a novel Dishevelled-interacting protein. KSRP negatively regulates Wnt/beta-catenin signaling at the level of post-transcriptional CTNNB1 (beta-catenin) mRNA stability. Thus, Dishevelled-KSRP complex operates in Wnt regulation of beta-catenin, functioning post-transcriptionally upon CTNNB1 mRNA stability (Bikkavilli, 2010).

The multi-functional nature of Dishevelled proteins continues to be discovered. In the current study, KSRP was identified as a novel Dvl3-interacting protein. KSRP was shown to interact with the C-terminus of Dvl3. Mammalian cells express three Dishevelled isoforms (Dvl1, Dvl2 and Dvl3). The distinct function(s) of the three isoforms in Wnt signaling are not known. KSRP specifically interacts with Dvl3, but not with Dvl1 or Dvl2. Although the mammalian isoforms share the common domains such as DIX, PDZ and DEP domains, the greatest degree of variation is observed in their C-terminal regions. The C-terminal region of Dvl3 is a largely natively unordered region. Dvl3 displays a short stretch of histidine residues (621-674) that are unique to Dvl3. It is speculated that the docking of KSRP to Dvl3 might involve this region. However, in the absence of defined mutations (e.g., point mutations or small internal deletions in the interacting domains of Dvl3 and KSRP) and parallel expression experiments in vivo, functionally relevant versus fortuitous bases for all binding interactions cannot be readily distinguished (Bikkavilli, 2010).

KSRP is an RNA-binding protein that recruits RNA-decay machinery and promotes rapid decay of the selected mRNAs. KSRP has been shown to be capable of influencing CTNNB1 mRNA levels. KSRP is known also to regulate the biogenesis of a subset of miRNAs. In the present study, it was observed that KSRP docks to the C-terminus of Dvl3 and acts as a negative regulator of canonical Wnt/β-catenin signaling. In the absence of Wnt, the Dvl3-KSRP complex probably regulates the stability of Ctnnb1 mRNA to a limited degree. Ctnnb1 mRNA is detected in the Dvl3-KSRP complex. Upon treatment with Wnt3a, a rapid loss of Ctnnb1 mRNA from the Dvl3-KSRP complex was recorded. It is speculated that escape from the Dvl3-KSRP complex promotes the stabilization of Ctnnb1 transcripts. This Wnt-stimulated mRNA stabilization probably contributes to the rapid accumulation of β-catenin, triggering downstream events in the canonical Wnt/β-catenin pathway. Exit of Wnt-stimulated Ctnnb1 mRNA from the Dvl3-KSRP complex correlates with β-catenin accumulation. As early as 1 hour following Wnt3a stimulation, a sharp decline was observed for Ctnnb1 mRNA content in the Dvl3-KSRP complex (i.e., mRNA stabilization); a sharp increase in the β-catenin accumulation follows (i.e., protein translation). The increase in the Ctnnb1 mRNA content of the Dvl3-KSRP complex later in the time course (6-8 hours) might well represent a switching 'off' of the canonical Wnt pathway signal (Bikkavilli, 2010).

The observations from the current study suggest that β-catenin is regulated in two ways. At the mRNA level, Dvl3-KSRP complex promotes de-stabilization of Ctnnb1 mRNA. At the protein level, GSK3β-mediated phosphorylation regulates degradation of β-catenin. Wnt acts to oppose the ability of the Dvl3-KSRP complex to promote de-stabilization of Ctnnb1 mRNA and to oppose the degradation of β-catenin mediated by GSK3β phosphorylation. Post-transcriptional stabilization and translation of Ctnnb1 mRNA coupled with reduced phosphorylation-mediated degradation of the β-catenin protein in response to Wnt contributes to rapid accumulation of β-catenin. Hence, novel post-transcriptional as well as known post-translational responses are essential for Wnt regulation of canonical gene expression (Bikkavilli, 2010).

A novel Wilms tumor 1 (WT1) target gene negatively regulates the WNT signaling pathway

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/beta-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/beta-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/beta-catenin signaling are not completely understood. This study identified a gene, CXXC5, which was renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/beta-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/beta-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, these results demonstrate that the WT1 negatively regulates WNT/beta-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis (Kim, 2010).

Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos; Function of Dishevelled homologs

How cells integrate the input of multiple polarizing signals during division is poorly understood. Two distinct C. elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/β-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. CKI (KIN-19) plays a role not only in the Wnt/β-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, a model is established for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event (Walston, 2004).

During development, certain cell divisions must occur with a specific orientation to form complex structures and body plans. In many cases, the polarizing input for oriented divisions involves Wnt signaling. One example of such division involves neuroblasts in Drosophila, in which the first division of the pI sensory organ precursor cell is under the control of Frizzled (Fz) and Dishevelled (Dsh). The orientation of blastomere divisions in the early C. elegans embryo has also been shown to require Wnt signaling. In the 4-cell embryo, the EMS blastomere is induced by its posterior neighbor, the P2 blastomere. This induction has two consequences: it specifies the fates of EMS daughter cells and properly positions the mitotic spindle of EMS. Although both processes are under the control of Wnt signaling, they are controlled through divergent pathways. When EMS divides, the anterior daughter, MS, gives rise to progeny that are primarily mesodermal, and the posterior daughter, E, produces all of the endoderm. The fates of MS and E are controlled in part by a Wnt signaling pathway that regulates the activity of the Tcf/Lef transcription factor, POP-1, in conjunction with the β-catenin WRM-1. WRM-1 interacts with POP-1 through a cofactor, LIT-1, a NEMO-like kinase that is activated through a parallel mitogen-activated protein kinase (MAPK) pathway. Pathways that utilize a β-catenin to alter transcription are referred to as Wnt/β-catenin pathways. Removal of some components of the Wnt/β-catenin pathway alters the fates of the two EMS daughters. Although the fate of the EMS daughters is controlled by a Wnt/β-catenin pathway, the orientation of the EMS division is controlled by a different Wnt pathway (Walston, 2004).

In wild-type embryos, the EMS spindle initially aligns along the left/right (L/R) axis and rotates to adopt an anterior/posterior (A/P) orientation during the initial stages of mitosis. In embryos that lack the function of certain Wnt signaling components, the EMS spindle often sets up in the proper orientation but fails to rotate along the A/P axis until the onset of anaphase. In some cases, the delayed spindle rotates dorsoventrally (D/V) before it adopts the proper A/P alignment. The Wnt spindle orientation pathway that controls EMS orientation involves a Wnt (MOM-2), Porcupine (Porc; MOM-1), and Fz (MOM-5). GSK-3, the C. elegans GSK-3β homolog, has been reported to act positively downstream of the Fz receptor to regulate EMS spindle positioning, rather than as a downregulator of β-catenin accumulation as observed with Wnt/β-catenin signaling. Indeed, Wnt/β-catenin signaling components downstream of GSK-3 are not involved in controlling EMS spindle alignment, and EMS spindle alignment occurs independently of gene transcription. Pathways such as the one that positions the spindle in EMS, which utilize GSK-3 but are independent of transcription, are referred to as Wnt spindle orientation pathways (Walston, 2004).

Although many Wnt signaling components have been identified that participate in spindle orientation, the role of the Dsh family has not been clearly characterized. The Dsh family proteins transmit Wnt signals received from Fz receptors. The Dshs use three domains (DIX, PDZ, and DEP) to interact with different downstream proteins and activate multiple Wnt pathways specifically. The C. elegans genome contains three Dsh family genes that possess the three conserved domains: dsh-1, dsh-2, and mig-5. Transcripts of dsh-2 and mig-5 are at similar, enriched levels in the 4- and 8-cell embryo based on microarray analysis, while dsh-1 levels are low (Walston, 2004).

Another molecule involved in Wnt signaling is Casein Kinase I (CKI). CKI has been shown to prime β-catenin for degradation by phosphorylating it at a specific serine residue. Once primed, the β-catenin can be further phosphorylated and targeted for destruction by GSK-3β. CKI has also been shown to bind and phosphorylate Dsh and may assist in inhibiting GSK-3β when Wnt signaling is active. Loss of function of the CKIα homolog, kin-19, causes defects in the fate of EMS daughter cells. Although the role of CKI in spindle alignment has not been examined, CKIα localizes to centrosomes and mitotic spindles in vertebrate systems (Walston, 2004).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle. When a Src pathway member and a member of the Wnt spindle orientation pathway are removed simultaneously, the EMS spindle fails to rotate into the proper A/P position prior to division and remains misaligned throughout division. Removal of Src pathway members also enhances endoderm fate specification defects observed following removal of Wnt/β-catenin pathway members. Spindle orientation defects in dsh-2(RNAi);mig-5(RNAi) embryos have not been reported unless the Src pathway is also removed; however, only defects in cell division orientation have been reported, as opposed to abnormalities in initial spindle positioning (Walston, 2004).

In addition to regulating the orientation of the EMS division, four of the mom (more mesoderm) genes, mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo. Three of the four AB granddaughters, ABal, ABpl, and ABpr, divide with spindle orientations that are parallel to one another. ABar divides in an orientation that is roughly perpendicular to the other three, an event best viewed from the right side of the embryo, placing anterior to the right. When the function of one of the above mom genes is removed, ABar divides parallel to the other AB granddaughters, resulting in mispositioning of its daughter cells, such that ABarp, the wild-type posterior daughter cell, adopts a position that is anterior to its sister, ABara. The source of the polarizing cue(s) that orients the division of ABar is unclear. However, using blastomere isolations, it has been demonstrated that C, MS, and E are all competent to align the spindle and generate asymmetric expression of POP-1 within unidentified, dividing AB granddaughters, suggesting that one or more of these cells could produce signals that orient the division of ABar in vitro (Walston, 2004).

In this study, the roles of two Wnt signaling pathways involved in regulating the mitotic spindle are demonstrated. (1) The nontranscriptional Wnt spindle alignment pathway requires contact from the C blastomere to align the spindle of ABar. The three Dshs differentially participate in aligning the spindles of EMS and ABar and vary with respect to their interaction with the Src signaling pathway during spindle orientation. Moreover, while KIN-19 participates in endoderm induction through the Wnt/β-catenin pathway, it also acts in the Wnt spindle orientation pathway. (2) A Wnt/β-catenin pathway regulates the timing of spindle rotation in ABar, presumably by specifying the fate of neighboring blastomeres. Taken together, these studies indicate that spindle orientation during early development is a tightly regulated event, influenced by multiple cues transmitted via redundant pathways (Walston, 2004).

Wnt signals in the early embryo are transmitted from P2 to EMS to orient its spindle and to specify the fate of the EMS daughters. The orientation of the spindle relies on Wnt ligands, including MOM-2, that are secreted from P2 and activate MOM-5/Fz on the surface of EMS. This ultimately activates GSK-3, resulting in spindle alignment irrespective of gene transcription or other downstream Wnt/β-catenin components. The current analysis suggests that all three Dsh proteins are upstream of GSK-3 activation. Removal of the function of any of the dshs results in an incorrectly positioned EMS spindle, with varying penetrance. The strongest effect is seen in offspring of dsh-2 mutant mothers, suggesting that DSH-2 is primarily responsible for transducing the signal from MOM-5 to GSK-3 in EMS. Antibody staining shows an enrichment of DSH-2 at the area of cell-cell contact between EMS and P2, consistent with a MOM-2/Wnt signal activating DSH-2 at the cell cortex through the MOM-5/Fz receptor (Walston, 2004).

This analysis also shows that kin-19 contributes to the Wnt spindle orientation pathway in both EMS and ABar. Although KIN-19 participates in EMS fate specification, it has not been demonstrated to influence the orientation of the EMS spindle. Depletion of KIN-19 results in spindle misalignment in EMS and ABar. Additionally, KIN-19 localizes to centrosomes during mitosis: this has been shown to be important in establishing the initial polarization axis in the 1-cell embryo. How kin-19 operates within the pathway remains unclear. Because CKI family members have the ability to prime β-catenin for further phosphorylation by GSK-3, KIN-19 may act as a priming kinase for GSK-3-mediated phosphorylation of other unidentified target proteins. Based on the localization of KIN-19, these targets may be linked to the cytoskeleton, thereby affecting the physical alignment of the spindles of EMS and ABar (Walston, 2004).

This analysis shows that the same Wnt spindle orientation pathway that orients the EMS blastomere also aligns the spindle of the ABar blastomere. The results indicate that, as in EMS, this pathway does not require gene transcription to align the ABar spindle and that GSK-3 could be interacting directly or indirectly with the cytoskeleton (Walston, 2004).

All three dsh genes also act redundantly during ABar spindle orientation as well. Surprisingly, the data show that MIG-5 is the Dsh that is most important during ABar spindle orientation, contrary to the case for EMS spindle alignment, where DSH-2 is most important. The ABar spindle defects seen in dsh-2(or302) embryos suggest that DSH-2 also contributes significantly to ABar spindle orientation. DSH-1 seems to play only a minor role, since dsh-1(RNAi) does not result in ABar spindle defects unless performed along with mig-5(RNAi). This combination may remove enough total Dsh protein to prevent ABar from dividing correctly. In contrast, when dsh-1 function is removed in combination with that of dsh-2, the amount of MIG-5 present may be sufficient to maintain the total Dsh protein at a high enough level that the removal of dsh-1 function has no effect. Alternatively, the Dshs may have slightly different functions in regulating spindle orientation (Walston, 2004).

In Wnt signaling mutants, defective EMS spindle orientation is eventually corrected to the proper orientation, which is presumably due to the activity of the parallel src-1 pathway. In contrast, the Src pathway does not rescue spindle defects in ABar, although the src-1 pathway does influence ABar division. At this time, targets of SRC-1 in spindle orientation are unknown. It is possible that one or more of the Dshs are SRC-1 targets; however, the more severe phenotype of src-1 mutants in EMS suggests that other targets are also affected. Interestingly, in EMS and ABar, removal of src-1 function along with the function of either dsh-1 or mig-5 has very little additional effect on spindle polarity; however, when src-1 function is removed in dsh-2(or302) mutants, spindle misalignment is enhanced to nearly complete penetrance in EMS and ABar. Thus, while the three Dsh proteins act partially redundantly, there may be differences in how they impinge on other pathways (Walston, 2004).

In the 8-cell embryo, ABar contacts the C and MS blastomeres. Blastomere isolations have been used to demonstrate that C and MS can orient the spindle of unidentified AB granddaughters. They also demonstrate that AB granddaughters have random spindle orientation when presented with a mom-2 mutant C blastomere, but not with a mom-2 mutant MS blastomere. Using pal-1(RNAi) to alter the fate of C and laser killing of blastomeres to create steric hindrance within the embryo, ABar has been unambiguously identified. These results show that a loss of contact between C and ABar results in misalignment of its spindle in virtually all cases. Thus, contact with C is not only sufficient to align the spindle of an AB granddaughter but is also necessary to properly orient the ABar spindle through the Wnt spindle alignment pathway. These results further suggest that the polarizing activity of C is mediated by MOM-2/Wnt (Walston, 2004).

The orientation of the EMS spindle is not affected when Wnt/β-catenin signaling is abrogated through disruption of transcription or removal of WRM-1/β-catenin or POP-1/Tcf/Lef. In contrast, when wrm-1, lit-1, pop-1, or ama-1 function is removed, the ABar spindle is delayed in rotating into position. All of these treatments are known to affect the differentiation of the progeny of EMS. Moreover, MS has been shown to be capable of orienting the spindle of AB granddaughters in isolated blastomeres independent of MOM-2 function. Given the physical proximity of the blastomeres to ABar in the wild-type embryo, MS may produce a MOM-2-independent signal that ultimately affects positioning of the ABar centrosome further from C. The data further suggest that abnormalities in the fate of EMS daughters result in rotation defects. In wrm-1(RNAi) embryos, both EMS daughters become MS-like, and β-tubulin::GFP analysis reveals that the centrosomes of ABar do not rotate properly in many cases. If a signal that aids orientation of the spindle of ABar is normally secreted by MS, the two MS-like daughter cells specified in wrm-1(RNAi) embryos could produce competing signals that result in spindle rotation defects in ABar. Similarly, when both of the EMS daughters adopt an E-like fate, as in pop-1(RNAi), altered signaling from EMS daughters could again lead to a similar phenotype. In these cases, the centrosomal positioning presumably relies solely on the Wnt signal from C to eventually position the spindle in the correct orientation (Walston, 2004).

In conclusion, spindle orientation in the early C. elegans embryo is regulated through a Wnt spindle alignment pathway involving the Dshs and KIN-19 but independent of gene transcription. In addition, in ABar, the Wnt/β-catenin pathway regulates the timing of spindle rotation in a transcription-dependent manner, presumably indirectly by altering the fates of E and MS. The components of the Wnt spindle orientation pathway downstream of KIN-19 and GSK-3 are unknown; future work should be aimed at identifying these components and determining which Wnts are involved in specific inductive events (Walston, 2004).

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. This study shows that C. elegans syndecan (SDN-1; see Drosophila Syndecan) is required for precise orientation of a mitotic spindle in response to a Wnt cue. SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation (Dejima, 2014).

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, since neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. The C. elegans dsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. It is suggested that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ (Chang, 2005).

Wnt signaling positions neuromuscular connectivity by inhibiting synapse formation in C. elegans

Nervous system function is mediated by a precisely patterned network of synaptic connections. While several cell-adhesion and secreted molecules promote the assembly of synapses, the contribution of signals that negatively regulate synaptogenesis is not well understood. This study examined synapse formation in the Caenorhabditis elegans motor neuron DA9, whose presynapses are restricted to a specific segment of its axon. The Wntlin-44 localizes the Wnt receptor lin-17/Frizzled (Fz) to a subdomain of the DA9 axon that is devoid of presynaptic specializations. When this signaling pathway, composed of the Wnts lin-44 and egl-20, lin-17/Frizzled and dsh-1/Dishevelled, is compromised, synapses develop ectopically in this subdomain. Conversely, overexpression of LIN-44 in cells adjacent to DA9 is sufficient to expand LIN-17 localization within the DA9 axon, thereby inhibiting presynaptic assembly. These results suggest that morphogenetic signals can spatially regulate the patterning of synaptic connections by subdividing an axon into discrete domains (Klassen, 2007).

In the canonical pathway, Wnt signaling results in the inhibition of the destruction complex, thereby allowing the accumulation of cytosolic β-catenin, which can translocate to the nucleus and regulate transcription through the TCF/Lef family of transcription factors. Interestingly, previous research has implicated components of this pathway in the stabilization of postsynaptic glutamate receptors in C. elegans (Dreier, 2005). Mutations in the β-catenins bar-1(ga80) and wrm-1(ne1982ts), or in the TCF/LEF pop-1(q645), did not result in defects in DA9 presynaptic positioning. No DA9 synaptic defects were observed in the mutants for the effectors lrp-1(ku156)/Arrow, lit-1(or131ts)/NLK, pry-1(mu38cs)/Axin, and tap-1(gk202)/TAB-1. As with the canonical pathway, mutations in fmi-1(gm122)/Flamingo in the planar cell polarity pathway and unc-43(n498n1186)/CaMKinase in the calcium-dependent pathway do not phenocopy lin-44, lin-17, or dsh-1 mutants. Therefore, a positive identification of downstream signaling mechanisms may require the discovery of novel effectors (Klassen, 2007).

Dishevelled and noncanonical Wnt signaling in zebrafish

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. The key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. Three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, these results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development (Matsui, 2005).

PKCdelta is essential for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. The PKC family is subdivided into three subfamilies: the classical, novel, and atypical PKCs (cPKC, nPKC, and aPKC, respectively). cPKC is activated by Ca2+ and diacylglycerol (DAG), nPKC is activated by DAG but not by Ca2+, and aPKC is not activated by these molecules. Novel genes encoding delta-type PKC have been identified in the Xenopus EST databases. Loss of PKCdelta (a member of the nPKC subfamily) function reveals that it is essential for convergent extension during gastrulation. The relationship between PKCdelta and the Wnt pathway was examined. PKCdelta is translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKCdelta function inhibits the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKCdelta forms a complex with Dishevelled, and the activation of PKCdelta by phorbol ester is sufficient for Dishevelled translocation and JNK activation. Thus, PKCdelta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled (Kinoshita, 2003).

Xenopus PKCdelta has a highly conserved C1 domain, which binds to DAG and phorbol esters such as PMA, a functional analog of DAG. PKCdelta was translocated to the plasma membrane in animal cap cells in response to both Xfz7 and PMA. These results and other observations suggested that Xfz7 might activate PKCdelta through DAG on the plasma membrane, although there is no direct evidence that activation of the Wnt/Frizzled pathway produces DAG. However, heterotrimeric G proteins have been implicated in the Wnt/Frizzled pathway. It has been shown that certain heterotrimeric G proteins coupled with seven-transmembrane receptors activate phospholipase C-ß, which hydrolyzes phosphatidylinositol phosphate to produce DAG and inositol triphosphate. In addition, Xfz7 function is blocked by pertussis toxin, which inhibits the Gi family. Taken together, these findings suggest that Xfz7 probably activates PKCdelta through a heterotrimeric G protein that produces DAG. It will be important to determine which G protein is involved in this pathway and whether DAG is produced by G protein function (Kinoshita, 2003).

Xdsh and PKCdelta form a complex and the complex formation is not dependent on PKCdelta activity. In addition, the activation of PKCdelta is sufficient and necessary for the membrane localization of Xdsh in response to Xfz7. These findings suggest that Xfz7 may be involved in the translocation of the PKCdelta-Xdsh complex to the plasma membrane through the production of DAG. In other words, PKCdelta recruits Xdsh to the membrane in response to Xfz7 signaling. It will be necessary to determine which domain of Xdsh interacts with PKCdelta and vice versa. Preliminary work shows that a C-terminal fragment including the DEP domain of Xdsh coimmunoprecipitates with PKCdelta as well as the full-length Xdsh protein. This is consistent with the fact that this domain of Dishevelled is sufficient for its membrane translocation and function in the PCP pathway (Kinoshita, 2003 and references therein).

The Dishevelled protein is known to be hyperphosphorylated in response to Wnt and Frizzled. The loss of PKCdelta function blocks this phosphorylation of Xdsh. It has been shown that the phosphorylation and membrane localization of Xdsh are closely related. The simplest model is that DAG activates PKCdelta on the membrane, and PKCdelta phosphorylates Xdsh directly. PKCalpha has been shown to phosphorylate Xdsh in vitro. PKCdelta may have the similar activity. However, Dishevelled is known to interact with other kinases, such as casein kinases 1 and 2, Par-1, and PAK1/MuSK. PKCdelta may regulate such protein kinases and thus indirectly regulate Xdsh phosphorylation. It would be interesting to examine whether PKCdelta phosphorylates Xdsh directly, and to elucidate the role of Xdsh phosphorylation in its localization and in the activation of downstream signaling. Determination of the sites in Xdsh that are phosphorylated by Xfz7 signaling awaits further study (Kinoshita, 2003).

The following three results indicate that PKCdelta mediates the activation of JNK by Xfz7: (1) JNK activation by Xfz7 was inhibited by the loss of PKCdelta function. (2) The activation of PKCdelta by PMA was sufficient for JNK activation. (3) The gastrulation-defective phenotype of PKCdelta MO is rescued by active MKK7, which activates JNK. JNK has been implicated in the noncanonical Wnt pathway, but it is still unknown how Xdsh activates the JNK pathway. The membrane localization and/or phosphorylation of Xdsh may enable other proteins such as Rho to interact with Xdsh to activate the JNK cascade. It will be interesting and important to learn how JNK regulates convergent extension movements during gastrulation (Kinoshita, 2003).

NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling

During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Disheveled, a key mediator of Wnt/PCP signaling, is essential for the medial-lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. This study demonstrates that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, these findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis (Zhang, 2014).

Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. High-resolution 3D digital datasets of frog gastrulae, and morphometrics are presented that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, these tools were combined with time-lapse analysis of gastrulation, and both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling were examined. Remarkably, although inhibition of Xdsh signaling disrupts both convergent extension and blastopore closure, mesendoderm internalization proceeds very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, a surprising degree of dissociability was found between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species (Ewald, 2004).

Protein Interactions of Dishevelled

The adenomatous polyposis coli (APC) protein binds to the cellular adhesion molecule ß-catenin, a mammalian homolog of Armadillo. Overexpression of APC blocks cell cycle progression. When ß-catenin is present in excess, APC binds to another component of the Wingless pathway, glycogen synthase kinase 3ß, a mammalian homolog of Shaggy/Zeste white 3. APC is a good substrate for GSK3ß in vitro, and the phosphorylation sites map to the central region of APC. Binding of ß-catenin to this region is dependent on phosphorylation of GSK3ß (Rubinfeld, 1996). APC also binds to DLG, the human homolog of the Drosophila Discs Large tumor suppressor protein. This interaction requires the C-terminal region of APC and the PDZ domain repeat region of DLG. APC colocalizes with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function (Matsumine, 1996). Perhaps the PDZ domain of Dishevelled (involved directly in Wingless signaling in Drosophila) interacts with APC and thereby provides a link between DSH and the APC-GSK3-ß-catenin complex (Perrimon, 1996).

To test the potential involvement of frizzled homologs in Wnt signaling, the effects of overexpressing rat frizzled-1 (Rfz-1) were examined on the subcellular distribution of Wnts and of Dishevelled, a cytoplasmic component of the Wnt signaling pathway. Ectopic expression of Rfz-1 recruits the Dishevelled protein as well as Xenopus Wnt-8 (Xwnt-8), to the plasma membrane (but not the functionally distinct Xwnt-5A). Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes (siamois and Xnr-3) in Xenopus explants in a manner that is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together, greater induction of these genes is observed, indicating that Rfz-1 can synergize with a Wnt. The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner that discriminates between functionally distinct Wnts; this involves translocation of the Dishevelled protein to the plasma membrane, and works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex (Yang-Snyder, 1996).

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. Treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with the finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induces hyperphosphorylation of Dsh. Furthermore, mouse Dvl proteins affect downstream components of Drosophila Wg signaling as does Dsh; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels; hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II coimmunoprecipitates with Dvl proteins, and Dvl proteins are phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway (Lee, 1999).

Dvl-1 or Dvl-2 overexpression elevates the Arm protein levels. The epistatic analysis of the Wg pathway suggests that overexpressed Dvl-1 or Dvl-2 inhibits ZW3, which in turn causes the Arm protein level to elevate. To directly evaluate Dsh-, Dvl-1-, or Dvl-2-induced modulation of ZW3 activities, an in vivo assay for GSK-3beta was developed that uses the microtubule-binding protein, Tau, which is another good substrate for GSK-3beta since Drosophila ZW3 has been shown to be replaceable with rat GSK-3beta. With the anti-Tau antibody that recognizes all forms of Tau, the transfected Tau protein is detected as an apparently broad band, which actually consists of several species of Tau that have different mobilities. Endogenous ZW3 kinase activity in clone-8 cells is too low to produce detectable amounts of phosphorylated Tau. Co-transfection of ZW3 with Tau induces the phosphorylation of Tau in clone-8 cells. However, despite the expression of the same amount of Tau and ZW3 protein, no phosphorylated form of Tau is detectable in Clone-8/Dsh, Clone-8/Dvl-1, and Clone-8/Dvl-2 cells, indicating that overexpressed Dsh, Dvl-1, or Dvl-2 inhibits ZW3-mediated phosphorylation of Tau. The simplest explanation for this is that overexpressed Dsh, Dvl-1, or Dvl-2 inhibit ZW3 kinase activity by an unknown mechanism, although the possibility cannot be excluded that Dsh or Dvl overexpression activates phosphatase(s) antagonizing ZW3-mediated phosphorylation of Tau. Probably similar molecular mechanisms of ZW3/GSK-3 suppression are operative in cells stimulated with Wg/Wnt treatment and with Dsh/Dvl overexpression (Lee, 1999).

The Wnt signaling cascade is essential for the development of both invertebrates and vertebrates, and is altered during tumorigenesis. Although a general framework for Wnt signalling has been elucidated, not all of the components have been identified. A serine kinase, casein kinase I (CKI), was isolated by expression cloning in Xenopus embryos. CKI reproduces several properties of Wnt signals, including generation of complete dorsal axes, stabilization of beta-catenin and induction of genes that are direct targets of Wnt signals. Dominant-negative forms of CKI and a pharmacological blocker of CKI inhibited Wnt signals in Xenopus. Inhibiting CKI in Caenorhabditis elegans generated worms with a mom phenotype, indicative of a loss of Wnt signals. In addition, CKI binds to and increases the phosphorylation of dishevelled, a known component of the Wnt pathway. These data indicate that CKI may be a conserved component of the Wnt pathway (Peters, 1999).

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd, are described. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals (Wharton, 2001).

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Sufficiency tests have identified casein kinase I epsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests have shown that CKIepsilon is required in vertebrates to transduce Wnt signals. In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. However, in C. elegans studies, loss-of-function of a CKI isoform most similar to alpha produces the mom phenotype, indicative of loss-of-Wnt signaling. An evaluation of the ability of the various CKI isoforms to activate Wnt signaling found that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of gamma, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance an understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo (McKay, 2001).

Biochemical studies support the placement of CKI downstream of Dsh; CKI directly binds to Axin, which functions downstream of Dsh in the Wnt cascade. CKI could function downstream of Dsh yet still phosphorylate Dsh. For example, CKI and Dsh might associate in the absence of Wnt signaling. Upon ligand binding, Dsh could activate CKI, and the activated form of CKI could in turn phosphorylate Dsh. This phosphorylation might then disrupt the interaction of Dsh and CKI, releasing CKI to activate downstream components of the pathway. Alternatively, the phosphorylation of Dsh might simply be a marker of activation of CKI and not a requirement for Wnt pathway function. Indeed, while it has been shown that Dsh phosphorylation is significantly higher on the dorsal side of the embryo during the onset of dorsalizing events, it has also been concluded that phosphorylation of Dsh is not required for second-axis formation. This conclusion is based on deletion analysis involving large deletions of Dsh. Mapping and mutagenesis of specific Dsh phosphorylation sites coupled with functional analysis may be required to clarify the role of the Wnt/CKI-dependent phosphorylation of Dsh (McKay, 2001).

Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/ß-catenin signaling pathway, but its mechanism of action remains obscure. A mammalian homolog of Naked cuticle, mNkd, has been cloned. mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. mNkd has been shown to acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupts convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway (Yan, 2001).

The interaction of mNkd with mDvl was demonstrated in mammalian cells using coimmunoprecipitation experiments. mNkd was transiently expressed in Cos7 or HEK293 cells; endogenous mDvl proteins were immunoprecipitated from total cell lysates. Because the EF-hand is included in the region of mNkd that is associated with mDvl in yeast two-hybrid experiments and is highly conserved between Drosophila Nkd and mNkd, the requirement of the EF-hand in the association with mDvl was investigated. Based on the crystal structure of the third EF-hand of the Recoverin protein, mutations were made that either changed the consensus residues in the calcium-binding loop or deleted the entire calcium-binding loop together with the surrounding amino acids. These mNkd mutants were expressed in HEK293 or Cos7 cells. Coimmunoprecipitation experiments revealed that none of these mutations significantly impaired the ability of these mNkd proteins to associate with mDvl. These data show that mNkd associates with mDvl in mammalian cells and that the intact EF-hand is not required for the association (Yan, 2001).

The domain of Dishevelled that associates with mNkd was identified. Dishevelled is a highly conserved protein and contains three distinct domains. The N-terminal region has a DIX domain that is required for canonical Wnt signaling. The middle region of Dishevelled contains a PDZ domain that is known to bind GBP/Frat1 and CK1epsilon, both positive regulators of the canonical Wnt pathway. The C-terminal region contains a DEP domain that is crucial for regulating the PCP pathway. Fragments corresponding to different regions of Drosophila Dishevelled (Dsh) were expressed in E. coli as GST-fusion proteins. Equal amounts of each fragment were mixed with in vitro-translated mNkd in the binding buffer, precipitated with glutathione beads and separated by SDS/PAGE. mNkd associates with the DM fragment of Dsh that encompasses the PDZ domain with the adjoining N-terminal basic amino acid stretch. Notably, the PDZ domain alone is not sufficient for the association. Also, there is no association of mNkd with the DIX domain in the N-terminal region or the DEP domain in the C-terminal region of Dsh. Thus mNkd is associated with a region within Dsh shared with GBP/Frat 1 and CK1epsilon (Yan, 2001).

Because Dishevelled is a known positive regulator of the canonical Wnt pathway and mNkd is found here to be directly associated with Dishevelled, the role of mNkd in the canonical Wnt pathway was tested in mammalian cell culture by using a Wnt-1 ligand-responsive luciferase reporter assay. In multiple experiments, activation of the reporter by Wnt-1 was inhibited 75% by coexpression of mNkd in mammalian cells. Expression of wild-type mNkd, in the absence of Wnt-1, has no effect on the activity of the reporter. These data suggest that mNkd negatively regulates the canonical Wnt pathway and are consistent with the inhibitory effect that Drosophila Nkd has on Wingless signaling in genetic studies. Interestingly, the EF-hand mutants of mNkd show an impaired ability to inhibit the canonical Wnt pathway, although these mutants are all capable of binding to Dishevelled. These data suggest that the association of mNkd with Dishevelled alone is not sufficient to inhibit the canonical Wnt pathway, and that the intact EF-hand is required for the inhibitory function. Importantly, mNkd fails to inhibit the gene response elicited by overexpression of ß-catenin, a result that places mNkd upstream of ß-catenin in the canonical Wnt pathway (Yan, 2001).

The induction of secondary axes by ectopic expression of Xwnt-8 in the Xenopus embryo was used as an assay for the role of mNkd in the canonical Wnt pathway in vivo. Ventral blastomere injection of 5-10 pg of Xwnt-8 RNA induced secondary axes in over 50% of the embryos; most of these secondary axes contained anterior structures. Coinjection of 25 pg of mNkd RNA suppresses the effect of Xwnt-8 and results in fewer secondary axes. Coinjection of higher doses of mNkd (250 pg) resulted in even fewer secondary axes, only half of which contained anterior structures, indicating that mNkd inhibits the canonical Wnt pathway in vivo. The promoter of the Xenopus nodal-related-3 (Xnr-3) gene has been shown to be directly activated by the canonical Wnt/ß-catenin signaling in the Xenopus embryo, and expression of Xwnt-8 RNA into Xenopus animal caps activates transcription from a coinjected Xnr-3-luciferase reporter plasmid in vivo. Consistent with the results from mammalian cell culture (and the secondary axis assay), coinjection of mNkd RNA suppresses Xwnt-8 activation of the Xnr-3 promoter. These data indicate that mNkd is an inhibitor of the canonical Wnt/ß-catenin pathway both in vitro and in vivo. Together, these findings that mNkd interacts directly with Dishevelled and inhibits the canonical Wnt pathway upstream of ß-catenin suggest that mNkd is an intracellular antagonist of the Wnt pathway (Yan, 2001).

Activation of JNK seems to be an important step in the PCP pathway, and a vertebrate cognate of the Drosophila PCP pathway controls convergent extension movements during vertebrate development. In both Xenopus and Drosophila, hyperactivation of this pathway disrupts PCP signaling without affecting the canonical Wnt pathway. Consistent with its ability to activate JNK in vitro, mNkd overexpression inhibits the normal elongation of Xenopus embryos. The normal formation of anterior structures in these embryos indicates that the phenotype is not the result of ventralization, suggesting that mNkd inhibits convergent extension. To assess more directly the effects of mNkd on convergent extension, open-face Keller explants of the dorsal mesoderm were examined. Such explants made from control embryos elongate and change shape significantly, whereas explants made from embryos expressing mNkd fail to elongate. These effects are similar to those elicited by overexpression of other wild-type components of the planar cell polarity cascade, such as Xdsh and Xfz-8, indicating a role for mNkd in controlling the PCP pathway (Yan, 2001).

Because mNkd is an inhibitor of the canonical Wnt pathway, it was important to test whether the effects of mNkd on convergent extension were simply a consequence of the effects of mNkd on the canonical Wnt pathway. Expression of dominant-negative GSK-3ß, a strong activator of the canonical Wnt pathway in Xenopus, does not attenuate the inhibitory effects of mNkd on convergent extension. Taken together, these data suggest that mNkd inhibits convergent extension by overstimulating the PCP-signaling cascade, and that this effect is independent of its inhibitory role on the canonical Wnt pathway (Yan, 2001).

Recently, a number of genes have been identified that are induced by Wnt expression in mammalian cells. Whether mNkd-mRNA levels change when cells are treated with Wnt ligands was tested. BALB/c LI mouse liver epithelial cells were treated with Wnt-3A-conditioned medium for 8 h, 19.5 h, or 27 h, respectively. Levels of mNkd transcripts increase significantly in cells treated with Wnt-3a-conditioned medium for 19.5 h and 27 h compared with control treatments. Wnt-3a-conditioned medium also causes an increase in mRNA levels of mNkd in L cells. The mRNA levels of mNkd also increases in BALB/c LI mouse liver epithelial cells treated with lithium chloride, a known inhibitor of GSK-3ß. Thus, the ability of mNkd to inhibit the intracellular signaling of the canonical Wnt pathway, in conjunction with the result that mNkd is itself a downstream target of the canonical Wnt signaling, suggests that mNkd is an intracellular cell-autonomous negative-feedback regulator of the canonical Wnt pathway (Yan, 2001).

Genetic and biochemical studies have shown that Dishevelled controls cell polarity by acting as an upstream activator of the JNK pathway both in vivo and in vitro. Because mNkd is directly associated with Dishevelled, whether mNkd participates in the JNK pathway was tested. NIH 3T3 cells were transfected with expression constructs of mNkd and c-Jun, in which c-Jun served to monitor JNK activities. In this assay, expression of mNkd or mDvl alone induces a strong phosphorylation of c-Jun that was detected by blotting with an antibody specific for phosphoserine-63. These data show that mNkd has an effect similar to Dishevelled in activating the JNK pathway in mammalian cell culture assays (Yan, 2001).

Wnt signaling via the Frizzled (Fz) receptor controls cell polarity and movement during development, but the molecular nature of Wnt/Fz polarity signal transduction remains poorly defined. In human cells and during Xenopus embryogenesis, Wnt/Fz signaling activates the small GTPase Rho, a key regulator of cytoskeleton architecture. Wnt/Fz activation of Rho requires the cytoplasmic protein Dishevelled (Dvl) and a novel Formin homology protein Daam1 (see Drosophila DAAM). Daam1 binds to both Dvl and Rho, and mediates Wnt-induced Dvl-Rho complex formation. Inhibition or depletion of Daam1 prevents Wnt/Fz activation of Rho and of Xenopus gastrulation, but not of ß-catenin signaling. This study illustrates a molecular pathway from Wnt/Fz signaling to Rho activation in cell polarity signal transduction (Habas, 2001).

Because Dvl2 PDZ domain is required for Fz/Dvl signaling to Rho proteins associated with the PDZ domain, interacting proteins were sought using the yeast two-hybrid technique. The widely expressed human Daam1 protein contains 1078 amino acids, and belongs to the family of Formin homology (FH) proteins that have been implicated in cell polarity from yeast to human. Formin is the product of the limb deformity locus and is required for limb morphogenesis in mice. Daam1 shares 22% to 30% identity with, and thus is distantly related to, several known mammalian FH proteins. Like other FH proteins, Daam1 contains a central proline-rich FH1 domain and a more carboxyl FH2 domain, and represents a novel subfamily that includes a closely related Daam2, Xenopus and zebrafish Daam, and a Drosophila ortholog, dDaam. The Daam subfamily exhibits extensive similarity both within and outside the FH1 and FH2 domains, including the amino and carboxyl terminal regions. Since several FH proteins bind to Rho, Rac, or Cdc42, Daam1 may also bind Rho GTPases (Habas, 2001).

The Daam1 amino terminus binds to Rho-GDP or Rho-GTP, suggesting a role for Daam1 as a scaffolding protein to recruit Rho-GDP (via the amino terminus) and a Rho-GEF (via the C-Daam1 portion), thereby enhancing Rho-GTP formation. The Daam1 amino terminus binds Rho-GTP with apparently higher affinity, raising an intriguing possibility of positive feedback control, a theme common in cell polarization. Polarity establishment relies on signal amplifications that interpret a small difference in a polarity signal field into a polarized cellular response. DFz1 (Frizzled) exhibits a polarized localization that depends on Dsh function, suggesting a positive feedback loop. Rho-GTP binding to the Daam1 amino terminus may stabilize Daam1 in its activated state, or recruit/activate additional Daam1, thereby promoting an amplification of Rho activation. Such a feedback loop would resemble one in pheromone-induced polarity in yeast. The mating pheromone, via its serpentine receptor and the trimeric G protein, recruits and activates a GEF specific for Cdc42. Activated Cdc42, in turn, is required for the GEF localization, thereby leading to further and polarized Cdc42 activation. The possibility that Daam1 may function primarily in such a feedback control cannot be ruled out. In this scenario, Wnt/Fz signaling initiates Rho activation without Daam1, and the activated Rho together with Dvl recruits/activates Daam1 to amplify Rho activation. In any event, Daam1 function is essential for Rho activation triggered by Wnt/Fz signaling (Habas, 2001).

Daam1 is distantly related to several distinct mammalian FH proteins, such as FRL (30% identity), FHOS (27%), mDia1 (28%), and mDia2 (22%), whose functions in GTPase signaling remain to be fully understood. FRL and FHOS bind specifically to Rac in a nucleotide-independent manner, and an activated FHOS is antagonized by Rac and Rac mutants, leading to the suggestion that FRL and FHOS are scaffolding proteins linking Rac to other proteins. Members of the mDia subfamily of FH proteins (see Drosophila Diaphanous) bind to Rho-GTP (and Rho-GDP in some cases), and are proposed to be Rho targets. However, since actin fiber induction by the activated mDia can be blocked by inhibition of Rho in some instances, and the activated mDia can cause RhoA activation, the relationship between mDia and Rho, and between FH proteins and Rho GTPases in general, may be complex and needs further investigation (Habas, 2001).

Vertebrate gastrulation involves polarization and intercalation of dorsal mesodermal cells along the mediolateral axis (convergence), resulting in the elongation of the anterioposterior axis (extension). This morphogenetic process is governed by Wnt-11 PCP signaling. In Xenopus gastrula, endogenous Rho activation is detected mainly in dorsal tissue, and is abolished when Wnt-11/Fz/Xdsh signaling or Daam1 function is inhibited. Conversely, ectopic Wnt-11/Fz/Xdsh signaling or C-Daam1 activates RhoA on the ventral side. Thus, Wnt-11/Fz signaling, via Xdsh and Daam1, is necessary and sufficient for RhoA activation during gastrulation, consistent with the previous finding that interference of Rho function inhibits gastrulation. In an explant assay, inhibition or depletion of Daam1 perturbs morphogenetic movements, whereas C-Daam1 restores the movements even when Wnt-11/Fz or Xdsh is inhibited. Daam1 thus functions downstream of Wnt-11/Fz/Xdsh in governing gastrulation. Finally, inhibition or depletion of Daam1 in the embryo blocks gastrulation and phenocopies the morphogenetic defects caused by inhibition of Wnt-11, Fz, or Xdsh signaling (Habas, 2001).

A molecular pathway for the Wnt/Fz activation of Rho is suggested, which is referred to as the Wnt/Rho pathway to distinguish it molecularly from Wnt/ß-catenin and Wnt/Ca2+ pathways. A Wnt signal activates a Fz receptor, which translocates Dsh to the plasma membrane and promotes Dsh-Daam1-RhoA complex formation and RhoA activation, likely via the recruitment of a Rho-GEF by the Daam1 scaffolding protein. Activated RhoA generates polarized cytoskeleton remodeling via the ROCK kinase, and perhaps also induces changes in gene expression. The zebrafish knypek gene product, a glypican, facilitates Wnt signal reception, whereas LRP5/6, which is the Fz coreceptor for Wnt/ß-catenin signaling, participates in neither PCP signaling nor RhoA activation. Whether and how other PCP gene products function in the Wnt/Rho pathway or in parallel pathways remains to be elucidated (Habas, 2001).

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane. Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells is dependent on added Wnt5A protein and is accomplished by the multifunctional adaptor protein ß-arrestin 2 (ßarr2), which is recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of ß-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling (Chen, 2003).

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).

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, it has been determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bind to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibit canonical Wnt signaling and block Wnt-induced secondary axis formation in a dose-dependent manner, but do not block noncanonical Wnt signaling mediated by the DEP domain. Together, these results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl (Wong, 2003).

The interaction between Fz and Dvl is relatively weak; it is therefore hypothesized that the membrane-targeting function of the Dvl DEP domain is required to ensure signal transduction. The weak interaction between Fz and Dvl could allow signaling from Fz to be mediated by cytoplasmic proteins, e.g., Dpr/Frodo. Indeed, the Dvl1 PDZ domain uses a single recognition site to interact with Fz and Dpr/Frodo. In addition, because of the weak interaction, the local physiological condition and the local environment, which includes the local concentrations of Dvl and its regulatory effectors, should play a considerable role in the mediation of the molecular recognition of Dvl. This possibility may serve as an explanation for the following discrepancy: despite the 90% amino acid identity between Dpr and Frodo, Dpr negatively regulates Wnt signaling, whereas Frodo enhances Wnt signaling (Wong, 2003 and references therein).

Multiple homologs of Fz and Dvl are present in mammals. The differences in the sequences of the PDZ domains of Dvl1 homologs and the C-terminal regions of Fz receptors suggest that the binding affinities of each in the Fz-Dvl complexes should differ. Further studies to fully investigate such differences will provide insight into the signaling pathways that involve Fz and Dvl (Wong, 2003).

Wnt signaling pathways in vertebrates use the phosphoprotein Dishevelled (Dvl). The cellular responses to Wnt signaling may in part be modulated by Dvl-associated proteins, including Dishevelled interaction protein Dapper (Dpr). The zebrafish Dpr paralogs Dpr1 and Dpr2 have been cloned and characterized. Loss-of-function studies reveal that endogenous Dpr1 but not Dpr2 is required to enhance Wnt/ß-catenin activity in zebrafish embryos that are hypomorphic for Wnt8. Conversely, Dpr2 but not Dpr1 is required for normal convergence extension movements in embryos that are hypomorphic for Stbm or Wnt11, supporting a functional interaction of Dpr2 with Wnt/Ca2+-PCP signaling. In gain-of-function experiments, Dpr1 but not Dpr2 induces Wnt/ß-catenin target genes. Dpr1 synergizes with zebrafish Dvl2, and with the Dvl-interacting kinases CK1epsilon, Par1 and CK2, in activating target genes. It is concluded that two Dvl-associated paralogs, Dpr1 and Dpr2, participate in distinct Wnt-dependent developmental processes (Waxman, 2004).

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling (Lu, 2004).

Ryk siRNA mice have defects in axon guidance of craniofacial motor nerves, ophthalmic nerves, and other nerves, suggesting an essential role of Ryk in axon guidance. Although there is no obvious deficiency in dorsal root ganglion neurite outgrowth in Ryk siRNA transgenic mice, dorsal root ganglion explants isolated from Ryk siRNA mice exhibit defects in neurite outgrowth in response to Wnt-3a stimulation. The lack of deficiency in DRG neurite outgrowth in Ryk siRNA mice is probably because NGF and other growth factors are also involved in inducing neurite outgrowth in vivo. The fact that the Wnt-3a-induces neurite outgrowth of dorsal root ganglion explants is inhibited in Ryk siRNA mice provides strong evidence that there is a functional interaction between Wnt and Ryk in neurite outgrowth (Lu, 2004).

Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. A novel interaction is described between Dishevelled2 (Dvl2) and μ2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of μ2. Dvl2 mutants in the YHEL motif fail to associate with μ2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. It is suggested that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling (Yu, 2007).

Based on four independent lines of evidence, it is proposed that a tight association between Dishevelled and AP-2 is important for at least some of the known biological functions of Dishevelled. One involves the observation that Frizzled4 is rapidly internalized upon its activation by Wnt, a process that requires Dvl2. This rapid and efficient uptake is coupled to Frizzled degradation, presumably in lysosomes, and both processes are greatly hindered in cells expressing variants of Dvl2 that fail to interact with AP-2 by virtue of selected point mutations in the YHEL motif or the DEP domain. It is suggested that proper engagement of Dvl2 with AP-2 is a key step for Frizzled4 endocytosis and its eventual degradation. It is possible that, under certain conditions, Dvl2 engages productively with the endocytic machinery by associating with β-arrestin2, which in turn can bind to clathrin and AP-2, as shown by failure to internalize Frizzled4 in cells depleted of β-arrestin2 by siRNA treatment. It seems, however, that the interaction of Dvl2 and β-arrestin2 can be superseded, because a block is observed in Frizzled4 endocytosis upon expression of Dvl2 mutants in the tyrosine motif that, according to a pull-down assay, bind β-arrestin2 perfectly well (Yu, 2007).

The second line of evidence involves Wnt signaling during frog embryonic development. Frog Xdsh has important regulatory roles in the canonical β-catenin and the noncanonical PCP pathways. Experiments, carried out in developing embryos, show that Xdsh with single-point mutations in its YHEL motif induces dorsal axis duplication as well as does the wild-type Xdsh, indicating that the mutations have little or no effect on the function of Xdsh in regulating the canonical β-catenin pathway. In contrast, presence of the YHEL motif is required for proper regulation of the noncanonical PCP pathway. This conclusion is based on the observation that overexpression of the wild-type Xdsh interferes with gastrulation in embryos and with elongation in the animal cap assay, whereas these processes are largely normal with any one of the YHEL mutant forms of Xdsh expressed at similar levels (Yu, 2007).

The third and fourth lines of corroborating evidence were obtained by following the effects of the Xdsh/Dvl2 mutants on two independent molecular signaling assays, one based on the activation of JNK in frog embryos, one of the hallmarks of PCP signaling, and the other based on stimulation of the TOPFlash reporter assay in mammalian cells, an indication of signaling through the canonical Wnt pathway. Xdsh, but none of the YHEL mutants, stimulated JNK, reflecting their failure to activate the noncanonical pathway; in contrast, both wild-type and Dvl2 mutants stimulated equally the TOPFlash assay, reflecting their comparable signaling through the canonical pathway. A possible caveat to the interpretation of these results is the fact that they involved gain of function effects by overexpression of mutant Dishevelled, rather than strict replacement of endogenous Dishevelled with the mutant forms. The latter experiment is currently not feasible, given the functional redundancy among different members of the Dishevelled family (Yu, 2007).

Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). This study identifies additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, it is shown that casein kinase 1 (CK1) δ and CK1epsilon are crucial for Dvl phosphorylation by non-canonical Wnts. In response to Wnt-5a, CK1epsilon binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1epsilon, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1epsilon kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, it was show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, these data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/epsilon-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling (Bryja, 2007).

CK1δ/epsilon have previously been reported to be a Dvl-phosphorylating kinase acting in the β-catenin pathway. A recent report describes that overexpression of CK1epsilon potentiates canonical Wnt signalling and diminishes JNK activation induced by Dvl1 overexpression. This finding led to the suggestion that CK1epsilon modulates the signalling specificity of Dvl towards β-catenin. CK1δ/epsilon also mediate non-canonical signalling, suggesting that canonical or non-canonical specificities are not determined by CK1epsilon but rather by the ligand. Moreover, the previously findings could be alternatively explained by the fact that CK1epsilon-mediated phosphorylation diminishes the activity of axin in the MEKK1-JNK pathway at the expense of its function in canonical Wnt signalling. Although the involvement of CK1δ/epsilon in the signal transduction of a non-canonical Wnt has not been demonstrated to date, a role of CK1δ/epsilon in other biological processes driven by non-canonical Wnts has been described. These include convergent extension movements in Xenopus and functions regulated by planar cell polarity pathway in Drosophila (Klein, 2006; Strutt, 2006). Thus, these data, together with published reports, support the notion that CK1epsilon mediates non-canonical Wnt signalling. Interestingly, a recent report by Takada (2005) suggests that, in Drosophila cells another CK1 isoform, CK1alpha is playing a similar role to the one described here for CK1δ/epsilon in Wnt-5a-driven phosphorylation of Dvl. Thus, it remains to be investigated whether the involvement of individual CK1 isoforms in Dvl phosphorylation differs among species (Bryja, 2007).

Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, this study shows that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. It is proposed that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization (Bilic, 2007).

The scaffold protein Dvl was previously thought to act downstream of LRP6 because dsh overexpression activates ß-catenin signaling in Drosophila LRP6 (arrow) mutants and because the constitutively active Dfz2-Arrow fusion protein is inactive in dsh mutants. The explanation for this discrepancy may be that overexpressing Dsh/Dvl leads to artificial sequestration of Axin or that the protein has multiple functions in the Wnt pathway (Bilic, 2007).

Taken together, the results suggest that Dvl-mediated co-aggregation triggers LRP6 phosphorylation by CK1γ. In this model, upon Wnt signaling Dvl aggregates form at the plasma membrane, where they co-cluster LRP6 with other pathway components including Fz, Axin, and GSK3ß, in a 'LRP6-signalosome.' The role of Wnt would be to bridge LRP6 and Fz, which copolymerize on a Dvl platform. Clustering of LRP6 then provides a high local receptor concentration that triggers phosphorylation by CK1γ and Axin recruitment (Bilic, 2007).

Predictions of this model are as follows: (1) artificial oligomerization of LRP6 should activate the receptor and (2) oligomerized LRP6 should signal independent of Dvl. Indeed, forced oligomerization of LRP6 using a synthetic multimerizer is sufficient to induce Wnt signaling, and this oligomerization bypasses the need for Dvl. (3) Constitutively active LRP6 should signal independently of Dvl because its self-aggregation should bypass the need for Dvl polymers. This is also the case as shown in reporter assays with Dvl siRNA knockdown, which supports previous findings. (4) If LRP6 aggregation is a prerequisite for phosphorylation by CK1γ rather than its consequence, LRP6 aggregates should form even when the kinase is blocked. This is the case: Nonphosphorylated LRP6 aggregates were observed in response to Wnt treatment in cells transfected with dominant-negative CK1γ. The model of LRP6-signalosomes not only provides a mechanism for Wnt signal transduction but may also be relevant for the understanding of intracellular transport of maternal Wnt determinants in the fertilized Xenopus egg (Bilic, 2007).

Wnt5a signals through DVL1 to repress ribosomal DNA transcription by RNA polymerase I

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. This study shows that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects are specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) (see Drosophila Sirt7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo (Dass, 2016).

The Hippo pathway regulates Wnt/beta-catenin signaling

Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. This study shows that the Hippo pathway restricts Wnt/β-Catenin signaling by promoting an interaction between TAZ (a yorkie homolog, sharing approximately 50% sequence identity with YAp, with a similar topology, containing two central WW domains and a C-terminal transactivation domain) and DVL in the cytoplasm. TAZ inhibits the CK1δ/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/β-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear β-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear β-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control (Varelas, 2010).

The Hpo/Wts kinase, which in vertebrates is represented by MST and LATS, respectively, converges on the transcriptional regulator Yorkie in Drosophila, or the vertebrate homologs, TAZ and YAP. Recent studies have focused on the transcriptional roles for Yorkie, TAZ, and YAP in controlling growth and cell fate choice. However, these proteins are also found in the cytoplasm, where TAZ has been shown to associate with the plasma membrane and actin cytoskeleton. TAZ cytoplasmic localization is mediated by LATS-dependent phosphorylation of serine 89, which drives binding to the cytoplasmic retention factor, 14-3-3. Altogether, these findings raised the possibility that TAZ has cytoplasmic functions. This study demonstrates that one such function is to constrain Wnt signaling by inhibiting CK1δ/epsilon-mediated DVL phosphorylation (see model). Although the ability of CK1δ/epsilon to phosphorylate DVL has been well established, the precise role of this event in Wnt signaling remains to be fully understood. This study has observed that CK1δ/epsilon-independent signaling can contribute to Wnt-induced β-Catenin stabilization. Nevertheless, the data clearly show that DVL phosphorylation and Wnt pathway activation caused by the loss of TAZ expression requires CK1δ/epsilon. Abrogation of LATS expression, which leads to the nuclear accumulation of TAZ, reduced TAZ-DVL interaction, enhanced DVL phosphorylation, and promoted Wnt pathway activation in cells. Furthermore, it was shown that loss of hpo or wts function or enhanced expression of yki in Drosophila wing imaginal discs also modulates Wnt signaling. Thus, by promoting the cytoplasmic localization of TAZ, the Hippo pathway not only prevents nuclear TAZ activity, but also leads to inhibition of Wnt signaling by antagonizing DVL activation. This study has focused on TAZ, but it will be interesting to determine whether the related protein, YAP, has a similar activity in the Wnt pathway. Indeed, functional redundancy is suggested by the Taz/Yap double-knockout mice, which display early embryonic lethality (Varelas, 2010).

Wnts are known to act throughout mammalian kidney development, and hyperactivation of Wnt/β-Catenin signaling causes severe kidney defects. For example, loss of Apc function results in nuclear β-Catenin accumulation that leads to cystic kidneys, and transgenic mice expressing an activated mutant of β-Catenin also develop cysts. Taz mutant mice also develop polycystic kidneys, and increased cytoplasmic and nuclear β-Catenin was found in both cystic and precystic regions of Tazgt/gt kidneys, indicating Wnt pathway activation. Moreover, cysts formed in humans with ADPKD, caused by mutations in PC1 or PC2, display a gene expression profile consistent with Wnt pathway activation. β-Catenin associates with PC1 (Lal, 2008), and TAZ has been proposed to indirectly regulate the activity of PC2 via the SCF ubiquitin ligase β-TrCP. It was confirmed that TAZ and TAZ (1-393, ΔWW), the DVL-binding mutant that cannot rescue the effect of loss of TAZ on Wnt signaling, both interact with β-TrCP. Thus, it is concluded that TAZ modulates Wnt signaling via direct regulation of DVL; however, TAZ may also function indirectly to regulate the PC complex via β-TrCP. Whether TAZ may have other additional roles in Wnt signaling remains to be determined. Altogether, these results suggest that, in the kidney, TAZ restricts canonical Wnt signaling that otherwise would disrupt tubule morphogenesis and the formation of cysts (Varelas, 2010).

Defects in cilia are also known to be involved in the pathogenesis of polycystic kidney disease. Indeed, recent analyses of Taz mutant kidneys have revealed that Taz localizes to cilia, and that cystic regions in Taz −/− kidneys have underdeveloped cilia. Interestingly, primary cilia have recently been implicated in restraining Wnt signaling (Corbit, 2008), and DVL has been reported in cilia (Park, 2008). These findings raise the intriguing possibility that TAZ may link suppression of DVL activation in the kidney to ciliogenesis. Of note, no evidence was found that the MDA-MB-231 breast cancer cells used in this study possess cilia, indicating that cilia, per se, are not required for TAZ inhibition of Wnt signaling. Nevertheless, it will be interesting to examine the relationship between TAZ regulation of ciliogenesis and DVL modulation in kidney epithelia (Varelas, 2010).

The development of multicellular organisms requires cell-cell communication and the coordinated action of distinct signaling pathways for proper patterning of the body and organs. In the Drosophila wing imaginal disc, the Wg morphogen gradient coordinates the expression of an array of developmentally important genes. These studies reveal that the Hippo pathway functions to restrain Wg signaling, and that loss of hpo or wts increases the levels of Arm (β-Catenin) outside the normal expression domain and enhances expression of Wg target genes, particularly at a distance from the Wg source. Thus, Hippo pathway activation may, in effect, serve to restrain the sensitivity of cells to Wg signals, thus sharpening the Wg response gradients to promote appropriate tissue patterning events. How the Hippo kinase cascade is regulated by upstream pathways remains unclear. However, genetic studies place a protocadherin, Fat, and an ERM domain protein, Expanded, upstream of Hpo/Wts kinases. Furthermore, and in accordance with analysis of hpo and wts mutants, fat and expanded mutants display enhanced Wg target gene expression independent of Wg ligand production. The results suggest that this occurs via a direct molecular link. In this regard, it is intriguing to note that mice null for Fat4, a mammalian homolog of Drosophila fat, also develop polycystic kidneys. Direct communication between the Hippo and Wnt pathways may thus be an ancient mechanism that coordinates morphogen signaling with tissue growth control in animals (Varelas, 2010).

This study shows that TAZ inhibits Wnt signaling via DVL, providing for a direct molecular link between the Hippo and Wnt signaling pathways. Other studies in Drosophila have implicated the Hippo pathway as a positive regulator of Notch signaling, and an essential role for TAZ in mediating TGFβ-dependent Smad signaling has been shown. Furthermore, TAZ is induced by BMP signaling to regulate mesenchymal cell differentiation. Altogether, these studies suggest a critical and direct role for TAZ in integrating signals from numerous morphogen signaling pathways (Varelas, 2010).

Lats2 Modulates Adipocyte Proliferation and Differentiation via Hippo Signaling

First identified in Drosophila and highly conserved in mammals, the Hippo pathway controls organ size. Lats2 is one of the core kinases of the Hippo pathway and plays major roles in cell proliferation by interacting with the downstream transcriptional cofactors YAP and TAZ. Although the function of the Hippo pathway and Lats2 is relatively well understood in several tissues and organs, less is known about the function of Lats2 and Hippo signaling in adipose development. This study shows that Lats2 is an important modulator of adipocyte proliferation and differentiation via Hippo signaling. Upon activation, Lats2 phosphorylates YAP and TAZ, leading to their retention in the cytoplasm, preventing them from activating the transcription factor TEAD in the nucleus. Because TAZ remains in the cytoplasm, PPARgamma regains its transcriptional activity. Furthermore, cytoplasmic TAZ acts as an inhibitor of Wnt signaling by suppressing DVL2, thereby preventing beta-catenin from entering the nucleus to stimulate TCF/LEF transcriptional activity. The above effects contribute to the phenotype of repressed proliferation and accelerated differentiation in adipocytes. Thus, Lats2 regulates the balance between proliferation and differentiation during adipose development. Interestingly, this study provides evidence that Lats2 not only negatively modulates cell proliferation but also positively regulates cell differentiation (An, 2013).

Degradation of Dishevelled

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development (Simons, 2005).

Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry proteins have been identified associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the BTB protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, these data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway (Angers, 2006).

Wnt signaling plays pivotal roles in the regulation of embryogenesis and cancer development. Xenopus Dapper (Dpr) was identified as an interacting protein for Dishevelled (Dvl), a Wnt signaling mediator, and modulates Wnt signaling. However, it is largely unclear how Dpr regulates Wnt signaling. Evidence is presented that human Dpr1, the ortholog of Xenopus Dpr, inhibits Wnt signaling. The regions responsible for the Dpr-Dvl interaction have been identified in both proteins; the interaction interface is formed between the DEP (Dishevelled, Egl-10, and pleckstrin) domain of Dvl and the central and the C-terminal regions of Dpr1. The inhibitory function of human Dpr1 requires both its N and C terminus. Overexpression of the C-terminal region corresponding to the last 225 amino acids of Dpr1, in contrast to wild-type Dpr1, enhances Wnt signaling, suggesting a dominant negative function of this region. Furthermore, Dpr1 induces Dvl degradation via a lysosome inhibitor-sensitive and proteasome inhibitor-insensitive mechanism. Knockdown of Dpr1 by RNA interference up-regulates endogenous Dvl2 protein. Taken together, these data indicate that the inhibitory activity of Dpr on Wnt signaling is conserved from Xenopus to human and that Dpr1 antagonizes Wnt signaling by inducing Dvl degradation (Zhang, 2006).

Nuclear localization is required for Dishevelled function in Wnt/β-catenin signaling

Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined. This study reports that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein. These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/β-catenin signaling pathway (Itoh, 2005).

Regulation of Lethal giant larvae by Dishevelled

The establishment of polarity in many cell types depends on Lgl, the tumour suppressor product of lethal giant larvae, which is involved in basolateral protein targeting. The conserved complex of Par3, Par6 and atypical protein kinase C phosphorylates and inactivates Lgl at the apical surface; however, the signalling mechanisms that coordinate cell polarization in development are not well defined. This study shows that a vertebrate homologue of Lgl associates with Dishevelled, an essential mediator of Wnt signalling, and Dishevelled regulates the localization of Lgl in Xenopus ectoderm and Drosophila follicular epithelium. Both Lgl and Dsh are required for normal apical-basal polarity of Xenopus ectodermal cells. In addition, the Wnt receptor Frizzled 8, but not Frizzled 7, causes Lgl to dissociate from the cortex with the concomitant loss of its activity in vivo. These findings suggest a molecular basis for the regulation of cell polarity by Frizzled and Dishevelled (Dollar, 2005).

In multicellular organisms polarity is a fundamental property of cells, one that is required for asymmetric division, changes in cell shape, adhesion and migratory behaviour. Recent studies have indicated that the core mechanisms of cell polarization are conserved. In Drosophila epithelial cells, sensory organ precursors and neuroblasts, as well as in mammalian cells, the apical Par (partitioning defective) complex, which consists of the PDZ-domain-containing proteins Par6 and Par3, and atypical protein kinase C (aPKC), regulates apical-basal polarity and proper localization of cell fate determinants. Activation of aPKC in the Par6 complex results in the phosphorylation of Lgl and its dissociation from the cortex. In neuronal precursors, Lgl is required for the asymmetric targeting of cell fate determinants. Lgl binds Syntaxin 4, a component of the basolateral exocytic machinery, and the yeast Lgl homologues Sro7 and Sro77 are required for polarized exocytosis. These findings support the view that Lgl controls cell polarity through basolateral targeting (Dollar, 2005).

In developing tissues, individual cells, although capable of intrinsic polarization, must polarize correctly in the context of their environment by responding to extracellular polarizing cues. In fact, both coordinated polarization of cells in the plane of epithelial tissue and the alignment of intercalating cells during vertebrate gastrulation require Frizzled (Fz) and Dishevelled (Dsh), which are components of the Wnt signalling pathway. In addition, members of the Wnt pathway have been implicated in many processes that also involve the Par6-Par3-aPKC pathway, including asymmetric division of Drosophila sensory organ precursors and mitotic spindle orientation in Caenorhabditis elegans blastomeres. Despite these functional similarities, an understanding of the biochemical communication between these two pathways remains limited (Dollar, 2005).

This study has identified a Xenopus homologue of Lgl as a protein that interacts with Dsh in a yeast two-hybrid screen, raising the possibility that Lgl is regulated by the Wnt pathway. To assay Lgl activity, the effect of overexpressing Lgl in Xenopus embryonic ectoderm was determined. Animal pole blastomeres express Lgl and have a well-defined apical-basal polarity, including the apical restriction of aPKC, a negative regulator of Lgl, suggesting that they are an appropriate in vivo system in which to examine Lgl activity. A full-length cDNA encoding Xenopus Lgl was obtained and expressed as a fusion protein with green fluorescent protein (GFP) or Myc in Xenopus embryos. Just before gastrulation, a marked change in pigment distribution was observed in superficial ectoderm cells overexpressing Lgl1. This phenotype was observed in most embryos injected with either GFP-Lgl1 or Myc-Lgl1 RNA and was dependent on dose, because the pigment redistribution became more evident when larger amounts of RNA were injected (Dollar, 2005).

To see whether these pigment changes reflected cell polarity defects, embryos expressing GFP-Lgl1 were cross-sectioned and double immunostained for GFP and either aPKC, an apical marker, or occludin, which marks the basolateral membrane and developing tight junctions in stage 10 embryos. Cells expressing GFP-Lgl1 showed a marked change in polarity as compared with uninjected cells, as judged by the loss of apical aPKC and the spread of occludin to the apical surface. Most GFP-Lgl1 was localized to the basolateral membrane, consistent with conserved regulation by endogenous aPKC. Some GFP-Lgl1 was detected at the apical surface, however, suggesting that the observed effects were due to ectopic Lgl1 activity (Dollar, 2005).

To determine the localization of endogenous Lgl1 in these cells, antibodies were raised against Xenopus Lgl1, which showed that it was restricted to the basolateral membrane. In addition, an Lgl1 construct in which conserved aPKC phosphorylation sites were mutated localized mostly to the apical surface and caused identical pigmentation and polarity changes when smaller amounts of RNA were injected. These and other observations indicate that the overexpression phenotype may result from ectopic Lgl1 activity at the apical surface. This interpretation is consistent with the ability of a mouse homologue of Lgl to inhibit tight junction formation in tissue culture cells (Dollar, 2005).

To confirm that Lgl and Dsh interact in vivo in Xenopus embryonic cells, immunoprecipitations were carried out with lysates from embryos expressing tagged proteins. The fragment of Lgl identified in the yeast screen (HA-LglC) specifically co-precipitated with full-length Myc-Xdsh. Similarly, in a complementary experiment full-length GFP-Lgl1 co-precipitated with a region of Xdsh containing the DIX domain (Xdsh-N). In addition, endogenous Dsh associated with both Lgl constructs. Because most Lgl1 and Dsh molecules do not colocalize at their steady-state levels in the cell, the two proteins are likely to interact transiently, reminiscent of the regulation of Lgl by Par6-aPKC (Dollar, 2005).

To evaluate whether Dsh is required for Lgl function, the activity of GFP-Lgl1 was examined in embryos injected with a morpholino antisense oligonucleotide (XdshMO) that has been shown to reduce specifically endogenous Xdsh. Depletion of Xdsh completely suppressed ectopic pigmentation caused by Lgl1 RNA, indicating that Dsh is required for Lgl1 activity. Consistent with this, an Lgl1 construct lacking the region involved in Dsh binding did not cause the pigmentation changes characteristic of full-length Lgl1, although it was expressed in similar amounts (Dollar, 2005).

Whether Dsh is required for the subcellular localization of Lgl was examined. The membrane localization of GFP-Lgl1 was abolished in embryos injected with XdshMO. Western blot analysis showed that Myc-Lgl1 protein was decreased in embryos depleted of Dsh in a dose-dependent manner. This decrease was specific to Lgl1, because XdshMO did not alter the amount of GFP, which was coexpressed in the same embryos as an internal control. In addition, XdshMO did not have the same effect on the expression of Myc-LglDeltaC. In a complementary experiment, overexpression of Dsh RNA in blastula ectoderm resulted in an increase in endogenous Lgl1. These observations indicate that Dsh may be involved in regulating the localization and stability of Lgl. It was confirmed that Dsh is required for localization of Lgl1 to the membrane by examining the distribution of endogenous Lgl1 in XdshMO-injected cells, using GFP as a lineage tracer. Almost no basolateral Lgl1 was detected in GFP-positive cells that received XdshMO. It is concluded that Dsh is required for Lgl1 localization and activity in vivo, possibly by stabilizing Lgl (Dollar, 2005).

To assess whether the regulation of Lgl by Dsh is conserved in other organisms, the distribution of Lgl was examined in dsh-null mutant clones in Drosophila. To avoid a possible compensatory effect of maternal Dsh, the requirement for Dsh was tested in the follicle cell epithelium, which forms around the oocyte relatively late in development. In follicle cells, Lgl is localized to the lateral membrane and in the nucleus. Mutant dsh follicle cell clones were generated with the FLP/FRT system. It was found that Lgl was delocalized from the membrane of dsh mutant cells. These results extend the observations made in Xenopus ectoderm to the fly follicular epithelium and show that Dsh has a conserved role in subcellular localization of Lgl (Dollar, 2005).

Next whether Lgl and Dsh are required for apical-basal polarity in embryonic ectoderm was tested, because the findings predicted that depletion of Lgl1 and Dsh would cause similar apical-basal polarity defects. A morpholino oligonucleotide was designed that specifically decreased Lgl1 protein in vivo by gastrulation stages (LglMO). Embryos injected with LglMO or XdshMO, together with GFP RNA as a lineage tracer, were sectioned and stained with antibodies to aPKC, occludin, beta-catenin and beta1-integrin, along with antibodies against GFP. Both Lgl1 and Dsh depletion resulted in a loss of apical aPKC and in ectopic accumulation of occludin at the apical surface. By contrast, the basolateral localization of beta-catenin and beta1-integrin was unaffected by Lgl and Dsh depletion. These results show that Lgl1 is required for some, but not all, aspects of apical-basal polarity of the ectoderm. The similar defects seen in Dsh-depleted cells are probably due to the role of Dsh in Lgl regulation. Most importantly, these findings establish that Dsh, as well as Lgl1, is involved in apical-basal epithelial polarization (Dollar, 2005).

Because Dsh is an essential mediator of Fz signalling, whether Fz signals can influence Lgl activity was examined. Overexpression of Fz receptors has been shown to mimic aspects of active signalling. Fz8, Fz7 and Fz3 RNAs were co-injected with Myc-Lgl1 or GFP-Lgl1 RNA into animal blastomeres of four-cell Xenopus embryos. It was found that Fz8, but not Fz7 or Fz3, RNA completely suppressed Lgl1-dependent pigment redistribution, indicating that Fz8 specifically interferes with Lgl function. Moreover, the membrane localization of GFP-Lgl1 was markedly altered by coexpression of Fz8, but not by Fz7. All three Fz RNAs were equally efficient in interfering with morphogenetic movements in later development, consistent with similar amounts of protein expression. Overall amounts of Myc-Lgl1 were slightly reduced by coexpression of Fz8, but not Fz7. In contrast to Dsh-depleted embryos, however, Myc-Lgl1 was still highly expressed in these embryos, indicating that Fz8 primarily affects Lgl localization (Dollar, 2005).

Lastly, the delocalization of Lgl in response to Fz8 was not limited to overexpressed Lgl, but was also observed for endogenous Lgl. Different Fz receptors seem to function in a compartmentalized manner in the cell, as suggested by their differential distribution along the apical-basal axis of Drosophila epithelial cells. This raises the possibility that in the assay Fz8 is acting in a dominant-negative manner by sequestering Dsh from its required subcellular localization. It was found that Fz8, as well as Fz7 and Fz3, recruits Dsh-GFP to the basolateral surface, however, supporting the notion that the effect of Fz8 on Lgl is receptor-specific. It was concluded that individual Fz receptors can differentially influence the intracellular distribution and activity of Lgl (Dollar, 2005).

The control of Lgl by Dsh suggests a general mechanism for the coordinated regulation of cell polarity by extracellular signalling that is based on existing apical-basal polarity cues. For example, localized Fz-Dsh signalling could provide positional cues for the Par3-Par6-aPKC complex by locally depleting Lgl, which acts antagonistically in the formation of this complex. Consistent with this model, in Drosophila embryos Fz and Dsh colocalize with the apical Par complex in ectoderm cells and restrict the Par complex to the posterior cortex in sensory organ precursors. A partial disruption of apical-basal polarity in the plane of epithelial tissue by localized Fz-Dsh signalling could contribute to planar cell polarity, whereas a more complete disruption could result in epithelial-mesenchymal transformation and altered cell movements that are crucial for morphogenesis. Further studies are warranted to examine the interactions between the molecules involved in regulating planar and apical-basal polarity and those controlling morphogenetic movements in development (Dollar, 2005).

Dishevelled in sea urchins

ß-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, ß-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. In-vivo measurements of the half-life of fluorescently tagged ß-catenin in specific blastomeres has been used to demonstrate a gradient in ß-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3ß-mediated phosphorylation of ß-catenin. Calculations show that the difference in ß-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. Dishevelled (Dsh), a key signaling protein, is required for the stabilization of ß-catenin in vegetal cells; evidence is provided that Dsh undergoes a local activation in the vegetal region of the embryo. GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus (Weitzel, 2004).

Dishevelled, gastrulation and convergent extension movements in zebrafish and Xenopus

Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. The zebrafish silberblick (slb) locus is shown to encode Wnt11 and Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head. The requirement for Slb/Wnt11 is cell non-autonomous, and the results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. The slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway, suggesting that, as in flies, Wnt signaling might mediate morphogenetic events through a divergent signal transduction cascade. These results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation (Heisenberg, 2000).

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrates that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules toward the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus (Miller, 1999).

Different components of the Wnt signaling pathway, including several Wnts, Dishevelled, ß-catenin and dominant negative GSK3ß have been shown to induce complete secondary body axes in injected frogs. These studies support the idea that Wnt signaling is essential for dorso-ventral axis determination in Xenopus embryos. It is thought that Wnt signaling is also involved in specification of the anterior-posterior axis in vertebrates. This hypothesis is supported by experiments demonstrating that overexpression of Xwnt3a posteriorizes Xenopus ectodermal explants treated with noggin or follistatin and that Wnt3a mutant mice have deficiencies in posterior patterning. Xenopus Dishevelled (Xdsh) was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of Xdsh mRNA reveal a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activate anterior neuroectodermal markers (XAG1 and Xotx2) whereas the higher doses induce more posterior neural markers, such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contain maximal amounts of HoxB9 transcripts and develop notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activates anterior neuroectodermal markers, but fails to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage reveals activation of several organizer-specific genes that have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). While Goosecoid, Xlim1 and Xwnt8 are not induced in these explants, another early marginal zone marker, Xbra, is activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development. These findings argue against induction of the entire organizer by Xdsh in ectodermal cells, since certain organizer-specific genes (goosecoid and Xlim1) are not activated at any tested dose of Xdsh mRNA. Embryonic cells do not show sharp thresholds in response to different doses of Xdsh at early stages. In order to generate a complete spectrum of AP fates, neural inducers are likely to synergize with additional factors affecting pattern formation, e.g. Xbra and Xotx2. Thus different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis. It is unlikely that Xdsh activity represents a morphogen gradient in embryos since no localization of Xdsh transcripts can be seen in early gastrula stage. Since Xwnt8 fails to fully mimic the effects of Xdsh on ectodermal explants, it is likely that the activation of Xdsh does not solely reflect Wnt-mediated signaling but requires other signaling factors (Itoh, 1997).

The role of Drosophila dishevelled in Xenopus Wnt signaling has been tested. Xenopus embryos ectopically injected with Drosophila DSH mRNA developed duplicated axes similar to those seen in embryos injected with Wnt mRNAs. Thus the intracellular response to the Wnt signal has been conserved during evolution to such an extent that its components may be interchanged between distantly related species (Dominguez, 1995 and Rothbacher, 1995).

Injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation (Sokol, 1995).

The Dishevelled (Dsh) protein is required for Drosophila cells to respond to Wingless, Notch and Frizzled signals, but the molecular mechanisms of its action are not well understood. Using the ability of a mutant form of the Xenopus homolog of Dsh (Xdsh) to block Wnt and Dsh signaling in a model system, this work attempts to clarify the role of the endogenous Xdsh during the early stages of vertebrate development. A mutant Xdsh (Xdd1) with an internal deletion of the conserved PDZ/DHR domain was constructed. Overexpression of Xdd1 mRNA in ventral blastomeres of Xenopus embryos strongly inhibits induction of secondary axes by the wild-type Xdsh and Xwnt8 mRNAs, but does not affect the axis-inducing ability of beta-catenin mRNA. These observations suggest that Xdd1 acts as a dominant-negative mutant. Dorsal expression of Xdd1 causes severe posterior truncations in the injected embryos, whereas wild-type Xdsh suppresses this phenotype. Xdd1 blocks convergent extension movements in ectodermal explants stimulated with mesoderm-inducing factors and in dorsal marginal zone explants, but does not affect mesoderm induction and differentiation. It is concluded that a vertebrate homolog of Dsh is a necessary component of Wnt signal transduction and functions upstream of beta-catenin. These findings also establish a requirement for the PDZ domain in signal transduction by Xdsh, and suggest that endogenous Xdsh controls morphogenetic movements in the embryo (Sokol, 1996).

Although cell movements are vital for establishing the normal architecture of embryos, it is unclear how these movements are regulated during development in vertebrates. Inhibition of Xenopus Dishevelled (Xdsh) function disrupts convergent extension movements of cells during gastrulation, but the mechanism of this effect is unclear, since cell fates are not affected. In Drosophila, Dishevelled controls both cell fate and cell polarity, but whether Dishevelled is involved in controlling cell polarity in vertebrate embryos has not been investigated. Using time-lapse confocal microscopy, it has been shown that the failure of cells lacking Xdsh function to undergo convergent extension results from defects in cell polarity. Furthermore, Xdsh mutations that inhibit convergent extension correspond to mutations in Drosophila Dishevelled that selectively perturb planar cell polarity. Finally, the localization of Xdsh at the membrane of normal dorsal mesodermal cells is consistent with Xdsh controlling cell polarity. These results show that polarized cell behavior is essential for convergent extension and is controlled by vertebrate Dishevelled. Thus, a vertebrate equivalent of the Drosophila planar cell polarity signaling cascade may be required for normal gastrulation (Wallingford, 2000).

Cell polarity was examined in Keller explants by determining the orientation of membrane protrusions from individual cells. Whereas lamellipodia were predominantly extended from the mediolateral ends of control cells, this polarity was severely disrupted in both a mutant form of Xdsh, Xdd1, and Xdsh-expressing cells. Analysis of cell elongation and orientation of the long axis provided additional indicators of cell polarity in the Xenopus dorsal marginal zone (DMZ). As cells begin convergent extension, they align and elongate in the mediolateral axis; the mean length-to-width ratio (LWR) of control cells is 1.97. Cells expressing Xdd1 (LWR = 1.65) or Xdsh (LWR = 1.57) are not elongated. These LWRs are similar to those of DMZ cells before gastrulation or animal cap cells, which do not converge and extend. Thus, cell elongation is correlated with normal, but not inhibited, convergent extension. Polarity is severely disrupted in Xdd1-expressing explants and in Xdsh-expressing explants. These data show that cell polarity is severely disrupted in Xdd1- and Xdsh-expressing DMZ explants. Although polarized cell behavior has been shown to be associated with convergent extension, these results show that experimental disruption of cell polarity is directly associated with failure of both cell elongation and convergent extension (Wallingford, 2000).

During amphibian development, non-canonical Wnt signals (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier?) regulate the polarity of intercalating dorsal mesoderm cells during convergent extension. Cells of the overlying posterior neural ectoderm engage in similar morphogenetic cell movements. Important differences have been discerned in the cell behaviors associated with neural and mesodermal cell intercalation, raising the possibility that different mechanisms may control intercalations in these two tissues. Targeted expression of mutants of Xenopus Dishevelled (Xdsh) to neural or mesodermal tissues elicits different defects that are consistent with inhibition of either neural or mesodermal convergent extension. Expression of mutant Xdsh also inhibits elongation of neural tissues in vitro in Keller sandwich explants and in vivo in neural plate grafts. Targeted expression of other Wnt signaling antagonists also inhibits neural convergent extension in whole embryos. In situ hybridization had indicated that these defects are not due to changes in cell fate. Examination of embryonic phenotypes after inhibition of convergent extension in different tissues reveals a primary role for mesodermal convergent extension in axial elongation, and a role for neural convergent extension as an equalizing force to produce a straight axis. This study demonstrates that non-canonical Wnt signaling is a common mechanism controlling convergent extension in two very different tissues in the Xenopus embryo and may reflect a general conservation of control mechanisms in vertebrate convergent extension (Wallingford, 2001).

In Drosophila, Dishevelled functions to transduce signals along at least two different Wnt pathways: the canonical Wnt/ß-catenin pathway and the planar cell polarity pathway. Both of these pathways are also used in the Xenopus mesoderm. Canonical Wnt signals induce dorsal cell fates in the early embryo, and ectopic Wnt signaling on the ventral side induces a secondary axis. During gastrulation, non-canonical Wnt signals similar to PCP signaling control convergent extension of the dorsal mesoderm. The data strongly indicate that a similar molecular mechanism is at work during convergent extension of the posterior neural ectoderm (Wallingford, 2001).

The distinction between canonical and non-canonical pathways is illustrated by the similar effects of Dishevelled constructs on neural convergent extension. Both severely inhibit convergent extension in the dorsal mesoderm. However, the two constructs have opposite effects in secondary axis assays: one is fully functional for canonical Wnt signaling, while one inhibits these signals. Since these two mutants of Xdsh have indistinguishable effects on convergent extension in both neural ectoderm and dorsal mesoderm, Xdsh-mediated control of convergent extension is likely to be independent of canonical Wnt pathways in both tissues (Wallingford, 2001).

This role for non-canonical Wnt signaling is particularly intriguing in the neural tube, where canonical Wnt/ß-catenin signaling plays an important role in specifying cell fates. For example, expression of one construct inhibits convergent extension, but does not significantly alter the A/P pattern of the neural tube. However, inhibition of canonical Wnt signaling in the neural tube has dramatic effects on this pattern. As such, Xdsh may function simultaneously via canonical and non-canonical pathways to control both cell fate and cell motility in the posterior neural ectoderm (Wallingford, 2001).

In Xenopus, Dishevelled (Xdsh) signaling is required for both neural tube closure and neural convergent extension, but the connection between these two morphogenetic processes remains unclear. Indeed normal neurulation requires several different cell polarity decisions, any of which may require Xdsh signaling. Two issues are addressed in this study: (1) which aspects of normal neurulation require Xdsh function, and (2) what is the role of convergent extension in the closure of the neural tube. Xdsh signaling is not required for neural fold elevation, medial movement or fusion. Therefore, disruption of Xdsh signaling provides a specific tool for uncoupling convergent extension from other processes of neurulation. Using disruption of Xdsh signaling, it has been demonstrated that convergent extension is crucial to tube closure. Targeted injection has revealed that Xdsh function is required specifically in the midline for normal neural tube closure. It is suggested that the inherent movement of the neural folds can accomplish only a finite amount of medial progress and that convergent extension of the midline is necessary to reduce the distance between the nascent neural folds, allowing them to meet and fuse. Similar results with Xenopus strabismus implicate the planar cell polarity (PCP) signaling cascade in neural convergent extension and tube closure. Together, these data demonstrate that PCP-mediated convergent extension movements are crucial to proper vertebrate neurulation (Wallingford, 2002).

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis. A noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila, participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, it is shown that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene, is an essential component for gastrulation cell movement. Xpk encodes an 835-amino acid protein with a single PET domain and three repetitive LIM domains in its N-terminal half. Both gain-of-function and loss-of-function of Xpk severely perturbs gastrulation and causes spina bifida embryos without affecting mesodermal differentiation. XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation (Takeuchi, 2003).

The possibility that XPK activates JNK was examined because JNK has been reported to act in the noncanonical Wnt pathway downstream of Dsh. To evaluate JNK activation, the phosphorylation of a target of JNK, c-Jun, was tested in HEK293T cells transfected with Xdsh, Xpk, or both cDNAs. Xpk alone failed to activate JNK, whereas Xdsh activates JNK in a dose-dependent manner. However, cotransfection of Xdsh with Xpk but not with ΔP/L (lacking the PET and LIM domains) cDNAs dramatically increase the level of c-Jun phosphorylation, even at Xdsh levels, which alone cannot activate JNK efficiently. This result suggests that XPK cooperates with Xdsh to activate JNK through its P/L domain. At a high level of Xdsh protein, Xdsh alone can activate JNK to a certain level, and interestingly, wild-type Xpk or ΔP/L but not P/L suppresses the JNK activation by Xdsh, suggesting that the part of XPK excluding the PET/LIM domain may act negatively to Xdsh-mediated JNK activation at high levels of Xdsh. This is consistent with the observation that ΔP/L and Xpk including the LIM domain counteract each other. These observations prompted a test of whether XPK interacts physically with Xdsh. To test this possibility, a GST-pull-down assay of tagged XPK and Xdsh was carried out in HEK293T cells. Xdsh and XPK are each efficiently precipitated with the other's GST fusion protein, indicating that XPK and Xdsh interacted physically with each other. The interaction between XPK and Xdsh is conserved among species; the Drosophila Prickle PET/LIM domain has been reported to bind Dsh. The yeast two-hybrid assay also demonstrates that the PET/LIM domains of XPK are sufficient to bind Xdsh (Takeuchi, 2003).

It is speculated that XPK might act as a scaffold for JNK activation, so whether XPK binds to JNK was tested. Neither the PET/LIM nor the ΔPET/LIM is sufficient to bind JNK, and only wild-type XPK can bind JNK significantly. Nevertheless, this further suggests that XPK forms a ternary complex with Xdsh and JNK. Although this possibility was examined, the formation of the ternary complex could not be demonstrated (Takeuchi, 2003).

It has been proposed that Prickle generates asymmetric Frizzled and Dishevelled localization in the Drosophila wing, through the suppression of Fz and Dsh localization at the proximal cell cortex. In this study, it is shown that XPK is a key component connecting Xdsh to JNK activation during Xenopus gastrulation. It has been predicted from Drosophila genetics that JNK is one of the downstream targets of the PCP pathway. These results reinforce the idea that the noncanonical Wnt (PCP) pathway regulates gastrulation cell movements in vertebrates through JNK activation. To further understand the pathway, attempts are currently being made to identify XPK-interacting components that regulate JNK activation (Takeuchi, 2003).

In addition to the canonical Wnt/ß-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila and the Wnt/calcium pathway in vertebrate embryos. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation. To further test this hypothesis, two zebrafish homologs were identified of the Drosophila PCP gene prickle (pk), both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/ß-catenin signaling. Like noncanonical Wnts, overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. These results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways (Veeman, 2003).

Association of Dishevelled with Eph tyrosine kinase receptor and ephrin mediates cell repulsion

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, Xenopus Dishevelled (Xdsh) is found to form a complex with Eph receptors and ephrin-B ligands and mediate the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affects the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induces the activation of RhoA and Rho kinase in the EphB2-overexpressing cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. It is proposed that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity (Tanaka, 2003).

Mutation in mammalian Dishevelled

Mice completely deficient for Dvl1, one of three mouse homologs of the Drosophila segment polarity gene Dishevelled, were created by gene targeting. Dvl1-deficient mice are viable, fertile, and structurally normal. Surprisingly, these mice exhibit reduced social interaction, including differences in whisker trimming, deficits in nest-building, less huddling contact during home cage sleeping, and subordinate responses in a social dominance test. Sensorimotor gating is abnormal, as measured by deficits in prepulse inhibition of acoustic and tactile startle. Thus, Dvl1 mutants may provide a model for aspects of several human psychiatric disorders. These results are consistent with an interpretation that common genetic mechanisms underlie abnormal social behavior and sensorimotor gating deficits and implicate Dvl1 in processes underlying complex behaviors (Lijam, 1997).

Murine Dishevelled 2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2-/- mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, ~90% of Dvl2-/- mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities are more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2-/- and Dvl1-/-;Dvl2-/- mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2%-3% of Dvl2-/- embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundantcy between Dvl1 and Dvl2 in some phenotypes (Hamblet, 2002).

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. Mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, it has been shown that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, a high degree of conservation was demonstrated between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows C-terminal DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea (Wang, 2006).

Dishevelled and Wnt/beta-catenin signaling

Frizzled receptors are components of the Wnt signaling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) were used and whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal is described. Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signaling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signaling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signaling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5, in which the tryptophan is substituted by a tyrosine (Umbhauer, 2000).

Frizzled (fz) functions as a 7-transmembrane receptor in the Frizzled-Dishevelled signal transduction cascade. It is involved in architectural control of development in species as divergent as Drosophila and vertebrates. Regulation of multicellular architecture requires control of cell alignment, but also involves an equilibrium among cell proliferation, differentiation, and apoptosis. Recently, modulation of the Frizzled-Dishevelled (Dvl) cascade has been related to apoptosis. However, the role of ß-catenin (a second messenger in the Frizzled-Dishevelled cascade) in programmed cell death is a matter of debate. To elucidate the role of this cascade in apoptosis, the effect of over-expression of fz1, fz2, dvl1, and ß-catenin was investigated. The signal transduction pathway and the involvement of ß-catenin were further investigated by using different inhibitors. These experiments were performed in different cell types: COS7, 293, and PC12. Overexpression of fz1, fz2, and dvl1 induce apoptosis in COS7 and 293 cells. ß-Catenin appears to be the mediator for this process since ß-catenin overexpression as well as lithium and valproate induce apoptosis. In contrast, lithium treatment does not result in apoptosis in PC12 cells. It is concluded that different components of the Frizzled-Dishevelled cascade can induce apoptosis, but that this effect is dependent on the cell type. ß-catenin transfection in 3T3 fibroblasts induces apoptosis independent of its transactivation function with LEF-1, suggesting that LEF is not involved in the apoptosis induced by ß-catenin overexpression. Further work will be needed to discover what the mechanism is by which ß-catenin influences apoptosis (van Gijn, 2001 and references therein).

Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. PP2A functions in Wnt signal transduction have been studied using early Xenopus embryos. The catalytic subunit of PP2A (PP2A-C) potentiates secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibits Dishevelled-mediated signal transduction, as does the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C does not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin is sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, it is proposed that PP2A regulates the Wnt pathway at multiple levels (Ratcliffe, 2000).

The Wnt/ß-catenin pathway plays important roles during embryonic development and growth control. The B56 regulatory subunit of protein phosphatase 2A (PP2A) has been implicated as a regulator of this pathway. Loss-of-function analysis of PP2A:B56epsilon during early Xenopus embryogenesis has been examined. PP2A:B56epsilon is required for Wnt/ß-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand. Maternal PP2A:B56epsilon function is required for dorsal development, and PP2A:B56epsilon function is required later for the expression of the Wnt target gene engrailed, for subsequent midbrain-hindbrain boundary formation, and for closure of the neural tube. These data demonstrate a positive role for PP2A:B56epsilon in the Wnt pathway (Yang, 2003).

Loss-of-function analysis in Drosophila and zebrafish suggests that PP2A:B56 family members play roles in the PCP pathway upstream of dsh. Although Frizzled and Dsh function in both the PCP pathway and the canonical Wnt/ß-catenin pathway, the requirement for PP2A:B56epsilon upstream of Dsh in Wnt/ß-catenin signaling has not been shown previously. The loss-of-function analysis of PP2A:B56epsilon presented here directly supports the hypothesis that PP2A:B56epsilon is required for Wnt/ß-catenin signaling. PP2A:B56epsilon is required for the expression of multiple Wnt/ß-catenin target genes, including Xnr5, Xnr6 during pre-MBT stages; Xnr3, siamois, noggin, goosecoid, cerberus and Xdkk1, during the gastrula stage, and en2 during neurulation. Furthermore, PP2A:B56epsilon is required for ß-catenin accumulation in cells that respond to endogenous Wnt signaling, such as dorsal blastomeres in blastula-stage embryos and in the neural ectoderm of early neurulae. In contrast, the constitutive turnover of ß-catenin in ventral blastomeres and in oocytes is not sensitive to PP2A:B56epsilon depletion. The requirement for PP2A:B56epsilon in canonical Wnt signaling is further supported by epistatic analysis. Dorsal gene expression is rescued by dsh and downstream Wnt signaling components but not by Wnt8b. Based on these observations, it is concluded that PP2A:B56epsilon is required for Wnt/ß-catenin signaling downstream of the Wnt ligand and upstream of dsh (Yang, 2003).

In contrast to observations with depletion of PP2A:B56epsilon, overexpression of PP2A:B56 family members inhibits Wnt/ß-catenin signaling. Other B56 family members, such as B56alpha, may have different functions in the Wnt/ß-catenin pathway. For example, overexpression of PP2A:B56epsilon in dorsal blastomeres leads to anterior truncation of embryos without altering dorsal gene expression, a phenotype that is distinct from the strong ventralization caused by PP2A:B56alpha overexpression. These observations are consistent with the suggestion that the two isoforms have different functions in the Wnt pathway. However, overexpressed PP2A:B56epsilon also inhibits secondary dorsal axis induction by positive modulators of Wnt/ß-catenin pathway. To explain this, PP2A:B56epsilon could regulate multiple steps in Wnt signaling, and loss-of-function at an upstream step may obscure an inhibitory role at a downstream step in the pathway. Alternatively, overexpression of PP2A:B56 or other subunits that interact with the PP2A complex could sequester essential components of the phosphatase complex and interfere with its normal function. It also remains unclear whether PP2A:B56epsilon regulates Wnt signaling through modulation of PP2A activity. Further experiments are needed in order to understand the mechanism by which PP2A:B56epsilon regulates the Wnt/ß-catenin pathway (Yang, 2003).

The Wnt/ß-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/ß-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits ß-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or ß-catenin, but has no effect on the signaling activities of a stabilized ß-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated ß-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, it is suggested that XRap2 is involved in Wnt/ ß-catenin signaling as a modulator of the subcellular localization of Dsh (Choi, 2005).

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-β-catenin signaling pathway requires both Gαo and Gαq heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gαo protein. Gαo propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-β-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-β-catenin signaling (Bikkavilli, 2008).

PTK7 recruits dsh to regulate neural crest migration

PTK7 regulates planar cell polarity (PCP) signaling during vertebrate neural tube closure and establishment of inner ear hair cell polarity; however, its signaling mechanism is unknown. This study demonstrates a new function for PTK7 in Xenopus neural crest migration and uses this system in combination with in vitro assays to define the intersection of PTK7 with the non-canonical Wnt signaling pathway that regulates PCP. In vitro, using Xenopus ectodermal explants, it was shown that PTK7 recruits dishevelled (dsh) to the plasma membrane, a function that is dependent on the PDZ domain of dsh, as well as on the conserved kinase domain of PTK7. Furthermore, endogenous PTK7 is required for frizzled7-mediated dsh localization. Immunoprecipitation experiments confirm that PTK7 can be found in a complex with dsh and frizzled7, suggesting that it cooperates with frizzled to localize dsh. To evaluate the in vivo relevance of the PTK7-mediated dsh localization, Xenopus neural crest migration was analyzed, since loss-of-function of PTK7 inhibits neural crest migration in whole embryos as well as in transplanted neural crest cells. Supporting the in vivo role of PTK7 in the localization of dsh, a PTK7 deletion construct deficient in dsh binding inhibits neural crest migration. Furthermore, the PTK7-mediated membrane localization of a dsh deletion mutant lacking PCP activity inhibits neural crest migration. Thus, PTK7 regulates neural crest migration by recruiting dsh, providing molecular evidence of how PTK7 intersects with the PCP signaling pathway to regulate vertebrate cell movements (Shnitsar, 2008).

Neural crest specification by noncanonical Wnt signaling and PAR-1

Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate (Ossipava, 2011).

Noncanonical Wnt ligands, such as Wnt5a and Wnt11, do not stabilizeα-catenin or activate TCF-dependent transcription, but regulate morphogenetic processes that involve changes n cell shape and motility, which are sometimes referred to as planar cell polarity (PCP). The signaling from Wnt5 or Wnt11 is thought to involve Ror and Ryk receptors, small Rho GTPases, Rho-associated kinase, c-Jun N-terminal kinases and intracellular calcium. Although noncanonical Wnt pathways have been shown to function in NC cell migration, their importance for NC specification has remained unclear (Ossipava, 2011).

Craniofacial defects in Wnt5a knockout mice, and in wnt11 (silberblick) and wnt5 (pipetail) zebrafish mutant embryos suggest possible roles for noncanonical Wnt signaling in NC development. The results of this study support the view that noncanonical signaling from Wnt11R is essential for NC specification in Xenopus embryos and that it might act by changing the localization and activity of the polarity kinase PAR-1 (Ossipava, 2011).

PAR proteins are conserved regulators of cell polarity that interact with several embryonic signaling pathways, including the Wnt pathway. PAR-1 associates with Dishevelled (Dvl, or Dsh) and participates in Frizzled-dependent Dvl recruitment. This study shows that PAR-1 is itself required for NC specification and can rescue NC defects in embryos with inhibited Wnt5 and Wnt11 signaling. These findings identify PAR-1 as a molecular target for noncanonical Wnt signaling and reveal an unexpected causal connection between cell polarization and the NC cell fate (Ossipava, 2011).

Regulation of planar cell polarity by Smurf ubiquitin ligases

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. This study reports an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. This work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE (Narimatsu, 2009).

Mechanistic analysis of PCP signaling has revealed physical interactions between otherwise differentially localized PCP components that include interactions between Vangl, Prickle and Dvl. This study has mapped interactions between Smurfs and components of the Wnt signaling pathway using a systematic screen and identified an interaction between Smurf and Dvl that is dependent on phosphorylation of Dvl. Accordingly, a DEP domain mutant that blocked Dvl phosphorylation also blocked Smurf interaction, as did a DEP mutation that is PCP-specific in the fly. These findings are in agreement with key roles previously defined for the DEP domain in PCP signaling in flies and vertebrates. Constitutive interactions were identified between Par6 and Dvl, consistent with previous observations in neurons, as well as an interaction between Par6 and Prickle. Together, these results suggest a model in which Par6 is engaged in a trimeric complex with Prickle and Dvl that upon phosphorylation-dependent recruitment of Smurf to Dvl in response to Fzd signaling leads to Prickle ubiquitination and degradation. Consistent with this, it was found that Wnt5a induced Smurf-Par6 interactions and that Prickle bound to Par6 was subject to Smurf-dependent ubiquitination and turnover that was also dependent on the Dvl binding domain of Par6. Moreover, in Smurf mutants increased levels of Pk1 protein the following two observations were made: (1) loss of the local asymmetric distribution of Pk1 in the neuroepithelium, and in the cochlea, (2) Pk1 that was normally localized in a medial crescent in the OHCs, extended to the lateral side (Narimatsu, 2009).

Dvl has also been shown to interact with the transmembrane protein Vangl, which also binds Pk and Pk can antagonize Dvl function. Moreover, Diego, an ankyrin repeat containing protein has been shown to directly compete with Pk for Dvl binding and can promote Dvl function in the PCP pathway via recruitment to membranes. Whether Diego functions primarily on Dvl, or plays a role in antagonizing Pk coassembled with Par6, perhaps in cooperation with Smurfs is unknown, but altogether these findings suggest that Pk and Dvl assemble via unique signaling complexes that respond to different extrinsic cues during PCP signaling. Consequently, while all PCP components might physically interact with each other, their asymmetric distribution may be maintained through dynamic modulation of complex membership (Narimatsu, 2009).

Although PCP and CE have been linked via shared components and a key role for noncanonical Wnt signaling, the relationship of the molecular pathways governing tissue polarity to CE has been less clear. In particular, during CE the neuroepithelium and the underlying axial mesoderm do not display the highly organized structures typically associated with PCP and tissue polarity and while Vangl and Dvl show asymmetric distribution in the cochlea, there is no evidence for asymmetric distribution of either protein during CE in the mouse. Regardless, Par6-dependent asymmetric localization of endogenous Pk1 was clearly detected in neuroepithelial cells; this was manifest at the cellular level, and in Smurf DKO mutants this asymmetry was lost. Furthermore, Par6 can also control the stable mediolateral extension of cell protrusions during CE. Localized Fzd signaling may thus regulate the asymmetric distribution of specific PCP components such as Pk1 in the absence of asymmetric distribution of all PCP pathway components. Vertebrates may thus broadly employ PCP signaling to control polarity at the local and even cellular level. Consistent with this, noncanonical Wnt signaling, Dvl and Par6 have all been shown to play key roles in regulating directed cell motility and the polarity of neurons (Narimatsu, 2009).

These results demonstrate a noncanonical Wnt signaling pathway in which Smurf is recruited to Par6 via Dvl to regulate the degradation of PCP pathway components that in turn controls the asymmetric distribution of Pk1. Signal dependent degradation of PCP components may thus allow for dynamic use of the pathway in a local manner during convergent extension, as well as in the establishment of tissue polarity in organized epithelia such as the inner ear (Narimatsu, 2009).

Developmental biology of Mammalian Dishevelled

A mouse homolog of the Drosophila dsh segment polarity gene has been isolated. The 695 amino acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. While no functional motifs have been identified, one region of Dvl-1 is similar to a domain of discs large-1. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with the highest levels found in brain and testis (Sussman, 1994).

A second Dishevelled homolog has been identified in mice. Dvl-2 is equally related to the Drosophila DSH sequence as to that of Dvl-1, but Dvl-2 is most similar to the Xenopus homolog Xdsh. Like the other genes, Dvl-2 is ubiquitously expressed throughout most of embryogenesis and is expressed in many adult organs. In fact all the components of the Wingless signal transduction pathway are ubiquitously expressed in flies and vertebrates, except for wingless itself. Dvl-2 can partially rescue the segmentation defects in Drosophila embryos devoid of dsh, while Dvl-1 fails to rescue Drosophila dsh mutants (Klingensmith, 1996).

The Dvl3 gene maps to mouse chromosome 16. The predicted amino acid sequence shares 64 and 62% identity to Dvl1 and Dvl2, respectively. The region of highest conservation between all three Dvl coding regions, at 97% identity, is noted at the PDZ domain (also termed the DHR domain or GLGF motif), a motif of 60 amino acids present in all dishevelled encoded proteins. In adult mice, Dvl3 expression is widespread with highest levels exhibited in brain, ovary, and heart. In embryos, Dvl3 is expressed in every tissue between 7.5 and 9.5 days postcoitum, and by 10.5 days postcoitum highest expression is seen in the dorsal root ganglia, somites, limb buds, branchial arches, heart, gut and throughout the developing central nervous system (Tsang, 1996).

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to an understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. The proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles. Its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding, resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle that lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways (Millar, 1999).

Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. The subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, was examined in the embryonic mouse kidney. Dishevelled associates with actin fibers and focal adhesion plaques in metanephric mesenchymal cells. Stimulation of Wnt signaling leads to profound changes in metanephric mesenchymal cell morphology, including disruption of the actin cytoskeleton, increased cell spreading, and increased karyokinesis. Upon activation of Wnt signaling, dishevelled also accumulates in and around the nucleus. Casein kinase Iepsilon colocalizes with dishevelled along actin fibers and in the perinuclear region, whereas axin and GSK-3 are only present around the nucleus. These data indicate a branched Wnt signaling pathway comprising a canonical signal that targets the nucleus and gene expression, and another signal that targets the cytoskeleton and regulates cell morphogenesis (Torres, 2000).

Dishevelled and microtubule stability

Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. The cellular localization and function of the mouse Dishevelled protein, DVL-1, has been examined. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3ß (GSK-3ß) and blocked by expression of GSK-3ß. These findings suggest that DVL-1, through GSK-3ß, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity (Krylova, 2000).

DVL-1 regulates MT stability as expression of DVL-1 increases the level of acetylated MTs and protects MTs from depolymerization by nocodazole in COS and neuroblastoma 2a cells. Similar results have been observed with microtubule-associated proteins such as Tau, MAP-1B, and MAP-2, which increase MT stability. Thus, DVL-1 acts as a MT stabilizing factor. How does DVL-1 regulate MT stability? Dishevelled proteins contain three conserved domains, a DIX domain also present in Axin; a negative regulator of the WNT signaling pathway; a PDZ domain present in a number of junctional proteins of the PSD-95 and Disc-large family, and a DEP domain present in the Caenorhabditis elegans protein Egl-10 and in Plecktrin. The PDZ domain is required for Armadillo stabilization through the canonical WNT pathway. In contrast, the DEP domain, but not the PDZ domain, is required for JNK signaling. The PDZ domain is also required for MT stabilization, while deletion of the DIX domain has a weaker effect. In contrast, deletion of the DEP domain does not affect the MT stabilizing function of DVL-1. The requirement of the PDZ domain suggests that DVL-1 may be acting through the canonical pathway. Consistent with these findings, DVL-1 stabilizes MTs through the inhibition of GSK-3ß, a downstream component of the canonical WNT pathway. This mechanism is supported by three findings: (1) expression of GSK-3ß blocks the MT stabilizing activity of DVL-1; (2) dnGSK-3ß enhances DVL-1 function; (3) lithium, an inhibitor of GSK-3ß, mimics the MT stabilizing activity of DVL-1. Inhibition of GSK-3ß by DVL-1 could lead to changes in the phosphorylation of microtubule-associated proteins such as Tau, MAP-1B, and MAP-2 that are direct targets of GSK-3ß. Studies of Tau and MAP-1B show that GSK-3ß-mediated phosphorylation decreases the ability of Tau to bind to MTs and decreases MT stabilization by MAP-1B. Thus, inhibition of GSK-3ß by DVL-1 may change the ability of MAPs to stabilize MTs. Although inhibition of GSK-3ß by lithium stabilizes MTs, DVL-1 has a stronger stabilizing effect than do high levels of lithium. Thus, it is likely that DVL-1 promotes MT stability via inhibition of GSK-3ß, but an alternative pathway may also contribute to this function of DVL-1. Dishevelled has recently been found to colocalize with actin fibers. More importantly, Dishevelled has been proposed to influence the actin cytoskeleton through the JNK pathway. Since MT dynamics is influenced by actin, Dishevelled could regulate MTs indirectly through the JNK pathway by changing the actin cytoskeleton. However, this mechanism seems unlikely, because the DEP domain of DVL-1, essential for the JNK pathway, is not required for MT stability. Consequently, these data suggest that MT stabilization by DVL-1 is mediated through GSK-3ß signaling and a parallel, as yet unidentified, pathway (Krylova, 2000).

In maturing neurons, endogenous DVL-1 colocalizes with axonal MTs that contain high levels of stable MTs. More importantly, DVL-1 increases the level of stable MTs in differentiated NB2a neuroblastoma cells and protects axonal MTs against nocodazole treatment. These findings suggest that neuronal DVL-1 may stabilize axonal MTs in vivo. Consistent with this view, a higher proportion of endogenous DVL-1 becomes associated with MTs from adult brain samples, which are enriched in stable MTs. Moreover, DVL-1 sediments along with cold stable MT fractions; these fractions represent the most stable pool of MTs. Thus, the localization of DVL-1 to MTs correlates with increased MT stability in maturing axons. Further studies are needed to address the in vivo role of DVL-1 in microtubule dynamics in developing neurons (Krylova, 2000).

Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling

The vertebrate planar cell polarity (PCP) pathway controls polarized cell behaviors rather than cell fate. Disruption of Xenopus laevis orthologs of the Drosophila PCP effectors inturned (in) or fuzzy (fy) affects not only PCP-dependent convergent extension but also elicits embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling result from a broad requirement for Inturned and Fuzzy in ciliogenesis. These proteins govern apical actin assembly and thus control the orientation, but not assembly, of ciliary microtubules. Finally, accumulation of Dishevelled and Inturned near the basal apparatus of cilia suggests that these proteins function in a common pathway with core PCP components to regulate ciliogenesis. Together, these data highlight the interrelationships between cell polarity, cellular morphogenesis, signal transduction and cell fate specification (Park, 2006).


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