InteractiveFly: GeneBrief

diego : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - diego

Synonyms -

Cytological map position - 41C1-2

Function - signaling

Keywords - wing, eye, tissue polarity

Symbol - diego

FlyBase ID: FBgn0033552

Genetic map position -

Classification - ankyrin repeat protein

Cellular location - cytoplasmic



NCBI link:EntrezGene
BIOLOGICAL OVERVIEW

During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. diego, a tissue polarity gene, encodes a protein comprised of six ankyrin repeats that colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. It is suggested that Frizzled acts through Diego to promote local clustering of Flamingo, and that the clustering of Diego and Flamingo in one cell nonautonomously propagates to others. Localized Frizzled signaling would modify the properties of the Diego protein at proximal-distal boundaries, increasing Diego's ability to promote clustering of itself and Flamingo. A bias in Diego localization might then be enhanced via homophilic interactions between Flamingo molecules on adjacent cells. This model is consistent with the reciprocal effects that Flamingo and Diego have on each other's localization (Feiguin, 2001).

To isolate genes whose overexpression causes tissue polarity defects, lines harboring 'EP' P elements were crossed to flies expressing GAL4 under the control of the apterous promoter. EP elements contain upstream activation sequences that drive GAL4-dependent transcription from an adjacent P element promoter through genomic DNA next to the site of insertion. Activation of transcription by GAL4 in apterous-expressing cells results in the overexpression of random genomic sequences in cells that give rise to the thorax and dorsal wing blade. A screen of 2300 EP crosses was carried out for those that produced bristle polarity defects in the thorax (33/2300), and then these EP lines were reexamined for their ability to cause defects in hair polarity or number. Six were found that produced multiple wing hairs when overexpressed, but only one, EP(2)2619, was found whose overexpression causes predominantly hair polarity defects. The DNA downstream of the EP element was cloned by plasmid rescue, and when it was sequenced, was found to be homologous to an EST corresponding to clone LD08259 (CG12342 in the GadFly database) (Feiguin, 2001).

To ask whether expression of the gene represented by this EST actually causes the polarity phenotype, transgenic flies were produced containing the cDNA under the control of the UAS promoter and its expression was driven by crossing these transgenic flies to flies that expressed GAL4 under the control of the apterous promoter. Expression of LD08259 causes polarity defects similar to those observed when EP 2619 is crossed to apterous GAL4. These data confirm that the protein encoded by LD08259 causes tissue polarity defects when overexpressed. The gene represented by this cDNA is diego (Feiguin, 2001).

Is Frizzled activity required for polarized Diego localization? Diego localization was examined in clones of frizzled null tissue. Diego protein is undetectable at the cortex between frizzled mutant cells. In contrast, Diego accumulates to a higher level at boundaries between wild-type and frizzled mutant cells, although it is not possible to resolve whether Diego is present on both sides of the boundary. These data suggest that Diego accumulates at boundaries between cells with different levels of Frizzled signaling activity (Feiguin, 2001).

Frizzled mutant clones nonautonomously reorganize the polarity of hairs distal to the mutant tissue, and the effect is most pronounced on the medial side of the clone. Correspondingly, Diego localization in cells on the distal/medial side of frizzled clones is reoriented, suggesting that perturbations in Frizzled signaling can nonautonomously repolarize the distribution of Diego (Feiguin, 2001).

To confirm that Diego localizes to boundaries between cells with different levels of Frizzled signaling activity, its distribution was examined in wings expressing Frizzled under the control of ptc:GAL4. This produces levels of Frizzled protein that change with distance from the AP boundary. In these wings, Diego relocalizes to reflect the artificial gradient of Frizzled expression generated by Ptc:GAL4. In the cells posterior to Frizzled-overexpressing cells, Diego is also relocalized to reflect the nonautonomous disruption of polarity caused by Frizzled. Taken together, these data show that apposition of cells with different levels of Frizzled causes the accumulation of Diego at the boundary between cells. These data further show that the nonautonomous disruption of polarity produced by altering Frizzled signaling results in the mispolarization of Diego (Feiguin, 2001).

Does cortical localization of Diego depend on Flamingo? The distribution of Diego was examined in clones of Flamingo mutant cells. Diego is undetectable at the cortex of flamingo mutant cells and is also absent from both proximal and distal interfaces between wild-type and flamingo mutant tissue. These data suggest that Diego is localized to the cortex via Flamingo, either directly or indirectly. Like Diego, Flamingo also fails to accumulate at either the proximal or distal interface between wild-type and Flamingo mutant clones. This has been interpreted to mean that Flamingo is normally enriched both proximally and distally and that homophilic interactions are required for its polarized accumulation. Similarly, it can be argued that Diego is normally enriched on both proximal and distal sides of the cell and that stable cortical Diego localization occurs only where homophilic Flamingo interactions are possible (Feiguin, 2001).

Clearly, Flamingo is required for cortical accumulation of Diego. To ask whether it was also sufficient, that is, whether Diego simply accumulates wherever Flamingo is present, the distribution of Diego was examined in cells that overexpressed Flamingo. Although some Flamingo can be detected at cell boundaries when the protein is overexpressed, Flamingo accumulates most obviously in bright spots inside the cell. Since Flamingo is a transmembrane protein, these spots might either represent protein that is trapped in the secretory pathway or that has accumulated in the endocytic pathway. In Flamingo-overexpressing cells, Diego protein appears less abundant on the cortex overall, and what Diego remains is no longer restricted to proximal-distal cell boundaries. Diego does not colocalize with Flamingo inside the cell where Flamingo accumulates to the highest level. This indicates that other proteins besides Flamingo must promote the cortical localization of Diego. Colocalization of Diego and Flamingo upon transfection of S2 cells could not be seen, indicating that other proteins may regulate whether Diego and Flamingo are present in the same complexes. Nevertheless, at least at the cortex, dominant mislocalization of Flamingo by overexpression can relocalize Diego (Feiguin, 2001).

In wild-type cells adjacent to Flamingo-overexpressing cells, Diego disappears from the proximal-distal boundaries and appears to accumulate to a higher level at the boundary with the Flamingo-overexpression domain, perpendicular to its normal proximal-distal pattern. Furthermore, overaccumulation of Diego is also seen on the cell boundary opposite that which is in contact with the Flamingo-overexpressing cells. In some wings, reorientation of both Diego and Flamingo occurs up to three cell diameters away from the overexpression stripe. Thus, despite the fact that loss of Flamingo activity in clones has an essentially autonomous effect, depleting Diego and Flamingo only from the directly adjacent cell boundary, flamingo overexpression causes nonautonomous relocalization of both itself and Diego over several cell diameters (Feiguin, 2001).

Is Diego required for Flamingo localization? The distribution of Flamingo in wild-type versus homozygous diego mutant wings was compared. In all of the wild-type wings examined, the Flamingo protein is enriched on proximal-distal boundaries by 27 hr after puparium formation. In contrast, Flamingo in dgo380 wings localizes uniformly around the junctional region at this time, similar to its distribution in frizzled mutant wings. The ratio of Flamingo staining intensity on proximal-distal versus anterior-posterior cell boundaries was quantified in images from four wild-type wings and four dgo380 mutant wings. In wild-type wings, Flamingo is enriched between 2.5- and 3.2-fold on proximal-distal boundaries at 27 hr after puparium formation. In contrast, the Flamingo in dgo380 mutant wings is not significantly enriched (ratios ranged between 0.8 and 1.3). These data suggest that Diego promotes Flamingo polarization (Feiguin, 2001).

Flamingo sometimes polarizes its distribution in parts of dgo380 mutant wings at later times (29-30 hr after puparium formation), but the axis of polarity is often abnormal, presaging the whorls of misoriented hairs observed in dgo380 adult wings. In all cases, the effect of diego is specific to Flamingo and does not affect the distribution of cortical actin. Clonal analysis shows that diego loss of function does not affect Flamingo localization outside of diego clones. These data suggest that Diego increases the efficiency of Flamingo polarization and promotes its correspondence with the proximal-distal axis determined by Frizzled. The fact that Flamingo is localized similarly in frizzled and diego mutant cells may indicate that Frizzled acts through Diego to mediate Flamingo polarization (Feiguin, 2001).

To assess the effects of excess Diego protein on polarity, the effect of Diego overexpression on Flamingo localization was examined. When Diego protein is overexpressed with the ptc:GAL4 driver, it still localizes exclusively to the junctional region, but is present in large aggregates with no proximal-distal polarity. Flamingo protein accumulates in these cortical clusters, and its distribution is no longer polarized along the proximal-distal axis. These data suggest that Diego specifically promotes clustering of Flamingo. It is suggested that under wild-type conditions, Diego-mediated Flamingo clustering is regulated in response to Frizzled signaling and occurs only at proximal-distal boundaries (Feiguin, 2001).

Interestingly, Diego overexpression has nonautonomous effects on Flamingo localization in adjacent cells. In the first row of wild-type cells adjacent to Diego overexpressers, Flamingo is missing from proximal-distal boundaries and presumably relocalizes to maximize homophilic interactions with the clustered Flamingo on the Diego-overexpressing cells. Interestingly, Flamingo protein levels are also modestly elevated on the cell boundary opposite that of the Diego-overexpressing cells. As a result, the Flamingo in the second row of cells seems less unambiguously polarized to proximal-distal boundaries than in the cells at a greater distance from the overexpression stripe (Feiguin, 2001).

The ability of adjacent pupal wing cells to compare the activity of different tissue polarity genes and orient hairs accordingly is intrinsic to the mechanism of planar polarization. When artificial expression gradients are produced, Frizzled and Flamingo cause opposite effects on hair polarity; hairs point away from cells with higher Frizzled expression but toward cells with higher Flamingo expression. To ask how cells with differing levels of Diego behaved, the adult wings from UAS:dgo * ptc:GAL4 crosses were examined. Wing hairs were oriented from high- to low-expressing cells in ptc:GAL4;UAS:dgo wings -- similar to the effects caused by Frizzled and the opposite of those caused by Flamingo. These data suggest that Diego responds positively to Frizzled signaling and that the subsequent Diego-mediated clustering of Flamingo has negative effects on Frizzled signaling activity (Feiguin, 2001).

The mechanism by which Frizzled proteins mediates cell polarization events is not well understood. In wing epithelial cells, one consequence of Frizzled signaling is the polarization of Flamingo. Diego protein has been shown in this study to be required for normal polarization of Flamingo. Diego is itself localized at proximal-distal boundaries in response to Frizzled signaling. In the wing, proximal-distal localization of Diego and Flamingo proteins is interdependent. Perturbing the distribution of either protein disrupts that of the other. In the absence of Flamingo, Diego fails to localize to the cortex at all. However, in diego mutant cells, Flamingo is cortically localized but fails to polarize efficiently or accurately along the proximal-distal axis. The localization of each of these proteins can also be perturbed by overexpression of the other. When Diego is overexpressed, it accumulates in large nonpolarized junctional clusters that recruit high levels of Flamingo protein as well. In contrast, overexpression of Flamingo has a dispersive rather than a clustering effect on Diego in the same cell, causing it to delocalize from proximal-distal boundaries and assume a more uniform cortical distribution. Furthermore, in cells adjacent to Flamingo overexpressers, Diego is dominantly relocalized with Flamingo to face the overexpression domain. Clearly, Flamingo and Diego each affect the localization of the other (Feiguin, 2001).

It is suggested that Diego associates with the cortical membrane and helps to polarize the cell in response to Frizzled signaling. Diego's cortical localization and ability to cluster make this model more likely than one in which Diego promotes polarized delivery of the Flamingo protein from the Golgi apparatus. How might Diego and Flamingo polarize in response to Frizzled? Although one possibility is that Diego mediates polarization by directly interacting with components of the activated Frizzled pathway, the interdependence of Flamingo and Diego localization speaks against such a linear model. A more reasonable model is that localized Frizzled signaling might modify the properties of the Diego protein at proximal-distal boundaries, increasing its ability to promote clustering of itself and Flamingo. This initial bias in localization caused by clustering might then be potentiated via homophilic interactions between Flamingo molecules on adjacent cells. This model is consistent with the reciprocal effects that Flamingo and Diego have on each other's localization (Feiguin, 2001).

The ability of polarization signals to propagate from cell to cell is one of the most intriguing aspects of tissue polarity and is one of the least understood. It has been known for some time that locally perturbing Frizzled signaling has long-range effects on the polarity of the surrounding tissue. This repolarization is reflected, at the molecular level, by the altered distribution of Flamingo and Diego. Interestingly, in the absence of any direct disruption of Frizzled, Flamingo overexpression causes nonautonomous repolarization of Diego and Flamingo that propagates over several cell diameters. Both Flamingo and Diego accumulate ectopically to a high level at the interface between frizzled mutant and wild-type tissue. The data suggest that the accumulation of Diego and Flamingo at frizzled clonal boundaries might play an important role in propagating repolarization of these proteins into the surrounding tissue (Feiguin, 2001).

How might this propagation occur? Although it is easy to understand how Flamingo-mediated homophilic interactions could recruit Flamingo and Diego from a directly adjacent cell to the boundary with the overexpression domain, it is less clear how this would result in a corresponding accumulation on the opposite side of the cell. One possibility is that when Diego and Flamingo are polarized via Flamingo-mediated homophilic interactions, a locally acting intracellular signal is generated that prevents similar interactions nearby. Flamingo and Diego might then tend to accumulate on the opposite side of the cell, where such interactions were not discouraged. Increased levels of Diego and Flamingo on the opposite side of the cell might, in turn, recruit Diego and Flamingo from the next cell in line. Such a mechanism might be sufficient to propagate polarization of these proteins over many cell diameters (Feiguin, 2001).

Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/β-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. The role of a Dsh-binding PCP molecule, Diego (Dgo), was studied in both Wnt-Fz/β-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/β-catenin signaling. The data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell (Wu, 2008).

It has suggested that the KTXXXW motif in Fz C-tails is important for the activation of Wnt-Fz/β-cat signaling targets, but conversely other data implied that this motif was dispensable for Wnt-Fz/β-cat signaling. This issue was addressed in a physiological context. Expressing Fz under control of the tubulin (tub)-promoter fully rescues Fz-activity in fz, fz2 double mutant flies with respect to both Wnt-Fz/β-cat and Fz–PCP signaling. Importantly no dominant phenotypes result from tub-fz expression and thus the tubulin promoter presumably drives the Fz transgenes close to endogenous levels. All Fz C-tail isoforms defective in the Dsh interaction motifs in the C-tail and third cytoplasmic loop (M469R) failed to rescue Wnt-Fz/β-cat signaling, indicating that the Dsh interacting sites are important for Wnt-Fz/β-cat signaling in vivo (Wu, 2008).

The function of the KTXXXW C-tail motif in PCP signaling has previously not been determined. All KTXXXW mutations tested in vivo in this study failed to rescue the fz PCP mutant phenotypes, indicating that the Dsh interacting sites are required for both signaling pathways. These data suggest that there are no obvious differences in how Fz and Dsh interact with each other in the context of either pathway, and therefore that additional factors are likely involved to modulate the signaling outcome and to provide specificity (Wu, 2008).

Dgo is a core Fz/PCP signaling factor (Feiguin, 2001: Jenny, 2005). During the interactions of the core PCP factors, Fz, Dsh, and Dgo become localized to the distal end of pupal wing cells (or the R3-side of the R3/R4 border in the eye), suggesting that they form a functional complex (Axelrod, 2001; Das, 2004; Strutt, 2001). The localization of Dsh and Dgo depends on Fz (Axelrod, 2001; Das, 2004). Dgo localization also partially depends on Dsh and Dgo and Dsh interact physically (Jenny, 2005). Taken together, these data are consistent with the notion that Fz, Dsh, and Dgo are forming a functional complex during PCP signaling that promotes Dsh PCP-activity (Wu, 2008).

Co-expression of Dgo enhances Fz-mediated inhibition of Wnt-Fz/β-cat signaling in the wing. Furthermore, overexpressed Dgo sequesters more Dsh into the subapical junctional region (where PCP signaling takes place) in a Fz dependent manner, suggesting that a Dgo-Dsh association sequesters Dsh away from canonical Wnt-signaling. Thus, a Fz–Dsh–Dgo complex selectively acts in Fz/PCP signaling and is likely not active for the Wnt-Fz/β-cat pathway (Wu, 2008).

Does Dgo affect the levels of Wnt-Fz/β-cat signaling in a loss-of-function (LOF) scenario? Although dgo LOF alleles show only minor effects on Wnt-Fz/β-cat associated phenotypes, double mutant LOF combinations of dgo and nmo, a mild inhibitor of Wnt-Fz/β-cat signaling, show more robust defects (manifest in the observation that the nmoP allele, which does not show Wg GOF defects, in combination with dgo LOF alleles frequently displayed ectopic margin bristles). This indicates that in vivo Dgo can affect the levels of Wnt-Fz/β-cat signaling, but redundantly with other Wg-signaling inhibitors. It is interesting to note that while Dgo presumably acts at the level of Dsh, Nmo phosphorylates the nuclear transcription factors of the TCF family and thus inhibits their association with β-cat and/or the DNA (Wu, 2008).

How does Dgo negatively affect Dsh in Wnt-Fz/β-cat signaling? in vivo data suggest that Dgo acts mainly by sequestering Dsh away from the cytoplasmic and/or basolateral cell regions where Wnt-Fz/β-cat signaling is thought to take place. Thus, a Dgo influenced shift in Dsh subcellular localization, caused either by loss or excess of Dgo, makes the pathway sensitive to additional changes. When Dsh itself or other Wnt-Fz/β-cat signaling factors become more limiting, alteration of Dgo levels can have effects on Wnt-Fz/β-cat signaling strength (Wu, 2008).

Does Dgo affect overall Dsh levels? Studies with the vertebrate Dgo homologue Inversin have suggested that Inversin, the vertebrate Dgo homologue, can affect Dsh levels through ubiquitination and associated degradation in HEK 293T cells (Simons, 2005). It seemed thus possible that Dgo affected the overall Dsh levels: Dgo could stabilize Dsh at the subapical membrane but cause its destabilization in the cytoplasm. However, no evidence was seen for a destabilization mechanism in vivo or in HEK 293T cells. Thus, it seems that Dgo and Inversin do not share this biochemical property (Wu, 2008).

Diversin is a second Dgo-related vertebrate factor that can act as a repressor of Wnt-Fz/β-cat signaling (Schwarz-Romond, 2002; Simons, 2005). The Diversin and Dgo sequences C-terminal to the Ankyrin repeats do not share homologous domains, although clusters of high homology are present. Diversin is thought to inhibit Wnt-Fz/β-cat signaling through its interaction with Axin and CKIε (Schwarz-Romond, 2002). Dgo does not interact with Axin. Thus, it appears that both Diversin and Inversin can inhibit Wnt-Fz/β-cat signaling by (at least partially) different mechanisms from Dgo, suggesting that these features have diverged evolutionarily (Wu, 2008).

Taken together, the in vivo and cell culture data suggest that Dgo can negatively affect Wnt-Fz/β-cat signaling by trapping Dsh in a Fz/PCP specific complex that is inactive for canonical Wnt-Fz/β-cat signaling. Comparative analyses with Dgo, Inversin, and Diversin will be interesting to shed light on conserved mechanisms of action for these three related proteins (Wu, 2008).


REGULATION

Protein Interactions

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. Evidence is provided that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. The initial membrane association of Dgo depends on Fz, and Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi (Das, 2004).

A crucial region for PCP signaling in the eye is in rows 2-5 in the 3rd instar larval disc behind the morphogenetic furrow (MF). Four lines of evidence support this assumption: (1) cells that take part in PCP signaling (R3/R4) are specified as photoreceptor subtypes in this region; (2) Frizzled-Notch signaling-dependent transcription in the R4 cell is initiated in this region, as detected by the mdelta0.5 reporter for the E(spl)mdelta gene; (3) the sev-enhancer, which is active in R3/R4 cells in this region, can drive a PCP gene in order to fully rescue the respective mutant phenotype; and (4) in the region ahead of the MF to the first row behind it, the PCP proteins are uniformly apically localized in all cells, before they begin at row 2 to display the characteristic PCP protein localization pattern (Das, 2004).

Following their initial symmetric apical localization, the PCP factors become asymmetrically enriched across the respective cell boundaries in the proximodistal axis in the wing or the dorsoventral axis in the eye. Although several models have been proposed as to how these complexes might be formed and maintained, the mechanism behind the early aspect of PCP establishment remains largely unclear. The data suggest a complex mechanism that involves redundancy among several PCP genes (Das, 2004).

Based on the analysis of single mutant clones in the eye, only Fz and Fmi affect PCP gene localization in a general non-redundant manner (and Stbm affects Pk localization). The single and double mutant clone data indicate the following (Das, 2004).

  1. Fz is required for membrane localization of Dgo and this step precedes any apparent PCP signaling requirement. Fz also affects the apical localization of Dsh but not of Fmi, Pk, or Stbm significantly.
  2. Dgo alone does not affect the apical localization of other PCP genes, but instead it shares this function redundantly with Stbm and Pk.
  3. Pk alone does not affect the apical localization of other PCP proteins significantly, but does so in conjunction with Dgo and Stbm.
  4. Fmi is responsible for the apical localization of Fz.

In addition to these initial requirements for apical localization and maintenance, the subsequent asymmetric resolution of the respective PCP proteins to the R4 cell is affected and often delayed in mutant backgrounds (Das, 2004).

How is the initial apical localization of all these factors maintained? As outlined above, none of the single mutant PCP genes, except fz and fmi, has a significant effect on the whole complex. However, in double mutant clones for either dgo and pk, or dgo and stbm, localization of the PCP proteins is severely affected. Most strikingly, the apical localization of Fmi and Fz is affected in these double mutant combinations. In addition, the localization of Stbm and Dsh are also affected. This could be either a direct effect of Dgo and Pk or could be mediated through their effect on Fmi [as in fmi- tissue, Stbm and Dsh as well as Fz are reduced apically]. These data suggest that the cytoplasmic PCP proteins, which are initially recruited to the membrane by Fz (i.e. Dgo and Dsh) and Stbm (i.e., Pk), form a protein complex that is required to maintain Fmi apically. This interpretation is supported by the observation that Dgo physically interacts with Stbm and Pk, and thus possibly stabilizes the initial complex. Thus, these studies reveal that Dgo, Stbm and Pk are required to maintain apical Fmi localization, possibly through the physical interactions among themselves and possibly other PCP factors, during the early stages preceding PCP signaling (i.e., anterior to MF in eye). In turn, apical Fmi promotes the maintenance of an initial PCP complex at adjacent cell membranes to facilitate their signaling specific interactions (Das, 2004).

It is possible to speculate on further implications of these data. During later stages of PCP signaling, the localization of the PCP factors is resolved into two types of complexes on adjacent cell membranes. The differential localization of either Fz/Dgo or the Stbm/Pk complex in the neighboring cells (R3 versus R4) suggests that asymmetric localization of PCP factors is maintained across the border of the R3 and R4 cells in the eye and across proximodistal cell borders in the wing. In the eye, the PCP proteins analyzed in this manner indeed localize to specific sides of the R3/R4 cell border. Similarly, proximodistal localization in the wing correlates with the respective R3/R4-specific localization. For example, the localization of Fz and Diego in the distal side of a wing cell correlates with the localization on the R3 side of the R3/R4 border; conversely, Stbm localization to the proximal side of a wing cell correlates with its localization on the R4 side of the R3/R4 border. The localization to either the R3 or R4 side also corresponds to the genetic requirements in either cell, as established in mosaic analyses. Thus, since Dgo, which is initially recruited by Fz, localizes to R3 and the pk/stbm complex localizes to R4, it is likely that at later stages during PCP signaling (posterior to MF) Fmi localization is maintained and stabilized through feedback loops on both sides of the R3/R4 boundary (Das, 2004).

A prediction from such a scenario is that Fz/Dgo are performing this function in R3 and the Stbm/Pk complex in R4. Since Fmi is known to function as a homophilic cell-adhesion molecule, the removal of the feedback loop on one side could be overcome through the homophilic recruitment of Fmi from the other side. Only when both feedback loops are weakened on either side, can Fmi localization become affected. This is supported by the different effects of the respective double mutants posterior to the MF; those that affect both sides of the R3/R4 boundary, e.g., dgo and stbm (R3side/R4side) or dgo and pk (R3side/R4side) can cause Fmi delocalization, whereas double mutants affecting only one cell, e.g., pk and stbm (both R4side), have no significant effect (Das, 2004).

Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding

Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in Drosophila. This study provides mechanistic insight into Frizzled/Dishevelled signalling regulation. The ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling (Jenny, 2005).


DEVELOPMENTAL BIOLOGY

Pupal

The activity of the Frizzled signaling pathway is required during the six hours prior to hair formation for normal planar polarization. To better understand the cell biological function of Diego protein during this time, antibodies were generated to a region COOH-terminal to the ankyrin repeats. This antibody recognizes a band of the expected size on Western blots of wild-type pupal wings that is of similar mobility to the protein produced by GAL4-driven overexpression and is undetectable in the Dgo380 mutant (Feiguin, 2001).

Immunofluorescence analysis of pupal wings at 18 hr shows that Diego localizes in a spotty pattern all around apico-lateral junctions, with no apparent polarity. By 24 hr, however, Diego has become localized to proximal and/or distal cell boundaries; whether Diego is present on one or both sides of the boundary is impossible to resolve by light microscopy. The localization persists during hair formation (which occurs at ~30 hr). These experiments show that Diego becomes localized to the proximal-distal junctional region when the Frizzled signaling pathway is actively generating planar polarity (Feiguin, 2001).

The localization of Diego to proximal-distal boundaries resembles that of Flamingo, a homophilic adhesion molecule with seven-transmembrane domains. To investigate the extent of colocalization between Diego and Flamingo, pupal wings were stained with antibodies to both proteins. Neither protein is smoothly localized at proximal-distal boundaries; instead, the proteins are more abundant at specific spots along the membrane. Diego and Flamingo show a strong tendency to be especially concentrated at the same places. This suggests that the two proteins are present in the same specialized regions of the membrane (Feiguin, 2001).

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. The atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. These results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing (Das, 2002).

The initial asymmetrical enrichment of Fmi in both R3 and R4, and the subsequent enrichment in R4 only, raised the question of in which cell(s) of the precluster is fmi required for PCP establishment. Interestingly, the analysis of mosaic clusters revealed a requirement for fmi in both R3 and R4. An ommatidium always adopts the correct orientation when both R3 and R4 are fmi+. When either R3 or R4 (or both) are fmi-, the ommatidium selects chirality randomly or stays symmetrical. Significantly, all ommatidia with wrong or no chirality had fmi- R3 and/or R4 cells. Loss of fmi function in any other R cells in any combination has no effect on ommatidial polarity. These data indicate that fmi is necessary and sufficient in both the R3 and R4 photoreceptor precursors for normal polarity establishment (Das, 2002).

The genetic requirement of fmi in both R3 and R4 is unique, since other PCP genes are required only in either cell (fz and dsh in R3 and stbm and N in R4), and raised the question of how Fmi relates to these genes in function and expression. Thus, the expression patterns of other PCP proteins were examined in the eye (Das, 2002).

Since Fmi is initially expressed in both cells of the pair, its inhibitory role on Dl can only be allowed in R4 and thus needs to be regulated. How is this achieved? Diego (Dgo) is a good candidate for this role. The cytoplasmic Dgo protein depends on Fmi for membrane association and generally colocalizes with Fmi at all membranes in the eye disc. The genetic interactions with sev --> Fmi (resulting in Fmi overexpression in the R3/R4 pair) identify dgo as a strong enhancer, suggesting that it is suppressing Fmi function in this context. Mosaic analysis of dgo shows that it is required in R3 and thus might keep the inhibitory function of Fmi off in R3. Since Fmi is necessary, but not sufficient, for Dgo membrane recruitment, other factor(s) are also required. Since Dgo and Fmi colocalize also in R4, a factor is needed there to antagonize Dgo function. Strabismus (Stbm), since it is required in R4, is a candidate. Since fmi mutants are enhancers of an Stbm overexpression phenotype, Stbm could serve this function in R4 (Das, 2002).

How could this be achieved? (1) There are the distinct requirements for dgo in R3 and stbm in R4; (2) the differences in Fmi levels in early R3/R4 versus late R4 could account for its individual functions. High levels of Fmi in R4 could lead to a formation of a different complex than that formed in R3. For example, an Fz/Dsh/Fmi/Dgo complex would promote Fz signaling, whereas, in R4, since there is significantly more Fmi, a different Fmi complex would inhibit Fz signaling by possibly sequestering Dsh from the Fz complex (Das, 2002).


EFFECTS OF MUTATION

In order to determine whether loss of diego function causes tissue polarity defects, diego mutants were generated by imprecise excision of EP 2619. Six mutants were identified harboring deletions that removed parts of the diego transcript, but left sequences upstream of the P element intact. These diego mutants are homozygous viable with wing hair polarity defects that resemble those of the strongest viable flamingo/starry night allelic combinations. Bristle polarity is not strongly affected by dgo mutation (Feiguin, 2001).

Diego mutant eyes also display typical planar polarity defects; some ommatidia have inverted chirality, others are symmetrical, whereas others are misrotated. The diego mutant phenotype is reminiscent of that observed for other 'core' polarity genes like fz. Of the diego mutant alleles, dgo 380 (which harbors the largest deletion) produces the strongest eye phenotypes, and its phenotype is not stronger over a deficiency, indicating that it is probably a null allele. Consistent with this, no Diego protein is detected on Western blots of dgo 380 homozygotes. All dgo alleles appear to have similar wing phenotypes that are not stronger when hemizygous. This suggests that whereas all the diego alleles are at least strong hypomorphs for wing-specific functions, the shorter deletions may retain some ability to function in the eye. The fact that Diego is required for planar polarization of both eye and wing epithelia places it in the 'core' group of tissue polarity genes and suggests that it forms an integral part of the polarization machinery (Feiguin, 2001).


EVOLUTIONARY HOMOLOGS

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

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

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

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

Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways

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. This study reports that 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).

Ankrd6 is a mammalian functional homolog of Drosophila planar cell polarity gene diego and regulates coordinated cellular orientation in the mouse inner ear

The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. This study reports that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling (Jones, 2014).


REFERENCES

Search PubMed for articles about Drosophila diego

Axelrod, J. D. (2001). Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling. Genes Dev. 15: 1182-1187. PubMed citation: 11358862

Das, G., Reynolds-Kenneally, J. and Mlodzik, M. (2002). The atypical cadherin Flamingo links Frizzled and Notch signaling in planar polarity establishment in the Drosophila eye. Dev. Cell 2: 655-666. 12015972

Das, G., et al. (2004). Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes. Development 131: 4467-4476. 15306567

Feiguin, F., Hannus, M., Mlodzik, M. and Eaton, S. (2001). The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization. Dev. Cell 1: 93-101. 11703927

Jenny, A., et al. (2005). Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding. Nat. Cell Biol. 7: 691-697. PubMed citation: 15937478

Jones, C., Qian, D., Kim, S. M., Li, S., Ren, D., Knapp, L., Sprinzak, D., Avraham, K. B., Matsuzaki, F., Chi, F. and Chen, P. (2014). Ankrd6 is a mammalian functional homolog of Drosophila planar cell polarity gene diego and regulates coordinated cellular orientation in the mouse inner ear. Dev Biol. PubMed ID: 25218921

Schwarz-Romond, T., et al. (2002). The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the ß-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling. Genes Dev. 16: 2073-2084. 12183362

Simons, M. et al. (2005). Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat. Genet. 37: 537-543. PubMed citation: 15852005

Strutt, D. I. (2001). Asymmetric localization of frizzled and the establishment of cell polarity in the Drosophila wing. Mol. cells 7: 367-375. PubMed citation: 11239465

Wu, J., Jenny, A., Mirkovic, I. and Mlodzik,M. (2008). Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila. Mech. Dev. 125: 30-42. PubMed citation: 18065209


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date revised: 10 October 2014

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