The Drosophila wing is covered by an array of distally pointing hairs. This tissue planar polarity is regulated by the frizzled pathway. The function of the grainy head transcription factor is essential for the function of the frizzled pathway. grainy head mutant cells fail to localize planar polarity proteins at either the proximal or distal sides of wing cells and produce multiple hairs of abnormal polarity. Levels of the Starry night protein are strongly reduced in grainy head mutants in both larval wing discs and pupal wings, which is sufficient to account for much of the polarity phenotype. In addition, grh has frizzled pathway independent functions during the development of the adult cuticle (Lee, 2004).
grh function is required for several different processes during the differentiation of the adult Drosophila epidermis. These include the function of the fz dependent tissue polarity pathway, pigmentation, the timing of differentiation, epidermal hair morphogenesis and wing vein/blade specification. The Grh protein was originally isolated by virtue of its ability to bind to DNA in a sequence specific manner and to regulate the expression of target genes. These and later experiments led to the conclusion that grh functions as a transcription factor for development specific gene regulation. Experiments on vertebrate homologs of grh also suggest a similar cellular function. It is likely that it serves a similar function in the development of the adult epidermis (Lee, 2004).
The analysis of grh function in regulating gene expression appears complex. The first studies on grh argued that it acted as a positive regulator of Ddc and Ubx expression. Curiously, although Grh was isolated by virtue of its ability to bind to a sequence essential for the neuronal activation of Ddc, grh mutations alter the epidermal and not neuronal expression of Ddc. More recently it was found that grh positively regulates tll expression and negatively regulates ventral dpp expression (Lee, 2004).
The function of the grh transcription factor is shown in this study to be required for the function of the fz pathway in the wing. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of the Stan protein are dramatically decreased. Furthermore, Stan levels are increased in cells with two versus one copy of grh. Thus, stan expression is directly related to grh dose suggesting that stan might be a direct target of Grh. The direct relationship between stan expression and grh dose is seen in both pupal wing cells where Stan is localized assymetrically and in third instar wing disc cells where it is evenly distributed. Thus, it is concluded that the decreased levels of Stan protein in grh cells is not due to a failure of assymetric localization. Grh does not affect Stan stability; stan expression from the endogenous stan gene is altered, consistent with Grh having an important role in promoting stan transcription. It is suspected that this could be due to a direct interaction of Grh protein with stan genomic DNA. stan does not appear to be highly enriched in putative Grh binding sites but this may be a reflection of the variability in identified Grh binding sites not providing an ideal consensus site. It is also concluded that the decreased level of Stan protein is neither the cause or effect of the the delay in hair morphogenesis in grh cells. Thus far, all of the proteins that localize assymetrically are co-required for the asymmetric localization of the others, however only Stan is required for the apical accumulation of all of the other proteins. The alterations in tissue polarity protein localization seen in grh mutant cells could be explained entirely by the effect of grh on Stan expression. It remains possible however, that grh could be important for the expression of several or all members of the tissue polarity group. These experiments did not allow the assessment of possible changes in Fz or Vang levels due to altered expression of these genes, since the localization was examined of proteins produced from transgenes that did not utilize the normal promoters. Decreased levels of Dsh were not seen by antibody staining, however the staining background was relatively high in these experiments which could have hidden a modest effect on Dsh levels. The finding that Arm cortical localization is not altered in grh clone cells indicates that apical-basal polarity is not altered and suggests that gross cellular physiology is not altered in grh clones (Lee, 2004).
While it is possible that the grh mutant planar polarity phenotype could be due solely to a lack of stan expression in grh mutant cells, this may not be the case since there are a number of differences between the phenotypes of grh and stan clones. For example, the multiple hair phenotype of grh is much stronger than stan. There is also a difference in the non-autonomy of grh and stan clones. For mutations in both of these genes the domineering nonautonomy of clones is much weaker than that of fz or Vang. However, the weak domineering nonautonomy is seen much more frequently with grh than stan clones, suggesting that grh mutations alter the expression of additional tissue polarity genes or other cellular genes that interact with the planar polarity system. Genetic screens for enhancers or suppressors of the dominant negative grhFK2131 allele could be useful in identifying such genes (Lee, 2004).
grh has both fz pathway dependent and independent functions during wing development. Epistasis experiments showed that the ectopic wing vein, cuticle pigmentation, disturbed marginal bristle row and extreme multiple hair cell phenotypes of grh mutations are not altered in a null fz, in or mwh mutants. Thus, it is quite likely that some of the target genes whose transcription is altered by grh mutations are not part of the fz pathway (Lee, 2004).
grh cells are often dramatically delayed in hair morphogenesis. This is not seen in cells mutant for fz or stan and hence is unlikely to be an indirect consequence of a failure of stan expression or in the inactivation of the frizzled pathway. The time course of pupal development is controlled by ecdysone and it is possible that grh functions as part of the ecdysone cascade. The delay in hair morphogenesis could be due to a failure to induce the expression of genes such as kojak, where a loss of function results in a similar delay (Lee, 2004).
The grh multiple hair cell phenotype differs from that of downstream members of the fz pathway such as inturned, in not showing the typical fz/in abnormal polarity pattern and in the hairs being much more erect. The identity of the targets responsible for this phenotype are unkown. The grh hair phenotype is somewhat reminiscent of that seen with mutations in genes such as Rho kinase or crinkled (myosin VII) suggesting these or related genes as possible targets (Lee, 2004 and references therein).
The transcription of the Ddc gene has previously been shown to be regulated by grh and Ddc activity is required for melanization. Is ddc likely to be the target gene whose altered expression leads to the lowered pigmentation of grh clone cells? This is certainly possible but it seems unlikely to be the entire story. Ddcts2 flies raised at the restrictive condition have more profound pigmentation defects than grh clones. However, clones of ddc null alleles typically have a less severe pigmentation phenotype than grh clones due to partial rescue of the pigmentation phenotype by neighboring cells (i.e. ddc displays submissive cell non-autonomy). Based on these observations it is argued that grh must have other targets that contribute to the decreased pigmentation (Lee, 2004).
The data reported in this paper argue that grh has multiple functions during the development of the adult epidermis. In this context it is not clear to what extent grh functions in a permissive fashion to promote the expression of developmentally important genes and/or to promote changes in gene expression that are associated with the differentiation of the adult cuticle. The data are consistent with grh functioning in both ways. The requirement for grh for the expression of stan was seen at multiple stages consistent with grh having a permissive role. The effects on the timing of hair morphogenesis are consistent with, but do not demand an instructive role (Lee, 2004).
To address whether Stan has an intercellular adhesion activity, stan was expressed in Drosophila S2 cells that exhibit a very weak self-aggregating property. Cell aggregates are formed by transfection of the S2 cells with a cDNA construct that encodes the full-length Stan protein (Usui, 1999).
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/Starry night 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).
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).
Northern blot analysis shows that the large STAN mRNA is present in a number of developmental stages. It is most abundant in 6-9 hour embryos and more abundant in pupae than larvae. In situ hybridization was used to examine the expression of stan in pupal wings. STAN mRNA is present at relatively even levels in all regions of the pupal wing. This is consistent with the genetic experiments showing that stan mutations have a phenotype over most if not all of the wing (Chae, 1999).
Hairs in adult wings are derived from prehairs that emerge 30-36 hr after puparium formation (hr APF) at 25°C. To gain insight into where Stan functions, researchers stained wing epithelia for Stan at various pupal stages and found dynamic transitions in Stan's subcellular distribution. In 18 hr APF wings, immunofluorescence signals of Stan are present almost entirely at cell-to-cell boundaries. However, at 24 hr APF, Stan becomes redistributed; its localization appears to be biased toward the proximal/distal (P/D) cell boundaries in many cells. At 30 hr APF, the polarized pattern becomes most prominent; signals run zigzag, orthogonal to the P/D axis, indicating that Stan molecules are predominantly localized at the P/D boundaries rather than at the anterior/posterior boundaries (A/P boundaries). In every typical hexagonal cell aligned parallel to the P/D axis, the emergence of a prehair at its distal cell vertex, where two Stan-rich distal boundaries meet, could easily be confirmed. The zigzag pattern is seen throughout the dorsal and ventral surfaces of the wing. This biased localization of Stan provides a striking contrast to the honeycomb distribution of an epithelial classic-type cadherin, Shotgun. Along the apicobasal cell axis at the cell-cell junction, Stan is present apically and its distribution is at least partially overlapped with that of Shotgun concentrated at the adherens junction. Once prehairs have emerged and initiated outgrowth, the distribution starts to be depolarized, although a temporal coordination between the extent of prehair length and that of depolarization of Stan is not strictly fixed in this transition phase (30-36 hr APF). The staining pattern becomes almost nonpolar by 36 hr APF, when prehairs are shifting toward cell centers (Usui, 1999).
Strong Stan signals at P/D boundaries may be explained by a concentration of Stan at either the proximal or distal edge of each cell; alternatively, the protein could be abundant at both edges. To distinguish between these possibilities, mutant clones were generated using an almost protein-null allele, fmiE59, and an examination was made of whether Stan is localized at interfaces between stan+ cells (fmiE59/+ or +/+) and mutant cells (fmiE59/fmiE59) along clone borders. Along the borders of more than 50 clones observed, Stan was missing at all interfaces between stan+ and stan- cells, no matter where the interface was positioned. Assuming that Stan molecules bind to each other in a homophilic fashion in vivo as in vitro, the most plausible interpretation of this mosaic analysis would be that both of the two apposed cells need to produce Stan to localize this receptor at the interface and that retention of Stan at P/D boundaries is achieved through homophilic interaction between its ectodomains. If this interpretation is correct, it follows that Stan is present at both proximal and distal edges in normal epithelial cells (Usui, 1999).
The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP). Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh). In this study, endogenous Dsh was visualized in the developing wing. A portion of Dsh colocalizes with Fmi, and the distributions of both proteins are interdependent. Furthermore, Fz controls the association of Dsh with cell boundaries: this association is correlated with the presence of hyperphosphorylated forms of Dsh. These results, together with studies on Fz distribution, support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that the restricted localization of this complex directs cytoskeletal reorganization only at the distal cell edge (Shimada, 2001).
Dishevelled was visualized in the developing wing by using specific antibodies. Some of the Dsh molecules are present at cell-to-cell boundaries in third-instar wing discs and in wings 2 hr after puparium formation (hr APF). By 18 hr APF, a larger fraction of Dsh molecules appear to be associated with cell boundaries, and then they are redistributed preferentially at proximal/distal (P/D), but not anterior/posterior (A/P), boundaries. This asymmetrical pattern is detectable at 24 hr APF and is most prominent at 30 hr APF, just prior to the onset of prehair outgrowth. This conversion produces a zigzag pattern on the epidermal plane: this pattern is highly reminiscent of the distribution of Flamingo (Fmi), a seven-pass transmembrane cadherin. In fact, Dsh and Fmi appeared to colocalize until 30 hr APF, and in terms of the time course, generation of the asymmetrical pattern of Dsh is indistinguishable from that of Fmi. Dsh, like Fmi, was present apically along the apicobasal axis at cell boundaries; curiously, Dsh distribution appears to be more restricted than that of Fmi. Besides Dsh molecules at cell boundaries, diffuse or punctate signals are also seen in the cytoplasm. Once prehairs emerge and initiate outgrowth at around 32–34 hr APF, the Fmi pattern starts to become depolarized and becomes almost nonpolar by 36 hr APF. In contrast, the asymmetrical Dsh distribution appears to persist, and patchy signals are found at distal cell vertexes and in outgrowing prehairs (Shimada, 2001).
Fmi localization has been studied under various genetic conditions of fz that alter polarity. Dsh was examined under the same genetic conditions and it was found that Dsh, like Fmi, is redistributed to cell boundaries where there is an imbalance of Fz activity. One example was seen along borders of clones of cells homozygous for fzR52, a strong fz allele. Another example was shown in an experiment of graded fz expression. A fz gradient expression along the anterior-posterior axis of the wing reorients hairs from high to low levels of fz expression. In these wings, ectopic Dsh and Fmi accumulation at A/P cell boundaries, instead of P/D ones, prefigures prehair outgrowth in the anterior or posterior direction. A tight coupling of Dsh and Fmi mislocalization with altered polarity is also seen in areas distal to fzR52 clones. All of these results are consistent with the ideas that (1) an imbalance of Fz activity at boundaries is necessary and sufficient to localize Dsh and Fmi there, and (2) in the wild type, distributions of the two proteins at the P/D boundaries direct the cell to choose the distal edge for prehair development. Nevertheless, to rigorously demonstrate that the distributions of Dsh and Fmi play instructive roles in polarizing cells, one would need to design each molecule to mislocalize at A/P boundaries and then investigate how cells are reoriented (Shimada, 2001).
To investigate how colocalization of Dsh and Fmi at P/D boundaries is controlled, the subcellular distribution of one of the two was studied in the absence of the other. Fmi distribution is nonpolar in dsh1, and the requirement of Dsh for making the Fmi pattern asymmetric was confirmed by staining dsh1 or dshV26 clones for Fmi. Fmi is uniformly present at boundaries within these clones. Curiously, the intensity of Fmi signals seems to increase in the dsh mutant cells, and this increase implies the possibility that Dsh may be involved in the destabilization of Fmi. Inside the clone and at the clone border, Fmi remains at apical cell boundaries whether Dsh is colocalized or not. This is perhaps due to the intrinsic nature of Fmi as a transmembrane molecule. In contrast, the boundary localization of Dsh is dependent on Fmi, as demonstrated by the staining of fmi mutant clones for Dsh. Fmi is missing at cell boundaries inside the clones and at clone borders, and Dsh is hardly detected at those boundaries. At boundaries outside the clones, Dsh always coexists with Fmi. These results support a reciprocal dependence between Dsh and Fmi for subcellular localization until prehair formation and may imply a complex formation. Under the experimental immunoprecipitation conditions, however, evidence could not be provided for a physical association between Dsh and Fmi (Shimada, 2001).
Although it has been shown that Dsh is phosphorylated in response to Wingless in a cell culture system and in the embryo, posttranslational modification of Dsh has not been studied in the context of PCP signaling. Western blot analysis shows that a fraction of Dsh molecules in pupae is hyperphosphorylated and that those forms are hardly detectable in fz or fmi mutants. Thus, the absence of the hyperphosphorylated forms correlates with the loss of Dsh at cell boundaries and suggests that the hyperphosphorylation is either required for, or is a downstream readout of, the cell boundary localization. Besides reduction in the level of the hyperphosphorylated forms, the overall amount of Dsh also appears to decrease in fz or fmi mutants; an exception may be the most quickly migrating band, which most likely represents an unphosphorylated or poorly phosphorylated form (Shimada, 2001).
Where is Fz localized within the cell? It has been shown recently that the ubiquitous expression of Fz-GFP rescues a fz polarity defect and that Fz-GFP colocalizes with Fmi: these findings strongly suggest that endogenous Fz assembles with Dsh and Fmi at the P/D boundary. Furthermore, Fz-GFP distribution is regulated by Dsh and Fmi. Therefore, in the sense of subcellular localization, there seems to exist an interdependence between any two of Fz, Dsh, and Fmi. This triangular relationship can be summarized as follows: (1) in the absence of Fz or Fmi function, the intracellular protein Dsh cannot be attached to cell boundaries; (2) without Dsh function, boundary distributions of Fz and Fmi no longer become asymmetric along the P/D axis; and (3) Fz localization at cell boundaries is abolished by a loss of Fmi (Shimada, 2001).
Other data strongly suggest bilateral distribution of Fmi at the P/D boundary, and it is pointed out that this bilateral pattern per se does not explain how the distal cell vertex, not the proximal one, is selected for prehair formation. Importantly, Fz-GFP localization is unipolar; i.e., it is present only at the distal boundary. A distal concentration of Dsh molecules could be likely because Dsh positively relays Fz signaling, although the possibility of the bilateral localization still exists. It had been expected that one could answer this question by tracing Dsh signals in wild-type cells contacting proximal and distal borders of dsh mutant clones; what underlies this approach is the fact that dsh controls PCP in a cell-autonomous fashion. Given that Dsh is localized only at the distal cell edge and that dsh mutant cells do not affect Dsh localization in wild-type neighbors, Dsh signals could have been detected at interfaces between wild-type and mutant cells only along proximal borders of the clones. Unexpectedly, Dsh was not always localized at those cell boundaries along proximal borders; furthermore, 50% of wild-type cells in direct contact with proximal borders mispositioned Dsh at anterior/posterior boundaries. Therefore, dsh mutations appear to exert a one-cell nonautonomous effect on Dsh distribution, and this did not allow a conclusion about unipolar versus bilateral localization of Dsh. This one-cell nonautonomy could be due to misplaced Fz and Fmi molecules in dsh mutant cells, which might send an illegitimate message to wild-type neighbors. In any case, the local assembly of a tripartite signaling complex of Fz, Dsh, and Fmi in the cell most likely amplifies Fz signaling only at the distal cell vertex and induces cytoskeletal reorganization (Shimada, 2001).
It should be noted that in larval wing discs and early pupal wings, Dsh distribution is not asymmetric along the presumptive PD axis; nonetheless, it is associated with cell boundaries, and this association is dependent on Fz. Functional relevance of the cell boundary localization of Dsh has been suggested in vertebrate embryos, in which Dsh controls cell polarization in convergent extension movements. A fusion protein of a Xenopus Dsh homolog and GFP (Xdsh-GFP) is associated with boundaries in cells undergoing morphogenetic movement, but this protein remains in the cytoplasm of cells that are not undergoing such movement. It would be interesting to examine whether the boundary association of Xdsh-GFP or endogenous Xdsh, as well as distributions of Xenopus homologs of Fz and Fmi, are biased toward the direction of the cell movement (Shimada, 2001).
Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP (Axelrod, 2001).
To investigate a possible role for Dsh membrane association during Fz/PCP signaling in vivo, Dsh subcellular localization during PCP signaling was examined in the developing wing. Transgenes were produced that express a Dsh::green fluorescent protein (GFP) C-terminal fusion, driven by native dsh regulatory sequences. One or two copies of these transgenes rescue dshv26 null mutants to viability and produce wild-type PCP, indicating that they fully replace the function of endogenous Dsh in both Wg and PCP signaling (Axelrod, 2001).
The pattern of Dsh localization observed in pupal wings is reminiscent of that for Fmi, a seven-pass transmembrane cadherin required for PCP signaling. By 30 h apf, both are seen at the proximal-distal boundaries, though Dsh is strictly distal, whereas Fmi was proposed to be at both proximal and distal edges. Double labeling for Fmi and Dsh reveals significant colocalization in 30-h apf pupal wings, each demonstrating a zigzag pattern. However, at later times, the Dsh asymmetry persists, whereas the Fmi asymmetry decays. Transverse sections taken in wing-edge cells indicate that both are located at the most apical region of cell-cell contact, and that low levels of Dsh are also seen throughout the cytoplasm. Fmi localization depends on both Fz and Dsh, but not on Multiple wing hairs (Mwh), suggesting that Fmi functions downstream of Dsh. To study this relationship further, Dsh localization was examined in fmi mutant wings. At 30 h apf, little Dsh is associated with the membrane in fmi mutant wings. This reveals a reciprocal dependence between Dsh and Fmi for persistent membrane association, and suggests that Fmi does not simply function downstream of Dsh (Axelrod, 2001).
Fz and Fmi colocalize at proximal-distal boundaries at 30 h apf. Furthermore, the asymmetric pattern of Fz localization depends on Fmi, whereas the asymmetric pattern of Fmi localization depends on Fz. Taken together, these data are consistent with the possibility that Fz, Dsh, and Fmi function together, perhaps in a complex, during PCP signaling, with both Fz and Dsh localizing to the distal edge, and Fmi apparently localizing to both the proximal and distal edges of the cell. A mutual dependence for asymmetric localization exists between these three proteins (Axelrod, 2001).
During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).
The polarity gene stbm is required for R4 fate: whether Stbm protein also shows an asymmetrical localization in R3/R4 was investigated using a Stbm-YFP transgene. Stbm-YFP is apically localized in cells posterior to the furrow, and, subsequently, its distribution is similar but distinct from that exhibited by Fz-GFP. In row 4, a symmetric pattern is observed, with Stbm-YFP around R3/R4, except where these cells contact R2/R5, and enriched on the posterior face of R8. This symmetric pattern is maintained until the ommatidia are already rotated in row 6 and more posteriorly. Staining then fades around R3, except where R3 contacts R4. Mosaic analysis revealed that, in contrast to Fz-GFP, Stbm-YFP is enriched on the R4 side of the R3/R4 boundary from row 4 onward, i.e., Stbm is on the opposite side of the boundary with Fz (Strutt, 2002).
Therefore Fz, Dsh, Fmi, and Stbm localize to the apical region of the R3/R4 cell boundary, where they become asymmetrically distributed prior to or concomitant with R3/R4 fate determination. Normally, Fz/Dsh are enriched on one particular side of the cell boundary, in the presumptive R3 cell. However, in mosaic ommatidia where one or other cell is mutant for polarity genes, the assembly of the asymmetrical complexes can be reversed. In all conditions examined, the polarity of Notch signaling between R3/R4 is consistent with the polarity of the asymmetric complexes, with Notch activity being lowest in the cell where Fz/Dsh accumulate. Finally, evidence is provided that the domain of N, which is known to interact directly with Dsh, is required for efficient R3/R4 fate decisions (Strutt, 2002).
The adoption of the asymmetric pattern occurs in two phases. The first involves symmetric apicolateral localization of Fz, Dsh, Fmi, and Stbm in R3/R4 (and in all other cells except R2/R5); this is evident in ommatidial row 4. As in the wing, the initial apical recruitment of Fz is dependent on Fmi, and the recruitment of Dsh is in turn dependent on Fz. Subsequently, the distribution evolves rapidly into an asymmetric pattern. Adoption of asymmetry requires the function of dsh, stbm, and the LIM domain protein Prickle-Spiny-legs (Pk-Sple), and if any of these are missing, Fz distribution remains symmetric in ommatidial rows 5/6, and ommatidial rotation is delayed. It is likely that the asymmetry evolves through the same mechanisms as in the wing, where it has been proposed that an extrinsic signal leads to a small bias in Fz/Dsh signaling on either side of the cell boundary, which subsequently becomes amplified by feedback loops that lead to Fz/Dsh becoming concentrated on one side of the interface and Pk-Sple/Stbm on the other (Strutt, 2002).
One notable difference between the eye and the wing is that asymmetric Fz/Dsh distribution is eventually observed in stbm and pk-sple eye discs, but in both cases it occurs with a random bias and is delayed by about one to two ommatidial rows. This correlates well with the fact that the adult phenotypes of stbm and pk-sple exhibit a low incidence of achiral ommatidia. Conversely, in fmi, fz and dsh, negligible asymmetric protein localization occurs, and there is a relatively high proportion of 'achiral' ommatidia in the adult eye, suggesting that achirality is a result of poor asymmetric complex formation. In general, the aquisition of asymmetry also correlates with mDelta0.5 activity, particularly in pk-sple and sple mutations where its expression usually resolves into a single cell by row 10 (Strutt, 2002).
In the pupal wing, asymmetric localization of Fz/Dsh/Pk-Sple is proposed to involve a signaling feedback loop that amplifies an initially small bias in Fz/Dsh activity across the axis of each cell. In the eye, the N/Dl feedback loop was proposed to perform a similar function, amplifying an initially small difference in Fz/Dsh activity between R3/R4. With the observation that Fz/Dsh are also distributed in asymmetric complexes in the eye, it appears that both mechanisms are operating in R3/R4, although it is not clear why both would be required, since either alone should be sufficient to amplify small biases in signaling activity (Strutt, 2002).
One possible explanation is that use of both mechanisms increases the speed and robustness of the R3/R4 fate decision. A fast fate decision may be necessary because of the dynamic nature of eye patterning, in which the R3/R4 decision is only part of a complex series of events involving cell recruitment and movement to generate the final polarized ommatidium. It is also possible that a rapid decision is required because the extrinsic polarity cue is transient in nature. It is noted that the rapidity of the decision would be further enhanced if N/Dl signaling also influenced Fz/Dsh localization. While there is no direct evidence for this, it could explain the eventual, randomly polarized, asymmetric protein localization seen in stbm and pk-sple backgrounds in the eye. In this case, the inability of Fz/Dsh to efficiently localize asymmetrically in the absence of Stbm/Pk-Sple might lead to N/Dl making a stochastic decision that then leads to Fz/Dsh asymmetry. Conversely, in the pupal wing, where N/Dl are not active in planar polarity decisions, Stbm/Pk-sple activity would be absolutely required, since their absence would not be compensated for by the N/Dl feedback loop (Strutt, 2002).
Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).
To specifically test the involvement of cytoskeletal elements and adhesion as well as junctional components, candidate genes were tested for dominant interaction of the mild Star rotation phenotype. These genetic data argue for an involvement of E-Cadherin/shotgun, the atypical cadherin Flamingo (Fmi), the adherens junction protein canoe, non-muscle myosin II (zipper), the septin peanut, and capulet, a protein with actin and adenylate cyclase-binding ability (Gaengel, 2003).
Next, the expression of Fmi and Shotgun in ommatidial preclusters was examined during rotation. Strong LOF alleles of Egfr and its signaling components also affect cell proliferation, fate specification and survival, making the analysis of cell adhesion and junctional components in the context of rotation rather difficult. Thus localization of the cadherins and Arm/ß-catenin was examined in imaginal discs of the rotation-specific aosrlt allele (Gaengel, 2003).
Although the overall expression and localization of Shotgun and Arm/ß-catenin are largely unaffected, the localization of Fmi is changed in aosrlt discs. In WT, Fmi is initially present apically in all cells of the morphogenetic furrow and subsequently becomes asymmetrically enriched in the R3/R4 precursor pair. In and posterior to column 6, Fmi is expressed at the membrane of R4, and largely depleted from R3 membranes that do not touch R4, forming a horseshoe-like R4-specific pattern. In contrast, in aosrlt discs, Fmi restriction to the R4 precursor is generally delayed, and often not established even in columns 8-12, where high levels of Fmi are still seen around the apical membrane cortex of R3 and R4. Since Fmi is thought to act as a homophyllic cell-adhesion molecule, its increased presence on R3 membranes should have a direct effect on Fmi localization in neighboring cells and thus possibly the adhesive properties of the precluster. It is worth noting that although Fmi is required during PCP establishment and R3/R4 cell-fate specification, the delay in Fmi restriction to R4 has no significant effect on the R3/R4 cell-fate decision. Although Fmi interacts with Fz and Notch in this context, the R4-specific mDelta-lacZ marker does not differ significantly from WT and adult aosrlt eyes also display no defects in R3/R4 specification. Thus, it appears that the delay in Fmi localization specifically affects ommatidial rotation, probably through adhesion, and possibly explains the broad range of rotation angles in aosrlt and other Egfr pathway mutants (Gaengel, 2003).
Thus Egfr/Ras signaling plays a general role in the
regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).
The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. Four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi) were examined in this study, and what happens when these genes are either removed or overexpressed in clones of cells was examined. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, a model is presented that builds on an earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. It is conjectured that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient (Lawrence, 2004).
Evidence is discussed that cells compare their scalar readout of the level of X with that of their neighbors and set their own readout toward an average of these. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information, and that Vang assists in the receipt of this information. The comparison between neighbors is crucial, because it gives the vector that orients hairs: these hairs point toward the neighbor cell that has the lowest level of Fz activity (Lawrence, 2004).
Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins studied, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view -- it is suggested that these localisations may be more a consequence than a cause of planar polarity (Lawrence, 2004).
There are a number of simple systems in which isolated cells orient to a
polarising signal. These include the localized outgrowth, or 'schmooing' of
yeast in response to mating pheromone and directed migration of Dictyostelium cells up a gradient of cyclic AMP. Small
differences (as little as 1%-5%) in receptor activation across single cells are
sufficient to polarise them, a response that, in yeast and elsewhere probably depends on localised exocytosis. It is not known whether the polarisation of single,
isolated cells is a model for planar polarity of cells in an epithelium, but
it is likely that they share at least some of the mechanisms (Lawrence, 2004).
It has been proposed that, in the abdomen of Drosophila, morphogen
gradients (Hh in the A compartment and Wg in the P compartment) organise a
secondary gradient ('X'); the vector of X specifying the polarity of each cell.
Although the composition of X is unknown, at least three proteins, Fj, Ds and
Ft, are implicated. All three may be expressed, or be active, in bell-shaped
distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries. Ds and
Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in
the Golgi. Ds and Ft are integrated into the membrane, suggesting that
the X gradient itself may not be diffusible but instead might depend on
information transfer from cell to cell (Lawrence, 2004).
How does Hh set up the X gradient? Although changing the real or perceived
level of Hh does affect polarity, many clones (for example clones that lack
Smo, an essential component of Hh reception) show there is no simple
correlation between Hh concentration and polarity. For instance, large
smo- clones in the center of the A compartment are
polarised normally, even though they are blind to Hh. Also, while
smo- clones in some regions of the A compartment do
affect polarity, both mutant and wild-type cells are repolarised. Both
these observations argue for some transfer of information about polarity
between cells, a process that would be at least partly Hh independent. This
paper explores this process and is concerned with four genes (stan, fz,
Vang and pk) that probably act downstream of ds, ft and
fj (Lawrence, 2004).
Perhaps normal cells could transfer information from one to another (this
might be particularly important for nascent cells following mitosis) to help
keep the readout of X as a smooth gradient? To do this they might make a
comparison of their neighbors and modify this readout of X toward an average
of those neighbors. X might be read by a receptor molecule and the results
point to Fz being the most likely candidate. The results indicate that the
comparison itself requires the cadherin Stan. Thus, a cell would need to read
and compare (using Stan) the levels of X (recorded in the activity of Fz) in
neighboring cells. Then, in a way analogous to how a Dictyostelium
amoeba reads the vector of a cAMP gradient, a cell would determine its
polarity from the vector of Fz activity. The results suggest that Vang also
acts in this step, helping cells to sense the level of Fz activity in
neighboring cells (Lawrence, 2004).
Some of the results are discussed in terms of the model (Lawrence, 2004).
Clones that lack, or overexpress Fz cause local and consistent repolarisations of cells that extend from within the clone and affect normal wild-type cells outside it. Because simply removing the fz gene
from all cells randomizes polarity in the ventral pleura, it is self-evident
that these organised polarity reversals must result from an interaction
between the clone and the surrounding cells. It has been argued that Stan and Fz
act in this process, but how? Note that stan and fz are the
only mutants that have randomised hairs in the pleura, and the results
indicate that neither Stan nor Fz can function properly without the other.
Averaging might depend on the capacity of Stan to form homophilic dimers as
bridges between neighboring cells, with such Stan:Stan dimers serving as a conduit for information about the relative level of Fz activity in each cell. However,
with respect to non-autonomy, the results with the two genes differ:
How far does the non-autonomy spread into wild-type cells? This process can be stimulated. According to the model this range would
depend on the value of a single adjustable parameter, a that relates to how much a cell's scalar is read from
X. At one extreme for this parameter (a=0), when the scalar of a cell
depends only on X, a wild-type cell just posterior to a clone of
fz- cells would reset its scalar as it was before;
there could be no averaging and only that cell and its
fz- neighbor will be repolarised. Thus the
non-autonomy would be limited to one cell. At the other extreme
(a=1), any local disturbance produced by a clone would decay rapidly
because of averaging, and the repolarisation will tend to be lost altogether.
In between these extremes, the non-autonomy spreads more than one cell, but
over diverse values for this parameter, the range is near the amount usually observed (2-4 cell diameters) (Lawrence, 2004).
It has been observed that fz- clones have effects
over longer range in backgrounds such as ds- where the X
gradient might be flatter than normal. Similarly, cells are normally polarised
in large smo- clones in the middle of the A
compartment, where, because there can be no input from Hh, the X gradient
could also be flat. Both these results are consistent with the model, because
the range affected by averaging will increase (Lawrence, 2004).
Many of the proteins required for normal cell polarity, including Fz, Dsh,
Dgo, Pk, Vang and Stan are found to be asymmetrically localised in the
proximodistal axis of wing cells. This localisation is restricted to a brief period of just a few hours shortly before the wing hairs grow out, but, nevertheless it is
assumed to be mechanistically important to planar polarity. For example, non-autonomy could be explained if localised proteins were components of one or more molecular complexes that propagate polarity from cell to cell. In support of this, note that loss of any of these proteins, including the removal of both Pk and Sple, prevents the asymmetric localisation of the others (Lawrence, 2004).
But the results do not seem to fit with such a mechanism, mainly
because they provide evidence that polarity can propagate into cells that
lack, or fail to localise all of these proteins. In particular,
pk- cells are normally polarised throughout the P
compartment and can be repolarised in both compartments by sharp
discontinuities in Fz activity even in the pleura (where polarity is
randomized in fz- and stan- animals). At a minimum, these findings challenge the hypothesis that Pk itself is an essential component of a feedback amplification mechanism responsible for polarising cells.
Furthermore, if it is assumed that the observed failure of Fz, Dsh, Vang and Stan
to localize in pk- wing cells reflects a general
property, these results also challenge the idea that Fz, Dsh, Pk, Vang, Diego
and Stan must be able to accumulate asymmetrically in order for cells to
detect, and be polarised by, the X gradient, or by disparities in Fz activity.
Indeed, Adler (2002) has already hinted that there is no convincing evidence that the asymmetric localisation of these proteins actually functions in planar
polarity: 'the preferential accumulation [of proteins] along the...edges
of wing cells is a process that intuitively seems likely to be part of a core
system...but perhaps it is not and if not...this would leave rather
little in the core' (Lawrence, 2004).
Are wing cells polarised only briefly just prior to the hair outgrowth? The
reason for raising this possibility is that the proteins are apparently only
asymmetrically localised at that time. If this localisation were not causal,
as it is now suggested, it could be that the cells are polarised for all or most of
development -- again arguing that the ephemeral localisation of the
proteins is more a consequence than a cause of polarisation (Lawrence, 2004).
The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it
as R4. The spalt zinc-finger transcription factors
(spalt major and spalt-related) are part of the molecular
mechanisms regulating R3/R4 specification and planar cell polarity
establishment. In mosaic analysis, spalt genes have been shown to be
specifically required in R3 for the establishment of correct ommatidial
polarity. In addition, spalt genes are required for
proper localization of Flamingo in the equatorial side of R3 and R4, and for
the upregulation of Delta in R3. These requirements are very similar
to those of frizzled during R3/R4 specification. spalt genes are required cell-autonomously for the expression of
seven-up in R3 and R4, and seven-up is downstream of
spalt genes in the genetic hierarchy of R3/R4 specification. Thus,
spalt and seven-up are necessary for the correct
interpretation of the Frizzled-mediated polarity signal in R3. Finally, it has been
shown that, posterior to row seven, seven-up represses spalt
in R3/R4 in order to maintain the R3/R4 identity and to inhibit the
transformation of these cells to the R7 cell fate (Domingos, 2004).
Therefore, the results suggest that sal is required upstream or in
parallel to the Fz/PCP pathway for R3/R4 specification. Also, in support of
this model, sal expression is not affected in R3/R4, either in gain-
or loss-of-function experiments with members of the Fz/PCP and Notch signaling
pathways. sal is required cell-autonomously in R4 for normal
levels of mdelta0.5-lacZ expression. This requirement of
sal in R4 could be due to a defect in the activation of Notch
signaling [e.g. sal may be required for the expression of
Notch or Su(H)]. Alternatively, sal may be required
for transcriptional activation of E(spl)mdelta, in parallel to
Notch signaling. The latter possibility is favored, since the expression of a
transgenic line, where lacZ is under the regulation of 12Suppressor of Hairless multimerized-binding sites
[12Su(H)-lacZ], is not affected when R4 is sal–. The 12Su(H)-lacZ transgenic line is a reporter for Su(H)-dependent Notch signaling, and thus, sal is not required for the expression or activation of Notch, Su(H) or other components required for signaling. In addition, exogenous expression of a constitutively activated Notch (sev-Nact) can rescue mdelta0.5-lacZ expression in sal– clones. Altogether, these results suggest that sal acts in parallel to Notch signaling for the transcriptional activation of E(spl)mdelta. Finally, although there is a reduction of E(spl)mdelta expression when R4 is sal–, this does not correspond to chirality defects in mature ommatidia. This suggests that other genes may be redundant to sal in R4 for PCP establishment (Domingos, 2004).
In conclusion, these results demonstrate that sal is required in R3
to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates.
Ommatidia mutant for sal show defects that are very similar to those
observed in fz and dsh mutants, as judged by the loss of
asymmetric Fmi localization at the equatorial side of the R3/R4 precursors,
and by the lack of Dl and E(spl)mdelta upregulation within
the R3/R4 pair. In addition, sal is required upstream of svp
for normal R3/R4 specification. Finally, these results show that, posterior to
row seven, svp represses sal in R3/R4 in order to maintain R3/R4 identity and to inhibit transformation of these cells to the R7 cell fate (Domingos, 2004).
Cells in a variety of developmental contexts sense extracellular cues that are given locally on their surfaces, and subsequently amplify the initial signal to achieve cell polarization. Drosophila wing cells acquire planar polarity along the proximal-distal (P-D) axis, in which the amplification of the presumptive cue involves assembly of a multiprotein complex that spans distal and proximal boundaries of adjacent cells. This study pursues the mechanisms that place one of the components, Frizzled (Fz), at the distal side. Intracellular particles of GFP-tagged Fz move preferentially toward distal boundaries before Fz::GFP and other components are tightly localized at the P/D cortex. Arrays of microtubules (MTs) are approximately oriented along the P-D axis and these MTs contribute to the formation of the cortical complex. Furthermore, there appears to be a bias in the P-D MTs, with slightly more plus ends oriented distally. The hypothesis of polarized vesicular trafficking of Fz is discussed (Shimada, 2006).
It is proposed that Flamingo- and Fz::GFP-containing vesicles are transported preferentially toward the distal cell cortex along P-D-oriented MTs. It has been considered that a cassette of transmembrane proteins (Four-jointed, Dachsous, and Fat) functions upstream of Fz and confers an initial small asymmetry of Fz activity across the cell. This small imbalance is amplified through formation of the multiprotein complex across the P/D boundary, reinforces Fz signaling, and eventually leads to specification of prehair formation at the distal cell end. The polarized transport of Fz that was observed may reflect one of the outputs of the upstream cassette to set an initial bias of Fz activity and/or an intermediate step of the Fz signaling feedback amplification (Shimada, 2006).
Immuno-EM studies revealed intracellular vesicles that were associated with Fz::GFP or Flamingo (Fmi) on the AJ plane. The number of these vesicles per cell was an order of magnitude higher than that of Fz::GFP particles visualized by confocal microscopy. Only a subpopulation of the vesicles, one containing large numbers of Fz::GFP molecules, may have been detected by confocal microscopic observations. Although both the multiprotein complex of the cortical PCP signaling components and the DE-cadherin-catenin adhesion complex are located at AJ in an almost overlapping manner, this study supports the possibility that components of these two complexes were separated into distinct vesicles at their exit from the trans-Golgi network (TGN). Sorting of Fz::GFP, Fmi, and possibly Dsh as well from DE-cadherin should also take place when molecules on the plasma membrane are incorporated into recycling endosomes. Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for sorting apical and basolateral plasma membrane proteins. Wing epidermal cells appear to have a machinery to subdivide molecules that are targeted to AJ (Shimada, 2006).
The results of double staining for Fz::GFP and Fmi indicated that the majority of Fmi-containing vesicles are transported distally, not to the proximal boundary. Then how are Fmi, Stbm/Vang, and Pk distributed to the proximal cortex (Shimada, 2006)?
Fmi, Stbm/Vang, and Pk have already been distributed rather uniformly at the cell cortex in imaginal and early pupal epithelia before the polarized transport starts, and these molecules may be allowed to diffuse laterally within the plasma membrane. Fmi, which is transported to the distal boundary together with Fz and Dsh, can lock a counterpart on the proximal membrane of the adjacent cell through its homophilic binding property. Formation of this Fmi-Fmi bridge across the P/D boundary would anchor the Fz-Dsh complex at the distal cortex, increasing local activity and the copy number of Fz-Dsh. This slight input may initiate recruitment of Stbm/Vang-Pk on the opposing proximal cortex by means of mutual exclusion between Dsh and Pk, and by means of hypothetical ectodomain interaction between Fz and Stbm/Vang. Then the nascent asymmetric complex amplifies the imbalance of Fz activity by unknown mechanisms. This amplification could be involved in either facilitating loading Fz-Dsh and Fmi into vesicles at their exit from the TGN, accelerating the transport of the vesicles to the distal cortex, and/or restricting diffusion of Fz-Dsh and Stbm/Vang-Pk out of the distal and proximal membrane domains, respectively (Shimada, 2006).
In setting up the initial bias, the polarized transport may not necessarily require Stbm and Pk, whereas the subsequent amplification presumably depends on the formation of the cortical complex. This hypothesis is consistent with the observation that bright Fz::GFP particles were hardly seen in the dsh, fmi, or pk mutant backgrounds. It also explains why FzP278L::YFP particles did not move distally, because FzP278L is postulated to lose its interaction with Dsh and most FzP278L::YFP particles did not contain Fmi (Shimada, 2006).
Besides noncentrosomal MTs along the apicobasal axis, P-D-oriented MTs were unequivocally visualized in the AJ plane, and the important role of this MT array in localizing the cortical PCP proteins and the distal-oriented movement of Fz::GFP particles was shown. Assuming the importance of the P-D MT alignment, how is the Fz::GFP particle transported or recycled back along the MT track in terms of vesicular trafficking? The particles showed staggered trajectories when followed at 1 or 10 s intervals, and this back-and-forth motion of the Fz::GFP particle can be interpreted as follows: (1) individual Fz::GFP particles bind multiple motors of different classes, and (2) activities of the motors of opposing directionality are coordinated. This bidirectional transport has been shown for organelle trafficking; despite such back-and-forth motion, cargos can still achieve net transport on longer time scales. Another, not mutually exclusive, possibility could be that the particle/cargo motor complex may repeat cycles of running, falling off the MT track, and reengaging. Whether specific members of MT motors contribute to the Fz::GFP transport or not awaits further genetic as well as biochemical characterization of the particle (Shimada, 2006).
Given that either of the above possibilities of motor-driven motions is the case, why did the Fz::GFP particle move predominantly toward distal cell boundaries when followed at 1 min intervals? Attempts were made to trace the origin of this asymmetry to the polarity of individual P-D MTs and it was found that the wing cell had slightly more + end-distal MTs than + end-proximal ones. Nevertheless, it is an open question as to whether such a small difference can be causally related to the distally oriented transport and whether the subtle difference in MT polarity can be one of the critical parameters in operating the cortical feedback loop (Shimada, 2006).
How are noncentrosomal MTs aligned approximately along the P-D axis at the level of adherens junction? P-D-oriented MTs were still observed in the absence of Fz function, and this strongly suggests that generation of the P-D MT array is controlled by a mechanism upstream of, or separate from, the cortical complex. One clue for this mechanism involving MTs may be Widerborst (Wdb), a B' regulatory subunit of PP2A. It would be necessary to investigate how exactly MTs are disorganized when Wdb function is abrogated. Another clue may be a recent report that the presence of apical MTs is related to Dpp signaling activity, although a cytoskeletal function for Dpp remains to be shown (Shimada, 2006).
A large gap remains in cell biological understanding of the global cue that is considered to be provided by the cassette of transmembrane proteins (Fj, Ds, and Ft). It should be pointed out that each of Fj, Ds, and Ft controls the ratio of proximal-distal to anterior-posterior growth of appendages and that Ds and Ft have been recently shown to control the shape of the growing organs by regulating orientations of cell divisions in imaginal discs. This supports the hypothesis that Ds and Ft can orient the mitotic spindle, and hence MT organization, during wing morphogenesis. It would be intriguing to investigate whether the MT orientation and polarity observed in this study are also under the control of the upstream cassette, and to pursue a molecular connection between tissue shape and polarity (Shimada, 2006).
Viable stan mutants (particularly stan3/stan3 and
stan3/Df-stan) were used to examine the stan wing phenotype. As is the
case for other tissue polarity mutants, stan mutants do not show
a complete loss or randomization of hair polarity across the
wing. Rather, they show a stereotypic abnormal polarity pattern. The polarity patterns that
result from mutations in many tissue polarity genes are quite
similar, albeit not identical. This pattern has been called the fz/inturned
(in) pattern after two of the best-studied genes in fz pathway. The stan mutant wings also have this general pattern.
A second criterion that has been used to characterize tissue
polarity mutants is the frequency of wing cells that form more
than the normal one hair. Wings homozygous for stan3 have
relatively few multiple hair cells (an average of 1.03 hairs/cell
in a typical test region). This is similar to wings mutant for
fz (1.02 hairs/cell), dsh (1.01 hairs/cell) and pk (1.02 hairs/cell). In contrast, it is much lower than that seen in wings mutant for
inturned (in) (1.82 hairs/cell), fuzzy (fy) (1.92 hairs/cell) and
multiple wing hair (mwh) (3.94 hairs/cell). The process of hair morphogenesis in stan was examined pupal
wings. In wild-type wings, the prehairs that develop into the
adult cuticular hairs are formed in the vicinity of the distal-most
vertex of the cell and extend away from the cell in a distal
direction. In stan mutants, many cells form
prehairs at a relatively central location on the apical cell surface. Other cells form prehairs at abnormal locations
along the cell periphery. This is similar to what has been found
for mutations in the fz-like genes.
Thus, based on several phenotypic criteria stan can be placed in the
fz-like group of genes (Chae, 1999).
In both embryos and imaginal tissues, stan is broadly expressed in epithelia and the nervous system. Mutations of the stan gene were isolated and base substitutions have been identified in the protein coding sequences of two genetically null alleles, fmiE45 and fmiE59, neither of which give a signal in immunostained epithelia. The stan null mutations are embryonic lethal, and the mutant embryos show local disconnection of longitudinal axon fascicles in the central nervous system. This lethality and the axonal defect can be rescued by stan cDNA expression in the nervous system of the mutant, fmiE45/fmiE59, indicating that the role of Stan in axon outgrowth is essential for viability. In the rescued animals, epithelia and imaginal photoreceptor cells are devoid of stan expression, and those adults exhibit defects in planar cell polarity in three classes of structures: ommatidia, sensory bristles, and wing hairs. This finding classifies stan into the core group of tissue polarity genes that includes frizzled and dishevelled. In region D of the wing of the fmiE45/fmiE59 escaper, hairs are deflected from the P/D axis and oriented toward the posterior wing margin. This pattern alteration is similar to that reported for the same region of fz mutant wings. As reported in null mutants in all the known tissue polarity genes, most hairs of the stan mutant do not point in random directions but possess an abnormal, nondistal polarity, similar to that of their neighbors. This 'stream' pattern suggests that a stan-independent system is working to align adjacent cells (Usui, 1999).
Polarity disruption of the above structures is reproduced in homozygous stan (fmiE59) clones, and prehairs emerge near the cell center or at wrong locations along the cell periphery, which is again similar to a phenotype of fz mutations. In the core group, several mutations in fz and strabismus/Van Gogh (stbm/Vang) show non-cell autonomy in specifying hair polarity, and those nonautonomous effects range over ten cells when clones are large. In contrast, the stan null mutation behaves essentially in a cell-autonomous way (Usui, 1999).
It is known that most fz mutations, including fzR52, show non-cell autonomy in orienting wing hairs with perfect penetrance. In areas distal to but not proximal to fz mutant clones, hairs of fz+ cells swirl toward the clone; this is due to initiation of prehair formation at abnormal subcellular sites, for example, at posterior and proximal cell vertexes. The mis-selection of prehair sites appears to be prepatterned by mislocalization of Stan. The Stan zigzag pattern is distorted in areas distal to the clone; many zigzags run obliquely rather than orthogonally to the P/D axis, and at a subcellular level, Stan is present at A/P cell boundaries in some fz+ cells. Deformation of the Stan pattern is minimal in areas proximal to the clone (Usui, 1999).
Whether prehair morphogenesis always occurs in the vicinities of such Stan-rich A/P boundaries is difficult to determine because Stan localization is being depolarized once prehairs start outgrowth. Nevertheless, the directional nonautonomous effect of the fz mutation on Stan localization was observed in 20 out of 20 clones examined that were well isolated from one another, suggesting a tight coupling of Stan mislocalization with incorrect placement of prehair sites. This observation is consistent with the hypothesis that assembled Stan molecules play an important role in initiating prehair morphogenesis (Usui, 1999).
The sensory bristles of the fruit fly Drosophila are organized in a polarized fashion such that bristles on the thorax point posteriorly. These bristles are derived from asymmetric division of sensory organ precursors (SOPs). The Numb protein, which is localized asymmetrically in a cortical crescent in each SOP, segregates into only one of the two daughter cells during cell division, thereby
conferring distinct fates to the daughter cells. In neuroblasts, establishment of apical-basal polarity by the protein Inscuteable is crucial for orienting asymmetric division, but this is not the case for division of SOPs. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pl) of SOPs. Flamingo/Stan, controls the planar polarity of sensory bristles and the orientation of the SOP pl division. Both the loss of function and overexpression of stan disrupted bristle polarity. During mitosis of the SOP, the axis of the pl division and the positioning of the Numb crescent are randomized in the absence of Stan activity. Overexpression of Stan and Frizzled cause similar effects. The dependence of proper Stan localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity (Lu, 1999).
Loss of starry night/flamingo function disrupts the planar polarity of non-innervated hairs on the Drosophila wing. To investigate the role of stan in regulating the planar polarity of sensory bristles, the bristles on the adult notum (dorsal thorax) were studied in stan mutants. Null mutants of stan are embryonic lethal. A transheterozygous combination between fmiE59, a putative null allele carrying a nonsense mutation in the segment of the gene encoding the ectodomain of Stan, and fmi71, for which the molecular lesion has not yet been determined, gives viable adult flies that display abnormal polarity in both types of sensory bristles (macrochaetes and microchaetes). Instead of pointing posteriorly within the epithelial plane as in wild-type flies, these bristles point in different directions: posteriorly, laterally and, less frequently, anteriorly. Moreover, some bristles also point upwards. The macrochaetes tend to be less affected than the microchaetes, and bristles located at different positions on the notum are affected to different degrees. Although only a small percentage of microchaetes located at the anterior (10%) and dorsocentral (13%) positions exhibit abnormal polarity, a higher percentage at the posterior (57%) and lateral (61%) positions are affected (Lu, 1999).
A similar planar polarity phenotype is observed in fmiE59 mutant clones. As in fmiE59/fmi71 transheterozygotes, fmiE59 mutant clones generated at different positions on the notum exhibit position-dependent penetrance of bristle polarity phenotypes. Marking the mutant clones with the yellow marker allows for an examination of whether Fmi activity is cell autonomous. In most cases, bristles that display abnormal polarity are confined within stan mutant clones. Infrequently, a wild-type bristle immediately adjacent to a mutant clone exhibits abnormal polarity. Thus, unlike fz, which has a long-range distal non-cell-autonomous effect on the wing, stan acts largely cell autonomously on the notum to control the planar polarity of sensory bristles. A similar cell-autonomous function of Stan is observed for the polarity of non-innervated hairs on the wing (Lu, 1999).
Not only does loss of stan function disrupt bristle planar polarity, overexpression of stan causes similar effects. stan was overexpressed on the adult notum using the apterous gene to drive expression of the Gal4-encoded transcription factor and a stan transgene driven by the upstream activator sequence (UAS) to which Gal4 binds. The apterous gene is expressed throughout the parts of the wing and haltere discs that give rise to the dorsal surface of wing blades and haltere, and in the regions that form the notum, scutellum and wing hinge. The microchaetes in apterous-Gal4 driven UAS-stan flies are affected to similar degrees as in fmiE59/fmi71 transheterozygotes; the macrochaetes are more affected than those in fmiE59/fmi71 flies. On the wing, overexpression of stan with a patched-Gal4 driver alters the planar polarity of non-innervated wing hairs. The disruption of bristle planar polarity by both overexpression and loss of function of stan suggests that the planar polarity signaling pathway is sensitive to the level of Stan activity (Lu, 1999).
The planar polarity pathway is also sensitive to the level of Fz activity. Overexpression of fz using UAS-fz and apterous-Gal4 also disrupted bristle planar polarity, consistent with earlier experiments using fz constructs driven by the heat shock gene (hs) promoter. Besides the planar polarity phenotype, overexpression of fz also leads to the formation of ectopic macrochaetes. The apterous-Gal4 driven UAS-fz flies exhibit frequent (~80%) ectopic induction of scutellar bristles, a phenotype not observed in apterous-Gal4 driven UAS-stan flies. Further overexpression studies with Drosophila Frizzled 2 (Dfz2) and dominant-negative forms of Fz and Dfz2 have suggested that, in addition to its role in controlling planar polarity, Fz also functions together with Dfz2 in the Wingless pathway to pattern the sensory bristles (Lu, 1999).
The orientation of the SOP pI division axis and the positioning of Numb protein crescent on the cortex both respond to polarity cues that are regulated by Fz signaling. In fz or dishevelled (dsh) loss-of-function backgrounds, the SOP pI division axis and the positioning of the Numb crescent are randomized. These two processes were examined in the pupal notum in apterous-Gal4;UAS-fz flies, and it was found that both the spindle orientation and positioning of Numb crescent no longer follows the anteroposterior axis and becomes randomized. Nevertheless, the Numb crescent still overlays one pole of the misoriented spindles and the randomization of spindle orientation is restricted two-dimensionally within the epithelial plane, as is observed in fz or dsh loss-of-function mutants (Lu, 1999).
The frizzled gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is
needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones
could be a consequence of a failure in the propagation of this signal. This hypothesis was tested in two ways. In one set of experiments the domineering non-autonomy of fz and Vang Gogh (Vang) clones was used to assess the direction of planar polarity signaling in the wing.
prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, pk mutations alter the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy
is not due to an alteration in a global signal. In a second series of experiments, cells in the pupal wing were ablated. A lack of cells
that could be propagating a long-range signal does not alter hair polarity. It is suggested that fz and Vang clones result in altered levels of a locally
acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal (Adler, 2000).
The complementary nature of the domineering nonautonomy of fz and Vang clones is striking. It is true for
the anatomical direction of the non-autonomy (i.e. distal vs.
proximal); the relationship of the domineering non-autonomy to the clone (i.e. affected wild-type hairs pointing toward or away form the clone), and
for the interactions with pk mutations. It is suggested that the
domineering non-autonomy of fz clones is a consequence of
a failure of the clone cells to send a locally acting polarity
signal. The domineering non-autonomy of Vang clones
could be due to the Vang clone sending excess signal
(models that reverse this arrangement are also possible) (Adler, 2000).
A model for tissue polarity signaling in the wing is presented.
Early models to explain planar polarity in the insect
epidermis suggested it could be a reflection of the vector
of a concentration gradient
and this idea has remained popular. It is suggested
that a distal/proximal gradient of fz activity is produced in
the early prepupal wing (or wing disc). One way this could
be achieved is by a gradient of a Wnt (or other type of
ligand) resulting in a gradient of ligand bound Fz. Later in development cells would produce a
locally acting second signal in amounts proportional to Fz
activity. This hypothetical signal is referred to as Z and it is
suggested that ligand bound Fz activates more Z production
than unbound Fz. In this way a gradient of Fz
activity would be translated into a gradient of Z. Cells
would respond by initiating prehair morphogenesis on the
side of the cell where Z level was lowest. This would result
in hair polarity being oriented in the same direction as the
vector of the Z concentration gradient. This is consistent
with previous results showing that a directed gradient of fz
expression results in cells with higher Fz levels producing
hairs that point toward cells of lower levels. The absence of fz activity in clone cells would result
in no Z being produced by the clone and a local decrease in
Z levels that would cause surrounding cells to produce hairs
that point toward the clone as is observed. Such a model
can effectively incorporate the affects of pk and ds mutations
on the direction of fz domineering non-autonomy. Mutations
in these genes could alter the relationship between the
ligand bound state of fz and Z production. For example, in
a new antimorphic dominant pk allele, pkD wing unbound Fz receptor could act as a super-activator of Z production. This would lead to a reversed gradient of Z and to the reversal of both polarity and the direction
of fz domineering non-autonomy. This model can
also explain the observation that cells inside of a pk clone
display the same polarity as do cells in a similar position in
an entirely pk wing, since the alternative polarity caused by pk
mutations would be due to abnormal amounts of Z. Such a
model can also explain some of the results seen with Vang. The domineering non-autonomy of Vang could be due to Vang cells being constitutive for the
production of high levels of Z. This would lead to locally
elevated Z levels and cells surrounding Vang clones producing hairs that point away from the clone, as is observed.
The model can also explain the ability of pkD to enhance
the extent of fz domineering non-autonomy and suppress the
extent of Vang domineering non-autonomy. In the model
the level of Z will be higher in all regions of a pkD wing
since now both bound and unbound Fz receptor will be strongly
activating the production of Z. Thus, when a clone
of cells lacking functional Fz protein is produced, the difference between the amount of Z produced by the clone cells and their neighbors will be increased over that seen in an otherwise wild-type wing. The ability of pkD to inhibit the
extent of domineering non-autonomy of Vang clones can be
explained by the reduced difference in the level of Z
produced by the clone and neighboring cells (Adler, 2000).
At first glance the model cannot explain the suppression
of Vang domineering non-autonomy in a fz mutant background, because the clone should produce high levels of Z in a background where there is little or no
Z produced. One possibility is that in the absence of functional Fz no Z can be produced. A second possibility is that fz has multiple functions in wing tissue polarity and that an additional function is what suppresses the domineering non-autonomy of Vang. The model can also explain the relatively weak and
poorly penetrant domineering non-autonomy of pk clones.
The cells in such clones would produce aberrant amounts of
Z, however the difference between the normal and mutant
levels would be less than is seen in a fz mutant clone (that
produces no Z) or in a Vang mutant clone (that produces
constitutive high levels of Z). Thus, it is reasonable that pk
(and ds) clones would show weak domineering non-autonomy (Adler, 2000).
The results of the cell ablation experiments are also
consistent with the model. After surgery wound healing
took place in the pupal wing. To form a permanent hole it
was necessary for the healing to juxtapose neighboring
dorsal and ventral wing cells. In those cases the juxtaposed
cells were of similar position along the proximal/distal axis
and therefore would be expected to be producing similar levels of Z.
Thus polarity disruptions, equivalent to
a clone of fz cells that juxtapose cells that produce normal
levels of Z with cells that produce none, would not be seen.
The model also predicts that the domineering non-autonomy of fz clones should be greater in proximal regions and
weaker in the most distal regions of the wing. Some evidence for this sort of variation was found, although over a
large middle region of the wing no significant difference in the strength of domineering non-autonomy is seen. Perhaps the hypothesized gradient of Z is shallow in this region of the wing or the assay is not sensitive enough.
The model also predicts that the domineering non-autonomy
of Vang clones should be greater in distal and weaker in
proximal regions, but this was not seen.
The nature of the hypothesized factor Z and its receptor
are unknown. The function of the genes that encode these
factors should be required for cells to sense differences in fz
activity. An attractive candidate for the receptor is fz itself,
since previous experiments have indicated that fz has both cell
non-autonomous and cell-autonomous functions in the
development of wing tissue polarity. Two roles for fz are also suggested by experiments that found both Vang and starry night/flamingo are required for some, but not all fz functions. This could be explained by fz functioning
both upstream and downstream of Vang and starry night.
Alternative candidates for factor Z and its receptor are Delta
and Notch. These genes have been shown to be downstream
of fz in the eye and to interact with dishevelled (dsh) during wing development. An interesting feature of the model is
that the fz-dependent production of factor Z should be dsh
independent because dsh is acting cell autonomously.
It is worth noting that models suggested to explain the
development of ommatidial polarity also rely on two sets of
gradients along the polar/equator axis. Indeed, there are substantial similarities between the
models. One difference is that in contrast to this model for
the wing, in the eye fz is suggested to be essential only in the
read out of the secondary gradient (Adler, 2000).
The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity
decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points
distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. During cell polarization, the Frizzled receptor (visualized using Frizzled-Green fluorescent protein) is localized to the distal cell edge, probably resulting in asymmetric
Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent
trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential
receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across
the axis of a cell (Strutt, 2001).
To understand the asymmetric distribution of Fz-GFP, the distribution was studied in flies mutant for other genes involved in trichome polarity establishment. In clones of cells lacking starry night (stan) function, both the apical and PD localization of Fz-GFP is completely abolished. However, in cells lacking dsh function, in which Fz signal transduction is compromised, Fz-GFP apical localization is preserved, but there is no proximodistal (PD) localization, with a splotchy irregular distribution being seen instead. The same phenotype is observed for mutations in the prickle-spiny-legs (pkpk-sple) and Van Gogh (Vang) genes. This would be consistent with the trichome polarity phenotypes of these mutations being due to a failure of Fz localization (Strutt, 2001).
Genetic data indicate that the polarity genes in, fy, and mwh act downstream of Fz/Dsh, inhibiting trichome formation where Fz is not active. In agreement with this, mutations in these loci do not alter Fz-GFP distribution despite trichome polarity being disrupted (Strutt, 2001).
The localization of Stan has also been reported to be disrupted in cells lacking dsh function but not in those lacking mwh. Therefore, whether Fz-GFP and Stan remain colocalized in different mutant backgrounds was tested. In clones of cells lacking dsh function, it was found that both Fz-GFP and Stan remain predominantly apical, and although the distribution of both appears diffuse, they nevertheless show broad colocalization. Similarly, in an in background, both Fz-GFP and Stan remain apical and colocalized to the PD cell boundaries (Strutt, 2001).
It is concluded that one important function of Stan is to localize Fz apically in the cell during polarity establishment.
Stan may also play a role in localizing and/or anchoring Fz at the distal cell edge. However, in the
absence of fz autonomous polarity signaling activity, neither Fz-GFP nor Stan is localized to PD boundaries.
This leads to the speculation that the Fz receptor is responsible for receiving an extracellular polarity signal, and that
the interpretation of this signal drives the localization of Fz to the distal cell edge and Stan to the distal and
proximal cell edges, Stan thus acting downstream of Fz. Nevertheless, the data would equally well support Fz
acting downstream of Stan; the codependence of the localization of both proteins would support this possibility,
as would the observation that Stan has homology to G protein-coupled receptors. Thus far, no ligand has been identified for the Fz receptor in polarity signaling, and it is
conceivable that in fact Fz might be activated by association with another transmembrane receptor, a role for
which Stan is clearly a candidate (Strutt, 2001).
Both Fz-GFP and Stan localization are also downstream of fz nonautonomous signaling activity. One of the functions of Stan is to measure differences in fz activity between adjacent cells, as Stan accumulates on the boundary between fz+ and fz- cells. These findings have been extended to show that Stan in fact accumulates on the boundaries between cells with different levels of fz nonautonomous signaling activity. This observation is consistent with there being a long-range gradient of fz nonautonomous signaling activity across the pupal wing, with each cell having different levels of fz nonautonomous activity relative to its neighbors (Strutt, 2001).
An intriguing observation is that in the cells bordering a clone deficient in only fz autonomous signaling, Stan appears to show a preference for localizing on the cell boundaries lying perpendicular to the clone boundary. Thus, in cells bordering the proximal and distal clone edges, Stan is sometimes seen lying on the lateral cell boundaries rather than the PD cell boundaries. Similarly, the same is true for Fz-GFP localization on the boundary of stan clones. This phenomenon seems to suggest that if Stan or Fz-GFP cannot localize on one of the PD boundaries of a cell, they show a preference for not localizing on the opposite PD boundary. This, in turn, opens up the possibility that there is an intracellular communication mechanism that couples Stan/Fz-GFP accumulation on one cell boundary to that on the opposite cell boundary. Interestingly, such a mechanism would enable the propagation of a wave of Stan/Fz-GFP polarization across the wing, starting from a single row of polarized cells at one edge. Obviously, such a mechanism for propagation of polarity would obviate the need for an external ligand gradient and would also argue against the existence of a long-range gradient of fz nonautonomous activity. However, long-range gradient models and cell-cell communication models for the propagation of polarity need not be mutually exclusive, and both may operate side by side (Strutt, 2001).
Taking these observations together, the following model is put forward for Fz function in the polarization of single cells in the developing wing. Initially, unlocalized Fz is required for the long-range propagation of a polarity signal. Fz is then recruited apically in a Stan-dependent manner and becomes stably localized at the distal cell edge in a process requiring Fz signaling and the activities of Stan, Dsh, Pkpk-sple, and Vang. This Fz localization then restricts the site of trichome initiation to the distal cell vertex. It is possible that Fz signaling activates Stan molecules to bind both to Fz (in the same cell) and to Stan molecules in the adjacent cell, and so anchors Fz at the distal edge of the cell. Localization of Fz may lead to further increased Fz signaling (possibly through the effects of receptor clustering), which could, in turn, recruit more Stan and Fz. Over time, increased activity of clustered Fz receptors at the distal cell edge would lead to the majority of the Fz in the cell being recruited to this location. In heterologous systems, Fz activity leads to recruitment of Dsh to the cell membrane, so it is likely that Dsh is also present at the distal cell boundary. A precedent for Fz-dependent localization of a cytoplasmic protein during planar polarity establishment is provided by the observation that the Numb protein requires Fz activity for correct asymmetric subcellular localization during sense organ precursor cell divisions (Strutt, 2001).
The stable PD localization of Fz also requires Pk-Sple and Vang activity, with their loss having a similar effect on Fz-GFP localization as loss of Dsh activity. It is possible that, like Dsh, they are required for the transduction of the Fz signal, or they may be involved in the function of Dsh itself. Interestingly, Vang activity on only one side of the PD boundary is sufficient for Fz-GFP localization to occur. Further investigations of the biochemical activities of these proteins will be required to fully elucidate their roles in planar polarity establishment (Strutt, 2001).
The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. The genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes) were investigated. Previous data has shown that the inturned-like genes are epistatic to (function downstream of) the frizzled-like genes when the entire wing is mutant. Consistent with this are observations showing that the asymmetric accumulation of Fz, Dsh, and Fmi is not altered in in, fy, or mwh mutants. Those experiments were extended and the behavior of frizzled clones in mutant wings was examined. The domineering nonautonomy of frizzled clones is not altered when the clone cells are simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it is blocked when the entire wing is mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, both inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. inturned, fuzzy, and multiple wing hairs are required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes the consequences of altering the activity of frizzled-like genes was determined in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhances the phenotypes of hypomorphic inturned and fuzzy mutants. Results from several different experimental paradigms support the hypotheses that in and fy function downstream of fz, dsh, stan, pk, and sple in the wing and that they are required for the transduction of the fz signal to regulate hair morphogenesis and hence the actin and microtubule cytoskeletons (Lee, 2002).
To determine the relative positions of in, fy, and mwh with respect to several other fz-like tissue polarity genes, their ability to block the gain-of-function phenotypes that result from the directed expression of stan/fmi, pk, and sple were examined. ptc-GAL4 was used to overexpress pk. This results in a band of expression in the C region of the wing, with a decreasing gradient of expression away from the center of the ptc expression domain. This results in hairs pointing toward the midline in the C region of the wing. This phenotype is blocked by mutations in in, fy, and mwh. The resulting wings (e.g., ptc-GAL4/UAS-pk; in/in) show no effects from the directed expression of pk. Two different GAL4 drivers (ms1096-GAL4 and act-GAL4) were used to direct the expression of UAS-sple. Both of these drivers result in relatively even expression across the wing (although in ms1096 the level in the dorsal cell layer is higher than that in the ventral cell layer). This results in a reversal of hair polarity over much of the wing that resembles that seen in pkD. Once again it was found that mutations in in, fy, and mwh are able to block this gain-of-function phenotype. The omb-GAL4 driver was used to drive expression of UAS-stan. In the wing disc omb-GAL4 drives expression in a band located centrally along the anterior/posterior axis of the wing. However, in the pupal wing the expression pattern is more complicated during the time for tissue polarity development. During this time in the distal region of the wing, a series of alternating bands of expression and no expression is seen. Driving expression of stan using omb-GAL4 leads to a series of bands of polarity reversals; polarity is oriented from cells of low toward cells of high stan expression. Mutations in in, fy, and mwh are able to block the consequences of overexpressing stan. These data argue that in, fy, and mwh functioned downstream of fz, pk, sple, and stan in tissue polarity (Lee, 2002).
Driving expression of dsh using the ptc-GAL4 driver results in the formation of multiple hair cells in the proximal part of the ptc expression domain. In addition, ectopic bristles are also often found, presumably due to the role of dsh in canonical wg signaling. When UAS-dsh +/+ ptc-GAL4; in/in flies were examined, an additive multiple hair cell phenotype was found (i.e., stronger than either single phenotype) in the proximal part of the ptc domain and the presence of ectopic bristles. Equivalent results were found in UAS-dsh fy; fy ptc-GAL4 wings. The formation of the ectopic bristles was not surprising, since this is likely an effect of dsh on canonical wg signaling and in and fy are not thought to play a role in wg signaling. The additive multiple hair cell phenotype was unexpected and argues either that in and fy are not downstream of dsh in the fz pathway or that when overexpressed, Dsh can bypass the requirement for in and fy function (Lee, 2002).
The overexpression of fz just prior to hair initiation results in a multiple hair cell phenotype, which resembles that of in and fy (this is sometimes referred to as the late fz gain of function). When fz was overexpressed just prior to hair initiation in pupae that were mutant for a null allele of either in or fy an increased number of multiple hair cells was found. Thus, for the late fz gain of function, as for the overexpression of dsh, either in and fy are not required for fz signal transduction or when fz is overexpressed late the requirement for in and fy can be bypassed (Lee, 2002).
To test whether the frizzled-like genes could be regulating the inturned-like genes negatively or positively, the effects of both decreased and increased fz-like gene activity on hypomorphic alleles of in and fy were examined. In these experiments advantage was taken of the much higher number of multiple hair cells found in in and fy mutants compared to fz-like mutants. In all cases examined, mutations (null or hypomorphic) in fz, pk, Vang, stan, and dsh acted as strong enhancers of a weak in or fy phenotype as assayed by the frequency of multiple hair cells. These data are consistent with the fz-like genes acting as positive regulators of in and fy (Lee, 2002).
If there were a simple positive relationship between the fz-like genes and in and fy then it would be expected that the overexpression of fz-like genes should suppress hypomorphic alleles of in and fy. To test this fz was overexpressed from a hs-fz transgene and it was found that this enhances the multiple hair cell phenotypes of weak inII53 and fyJN12 alleles. These results argued that fz antagonizes the activity of in and fy. The genetic relationship between pk, sple, and fmi and weak alleles of in and fy was similarly tested. Overexpression of sple and stan from ms1096-GAL4 (or act-GAL4); UAS-sple and omb-GAL4; UAS-stan, respectively, in hypomorphic mutant backgrounds of in and fy results in an increase in the multiple hair cell phenotypes in the region of the wing, where expression is driven by the GAL4 enhancer trap. A similar enhancement was not seen for the overexpression of pk from ptc-GAL4 UAS-pk. These results suggest that sple and stan antagonize the activity of the in and fy. Notably, the gain-of-function polarity phenotypes of overexpressed pk, sple, and stan are blocked even with weak alleles of in and fy, confirming that these genes are required for the function of the fz pathway. It is clear that the fz-like genes do not act as simple positive or negative regulators of in and fy. The interaction is reminiscent of the observations that similar phenotypes result from either overexpression or a lack of function of fz-like genes (Lee, 2002).
The disruption of the microtubule cytoskeleton by treatment with vinblastine or colchicine results in many wing cells forming more than one hair. At the level of the individual cell these multiple hair cells resemble those found in in and fy mutants since the hairs are formed at the cell periphery and appear to be the result of independent initiation events. The altered polarity seen in in and fy mutant cells is not induced by vinblastine or colchicine. Immunostaining has not revealed any defects in microtubule organization in in or fy mutants. Thus, it seems unlikely that the in and fy phenotypes are due to disrupting the microtubule cytoskeleton and in this way indirectly affecting hair morphogenesis. However, it seemed possible that the disruption of the microtubule cytoskeleton could be inducing multiple hairs by interfering with in and/or fy function. It was reasoned that if that is the case then treatment of in or fy null mutant wing cells with vinblastine or colchicine would not produce a stronger phenotype than the in or fy null cells by themselves. Vinblastine was injected into in or fy mutant pupae and an additive response was found. That is, injected in or fy mutant cells produces a stronger multiple hair cell phenotype than does untreated mutant cells or cells from injected wild-type pupae. This result shows that the microtubule disruption phenotype cannot be due to a simple interference with in/fy function and argues that the microtubule cytoskeleton has a function that is independent of the fz pathway. The additive response in this experiment stands in sharp contrast with the lack of additivity in double mutants of in, fy, frtz, and mwh (Lee, 2002).
The hypothesis that in, fy, and mwh function downstream of fz and dsh originally came from the observation that mutations in in, fy, and mwh are epistatic to mutations in fz and dsh. Consistent with this hypothesis, the distal accumulation of Fz, Dsh, Fmi, and Dgo is not altered in in, fy, or mwh mutants. in, fy, and mwh are required for cells to either detect or respond to the lack of fz activity in a clone of neighboring mutant cells. The function of in and fy was required for cells to respond to a directed gradient of fz, stan, pk, or sple expression. Thus, using both of these experimental approaches it was found that in and fy are required for the function of the fz pathway. The simplest interpretation of this is that in and fy are essential downstream components of the fz pathway in the wing. An alternative explanation is that in and fy function in a parallel pathway and that the function of the fz pathway is dependent on this putative in pathway. There were a couple of surprising exceptional results. The overexpression of dsh or the late overexpression of fz induces the formation of multiple hair cells and it was found that these phenotypes are additive with an in or fy mutation. This could indicate that there are multiple pathways downstream of fz and dsh in the wing and that one of them does not require in or fy function. It is thought more likely that this is an example of overexpression bypassing a normal requirement for downstream proteins. It is suggested that In and Fy normally function as adapters to allow distally accumulated Fz and Dsh to stimulate the cytoskeleton and induce hair initiation in the correct part of the cell. When Fz or Dsh is overexpressed just prior to hair initiation the high cellular concentration might allow these proteins to bypass the need for In and Fy and to interact with and hyperactivate the hair initiation machinery (Lee, 2002).
In vertebrate embryos, a fz-based planar polarity pathway regulates convergent extension. Thus far there has been no evidence for a fz-based planar pathway having an analogous role in Drosophila. The eversion defect seen in fz mutant wings may be such an example. Some fz flies have a deformed wing that is short, fat, and often kinked proximally. This is seen in all strong and null fz alleles examined. This is due to a defect in wing disc eversion. In a normal pupa the wing everts so that it extends posteriorly and ventrally. In a fz mutant many pupae contain wings that extend anteriorly and sometimes dorsally. The wing eversion phenotype is another phenotype where in and fy appear to be epistatic to fz. The observations are somewhat puzzling as the defect in fz appears to be rescued by a mutation in in or fy. Thus, it is not clear that a simple inactivation of the fz pathway is responsible for the eversion phenotype. Perhaps for this phenotype there are multiple inputs into the fz pathway upstream of in and fy and the eversion defect is due to an imbalance in the input to in and fy. Blocking the pathway at in or fy would eliminate the function of the pathway completely and suppress miseversion (Lee, 2002).
The cellular mechanisms involved in the eversion of the wing are only poorly understood, but it is possible that convergent extension plays a role. It is also possible that the joint defects seen in fz mutants have a similar basis. In vivo observations on everting wings and joint morphogenesis would make an important contribution toward determining if convergent extension plays a role in these morphogenetic events. The development of in vivo imaging approaches for Drosophila pupae makes such experiments possible (Lee, 2002).
To study the roles of intracellular factors in neuronal morphogenesis, the mosaic analysis with a repressible cell marker (MARCM) technique was used to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. Individual neurons in the peripheral nervous system (PNS) develop clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster develops a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis reveals that Flamingo does not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limits the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype is partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Molecular analysis also indicates that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons (Sweeney, 2002).
In studies of neuronal morphogenesis, it is important to
differentiate the direct and indirect effects of the gene of
interest. Similar to other important regulators, Flamingo
functions in different cell types and at different developmental
stages. Evidence is provided
that Flamingo has a direct role in controlling dorsal dendritic
growth in postmitotic neurons. Although individual MD neurons in the dorsal cluster differ greatly in their dendritic fields, the defects caused by
flamingo mutations appear to be similar: mostly one process
of the mutant neurons overextends toward the dorsal
midline. Surprisingly, the general dendritic architecture of
these MD neurons is not affected dramatically. In
addition, these findings suggest that Flamingo is not a cell-type-specific regulator of dendritic morphology, nor does it
affect dendritic branching patterns in a global way. It seems
that Flamingo functions cell autonomously in controlling
dendritic fields of different MD neurons by limiting the
overextension of their dorsal dendrites. On the contrary,
other mutants identified, such as tumbleweed, appear to affect both dorsal and lateral dendrites in a more general way. The studies also
demonstrate that Flamingo function in neuronal morphogenesis
is independent of its function in precursor cells (Sweeney, 2002).
How does Flamingo mainly control dorsal dendrite extension
in postmitotic neurons? Neuronal morphogenesis of dorsal cluster MD neurons
can be separated into relatively discrete developmental
phases. These neurons always extend their axons first
toward the ventral nerve cord. The extension of dorsal
dendrites toward the dorsal midline ceases at 16-17 h AEL,
before lateral dendrites extend toward the adjacent segment
boundaries. If different development
phases are controlled by different mechanisms, Flamingo
may function mainly during dorsal dendrite extension.
Indeed, Flamingo also prevents precocious
initiation of dorsal dendrites: this contributes to the longer
dorsal dendrites before 16 h AEL in flamingo mutant
embryos. The failure to stop after 17 AEL also
contributes to the longer dorsal dendrites in flamingo
mutant embryos. Accordingly, the level of
flamingo mRNA expression decreases during late embryogenesis, and Flamingo expression also decreases in the first instar larvae. The
results presented here indicate that Flamingo has a function
during early neuronal differentiation to control the initiation
and extension of dorsal dendrites (Sweeney, 2002).
How does Flamingo function at the molecular level?
Flamingo contains seven transmembrane segments homologous
to a subset of G protein-coupled receptors, however,
whether Flamingo functions through G protein signaling
pathway is still unknown. The finding that the
extracellular domain of Flamingo is still present on MD
neuron membranes in flamingo72
mutant embryos indicates that the transmembrane segments are important for Flamingo function. In addition, mutational analysis suggests
that the amino acid sequence near the first EGF motif
is required for the proper subcellular localization and function
of Flamingo. Whether the first EGF motif is directly involved in protein-protein interactions or whether the mutation affects the folding and/or trafficking of Flamingo remains to be determined. It would be useful to
identify the proteins that interact with the first EGF motif
and other domains of Flamingo (Sweeney, 2002).
GFP labeling of single mutant neurons provides an opportunity
to study whether genes that control dendrite development
also affect other aspects of neuronal morphogenesis,
such as axon growth. Flamingo is required cell autonomously for promoting axonal growth. Since axons extend several hours earlier than dendrites, it is
possible that the perdurance of Flamingo prevents the
appearance of axonal p