Gene name - dishevelled
Cytological map position - 10B3-8
Function - signal transduction
Symbol - dsh
Genetic map position - chrX:11,249,914-11,252,759
Classification - DEP domain, DIX domain, PDZ domain
Cellular location - cytoplasmic
|Recent literature||Mannava, A. G. and Tolwinski, N. S. (2015). Membrane bound GSK-3 activates Wnt signaling through Disheveled and Arrow. PLoS One 10: e0121879. PubMed ID: 25848770
Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. This study examined the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. Targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. These results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol.
|Mund, T., Graeb, M., Mieszczanek, J., Gammons, M., Pelham, H. R. and Bienz, M. (2015). Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled Open Biol 5. PubMed ID: 26701932
Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1-3). This study shows that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. Notch intracellular domains were shown to be substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state (Mund, 2015).
|Weber, U. and Mlodzik, M. (2017). APC/CFzr/Cdh1-dependent regulation of planar cell polarity establishment via Nek2 kinase acting on Dishevelled. Dev Cell 40(1): 53-66. PubMed ID: 28041906
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. A post-mitotic requirement is indicated for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. This study has thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.
|Wang, Y., Naturale, V.F. and Adler, P.N. (2017).
Planar cell polarity effector Fritz
interacts with Dishevelled and has multiple functions in regulating PCP.
G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28258110
The Planar cell Polarity Effector (PPE) genes inturned, fuzzy and fritz are downstream components in the frizzled/starry night signaling pathway, and their function is instructed by upstream Planar Cell Polarity (PCP) core genes such as frizzled and disheveled PPE proteins accumulate asymmetrically in wing cells and function in a protein complex mediated by direct interactions between In and Frtz and In and Fy. How the PCP proteins instruct the accumulation of PPE protein is unknown. This study found a likely direct interaction between Dishevelled and Fritz and Dishevelled and Fuzzy that could play a role in this. It has been previously shown that mild over expression of frtz rescues a weak in allele. To determine if this is due to extra Frtz stabilizing mutant In or due to Frtz being able to bypass the need for In, a precise deletion of the inturned gene (inPD) was generated. Mild overexpression of Fritz partially rescues inPD, indicating that fritz has In independent activity in PCP. Previous studies of PPE proteins used fixed tissues, and did not provide any insights into the dynamic properties of PPE proteins. In this study, CRISPR/Cas9 genome editing technology was used to edit the fritz gene to add a green fluorescent protein tag. fritzmNeonGreen provides complete rescue activity and works well for in vivo imaging. Data show that Fritz is very dynamic in epidermal cells and preferentially distributed to discrete membrane subdomains ("puncta"). Surprisingly, it was also found in in stripes in developing bristles.
|Weber, U. and Mlodzik, M. (2017). APC/CFzr/Cdh1-Dependent Regulation of Planar Cell Polarity Establishment via Nek2 Kinase Acting on Dishevelled. Dev Cell 40(1): 53-66. PubMed ID: 28041906
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. This study indicates a post-mitotic requirement for the APC/CFzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/CFzr/Cdh1 substrate. This study has thus uncovered a pathway of regulation whereby APC/CFzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.
In Drosophila, Dishevelled is required in two distinct signaling pathways that share Frizzled receptors of similar structure, but have distinct intracellular signaling routes. Dishevelled phosphorylation is concurrent with Wingless pathway activation. Wingless serves to activate Dsh, which in turn inactivates Shaggy/Zeste white 3. In the absence of signaling, Armadillo is targeted for proteolytic degradation through phosphorylation by Shaggy. Arm accumulates in the cytoplasm and nucleus, where it activates Wg target genes by complexing with Pangolin/TCF transcription factors. Experiments suggest that upon Wnt signaling, part of the cytoplasmic pool of Dsh is phosphorylated and recruited to the membrane. Deletion analysis experiments have shown that the Dsh DEP domain is not required to induce Arm accumulation. The Dishevelled role in the tissue polarity pathway clearly indicates that, compared to its role in the Wingless pathway, different downstream components are involved. Rather, dominant genetic interactions and rescue experiments in the eye imaginal disc suggest that signaling is routed through a Rho/Rac and JNK/SAPK-like kinase cascade. Thus, Dsh is involved in two distinct Wnt signaling pathways, raising the question of how Dsh routes the information to the right effector cascade in each case (Boutros, 1999 and references).
dishevelled is an integral part of the wingless pathway. Wingless is secreted from anterior segmental compartments (for details, see wingless and engrailed), and is induced by Hedgehog, a protein made in posterior compartments. Wingless is required for the maintenance of engrailed synthesis, and for the accumulation of Armadillo protein (Klingensmith, 1994). Cells lacking dsh are unable to adopt the fates specified by Wingless.
Dishevelled is one element in a chain of interacting proteins that carry the wingless signal to sites within the cell, including the cell membrane and the nucleus. Phosphorylation is the most important biochemical mechanism for carrying signals between proteins. Dishevelled, a phosphoprotein, is synthesized uniformly throughout the embryo, but its level of phosphorylation varies with the level of wingless activity, (Yanagawa, 1995). Evidence that the serine threonine kinase shaggy/zeste white-3 is downstream of dishevelled suggests that it is not this kinase that is responsible for DSH phosphorylation, but that DSH phosphorylation is carried out by an as yet unidentified kinase (Yanagawa, 1995).
Phosphorylated DSH is correlated with high levels of cytoplasmic phosphorylated Armadillo. The function of this modification of ARM is not yet understood (Peifer, 1994), but certainly DSH and the wingless pathway are involved in cell surface integrity, acting through Armadillo on adherens junction stability (Klingensmith, 1994).
Most if not all biological organisms demonstrate an incredible (though imperfect) symmetry of structure and patterning. By symmetry is meant the positional repetition of a pattern on opposite sides of a dividing line or plane, distributed about a center or axis, resulting in geometric regularity. Symmetry is only one aspect of patterning. A second aspect, tissue polarity is exemplified by the pattern of epithelial hairs. For example, all the hairs of the wing surface point in the same direction. What is the biological basis of tissue symmetry and polarity? In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). PCP signaling controls the polarity of epithelial cells within a plane orthogonal to their apical-basal axis. One manifestation of this cellular polarity is the oriented organization of trichomes (cell hairs). In wild-type flies, the cell hair arising from each cell's distal vertex contributes to a parallel and specifically oriented array. Mutations in dsh, as well as additional genes including frizzled (fz), prickle (pk),inturned (in), fuzzy (fy),multiple wing hairs (mwh), and others all disrupt the polarity of the trichomes. The resulting mutant phenotypes include swirls and distortions of the hair polarity pattern, and in some instances, more than one trichome per cell. A putative signal transduction pathway has been proposed, which serves to polarize cells, allowing them to distinguish one side of the cell from the other, and to propagate this information from cell to cell. In this pathway, Frizzled, a seven-transmembrane protein (without apparent primary sequence homology to the G-protein-coupled receptors) acts as a receptor, functioning upstream of Dsh. Dsh then antagonizes the activities of Fuzzy, Inturned (both novel transmembrane proteins), and Mwh; in turn, it is proposed that these proteins regulate the cytoskeletal apparatus responsible for control of PCP. Mutations in RhoA affect this process, and experiments with dominant-negative mutants have implicated Cdc42 and Rac1 as additional effectors. Pk is proposed to function either in transmission of the signal to adjacent cells, or in interpretation of the directionality of the signal. Thus far, no ligand for the PCP pathway has been identified. Although a tentative signal transduction pathway has been proposed, the mechanism by which asymmetry is established in the responding cells is not understood (Axelrod and references, 1998).
In Drosophila, planar cell polarity signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. A heterologous system was used to examine the interaction of Dsh with Frizzled. mRNAs encoding Fz or Frizzled2 and a fusion of Dsh to green fluorescent protein (Dsh-GFP) were synthesized in vitro and injected into Xenopus embryos at the four-cell stage. Animal caps from stage 9 embryos, dissected to reveal the blastocoelar cells, were then examined by confocal microscopy. Dsh is recruited selectively to the membrane by Fz but not Frizzled2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. When Fz is expressed simultaneously with Dsh-GFP, Dsh-GFP shows a qualitative redistribution to the membrane or cell cortex. Under these conditions, localization of Dsh-GFP to filopodia present on the blastocoelar (free) surfaces of the animal cap cells was also noticed. Staining with phalloidin (and Dsh-GFP) revealed that the filopodia contain filamentous actin. It is interesting to note that although the filopodia stain with Dsh-GFP, little or no Fz localizes there; at the cell cortex, the Fz and Dsh-GFP show imperfect colocalization. Fz staining is localized predominantly to the plasma membrane, and to a lesser extent to intracellular membranes (probably ER and/or Golgi) in these cells. This suggests that while Fz may induce localization of Dsh-GFP to the membrane and filopodia, it may do so by a mechanism other than direct binding. Frizzled2, the Wingless receptor, fails to induce membrane localization of Dsh, even in the presence of a functional Fz2 ligand (Axelrod, 1998).
Drosophila Dsh is a modular protein of unknown function that is well conserved in relation to its vertebrate homologs. Alignment of family members reveals three conserved domains. The first, a DIX domain, is similar to a domain in murine Axin (see Drosophila Axin), a recently described modulator of the Wnt1 pathway. The second contains a PDZ domain; PDZ domains recognize and bind short motifs at the carboxyl termini of proteins (but may bind other motifs as well). PDZ domains can also form dimers. The third domain, called DEP, is conserved among a set of proteins that have in common the ability to regulate various GTPases, including both heterotrimeric G proteins and Ras-like small GTPases. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. A single amino acid substitution in the DEP domain of Dsh is shown to confer a loss of function for PCP signaling, yet the mutant protein is functional for Wg signaling. This single amino acid substitution, coded for by the dsh1 allele, allows for translocation to the membrane, but is thought to impair the ability of Dsh1 to associate with its target at the membrane. This altered membrane interaction diminishes the ability of Dsh1 to function in PCP signaling (Axelrod, 1998).
Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. For example, the individual domains Dsh(DIX) and Dsh deleted for the PDZ domain, are each dominant negative for Wg signaling but have no effect on PCP signaling. Overexpression of Zeste white3, or an activated Arm protein, both involved in Wingless signaling, also fail to produce any effect on PCP. It is proposed that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation. This analysis suggests that Dsh has different interactions in PCP and Wg signaling and predicts an additional genetic behavior (Axelrod, 1998).
If Wg and planar polarity signaling utilize Dsh in a common fashion, then ectopic activation of one pathway should be able to cross-activate the other by promiscuously activating Dsh. In contrast, if each pathway utilizes Dsh in a distinct fashion, then ectopic activation might sequester Dsh in pathway-specific complexes, rendering it unavailable and therefore titrating the activity of the other pathway. These possibilities could best be tested under conditions in which Dsh is limiting. Overexpression of Fz causes a dominant gain-of-function PCP phenotype, and this phenotype is sensitive to the dose of dsh. Can Wg cross-activate Dsh activity for PCP signaling, or can it sequester Dsh? To investigate this question, it was first necessary to know if the Fz-overexpression phenotype would be either enhanced or suppressed by Wingless overexpression. Ectopic expression of Wg suppresses the Fz overexpression phenotype, suggesting that activation of Wg signaling may titrate the amount of Dsh available for PCP signaling. The reciprocal experiment was performed by asking if ectopic activation of the PCP pathway could interfere with Wg signaling. Because the ligand for PCP signaling is unknown, Fz was overexpressed during embryogenesis, and the cuticle phenotype analyzed. These embryos develop with lawns of denticles and are reminiscent of wg-mutant embryos, or those expressing dominant-negative Dsh constructs. The results suggest that titration can occur in this direction as well. The possibility cannot be ruled out that the titration observed in these experiments results from a promiscuous interaction between Wg and Fz, although this interaction may not occur in vivo. The observations are equally consistent with the possibility that under these conditions, activity of one pathway titrates the Dsh level available for the other (Axelrod, 1998).
Dishevelled appears to interact with the Notch pathway, and this interaction provides a direct link between Notch and wingless pathways. The dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways (Axelrod, 1996).
Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).
How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).
The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).
Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).
Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).
To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).
In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier? and Eisenmann's Wnt Signaling). Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).
Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).
Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).
The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).
Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).
Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).
Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).
Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).
Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).
These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).
Bases in 5' UTR - 213
Bases in 3' UTR - 519
A deletional analysis of dsh has identified several conserved domains essential for activity including a so-called GLGF/DHR motif found in Drosophila discs-large(a tumor suppressor gene) and its relatives (Yanagawa, 1995), an opa repeat, and several PEST sequences (Klingensmith, 1994). The GLGF/DHR motif is also found in a vertebrate postsynaptic density protein (PSD-950) and an erythrocyte membrane protein (Klingensmith, 1994).
All Dishevelled proteins have three highly conserved domains. An N-terminal DIX (Dishevelled-Axin) domain extends for 80 amino acids and is also found in the Axin protein, which seems to be a scaffolding factor for Wnt signaling. Sequence analysis reveals that Dsh has a PDZ domain. Many other signaling proteins contain PDZ domains that can bind to a conserved stretch of amino acids at the C-termini of receptors to form homotypic complexes with other PDZ domains. Multi-domain PDZ domain proteins have been proposed to integrate signaling molecules into larger complexes. A third domain at the C-terminal part of Dsh, called the DEP (Dishevelled-EGL-10-Pleckstrin), can be found in signaling factors such as the RGS protein family, that are involved in G protein signaling. It is not yet known whether DEP domains play an active role in regulating G proteins. Although Dsh and its domains are highly conserved from Drosophila and C. elegans to mouse and human, Dsh does not have an apparent catalytic biochemical activity, nor have any clear interacting partners for Dsh been identified (Boutros, 1999).
date revised: 10 April 2008Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
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