wingless

Directional transport of extracellular Wingless

Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces. Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibire^ts (shi^ts) mutation to block endocytosis. shi encodes the fly dynamin homolog, a GTPase required for clathrin-coated vesicle formation. Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg-expressing cells, shi mutant embryos show high level accumulation of Wg around the wg-expressing cells. Structure/function analysis of the Wg molecule further supports the idea that active transport of the ligand is essential. Four mutations within wg have been isolated that specifically disrupt Wg transport without abolishing signaling activity. These mutant molecules generate a restricted response within the segment, as assayed by both cuticular pattern elements and molecular events. Homozygous mutant embryos produce naked cuticle but little denticle diversity, and show narrowed domains of Wg protein distribution and Arm stabilization. Three of these four mutations are single amino acid substitutions; each affects a residue that is highly conserved throughout the Wnt family, suggesting that ligand transport may be an important general aspect of Wnt function (Moline, 1999 and references).

To assess the functional consequences of this broad Wg distribution, a means has been devised of perturbing endocytosis in spatially restricted domains within the embryo. A transgene expressing a dominant negative form of shibire (shi), the fly dynamin homolog, was constructed. When this transgene is expressed using the GAL4-UAS system, Wg protein distribution within the domain of transgene expression is limited and Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. These results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment (Moline, 1999).

Interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. It is proposed that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment (Moline, 1999).

Using en-Gal4-driven shi^D expression to reduce posterior movement of Wg suppresses the phenotype of the segment polarity mutation, naked. nkd mutant embryos secrete denticle belts that have essentially normal denticle type diversity but that are replaced to varying degrees by naked cuticle. This excess naked cuticle depends upon Wg activity levels. The wg;nkd double mutant shows no naked cuticle across the ventral region and reducing the dosage of wg in a nkd mutant restores denticle belts. Thus wild-type nkd gene function appears to be involved in limiting Wg signaling activity within the segment. Consistent with this idea, Wg target genes become ectopically expressed in nkd mutant embryos. The en expression domain expands 2-3 cell diameters during stage 9, and an ectopic stripe of wg expression arises at stage 10, in the row of cells posterior to this expanded en domain. The posterior expansion of en expression suggests that nkd might play a role in restricting the movement of Wg protein in a posterior direction. Indeed, when nkd mutant embryos were generated that express shi^D at moderate levels in the en domain, there was a dramatic reduction in the amount of naked cuticle specified. These embryos are very similar in appearance to wild-type embryos in which en-Gal4 drives shi^D expression except that the nkd mutant head defect is not fully rescued. en-Gal4-driven shi^D expression also prevents the ectopic activation of en expression in nkd mutants. The stripes of en expression in the thoracic and abdominal segments are restored to the normal width, although some expansion is still observed in the head segments (Moline, 1999).

Since wild-type Wg signaling activity is required for stabilization of en expression, En stripes of normal width indicate that sufficient functional Wg contacts both rows of en-expressing cells to produce normal target gene regulation. This result demonstrates that expression of shi^D does not interfere with Wg signal transduction and supports the idea that moderate level shi^D expression reduces, but does not eliminate, transport of Wg across the affected domain. In contrast, embryos expressing high level shi^D in the en domain show a narrowed stripe of En antibody staining, suggesting that Wg can no longer traverse the first row of en-expressing cells to stabilize en in the second row. However, because of the severe effects of a more complete endocytotic block, these embryos do not secrete cuticle properly and so the effects on cuticle pattern are not interpretable (Moline, 1999).

During early stages of wild-type embryogenesis, Wg protein can be detected at high levels in cells both anterior and posterior to the wg-expressing row of cells. Diversity of denticle types, as well as stabilization of en expression in the adjacent cells, are specified by Wg activity during these early stages of embryonic development. By mid-stage 10, when Wg is no longer required for denticle specification or en stabilization, the Wg protein distribution shifts and Wg appears to be excluded from the en-expressing cells. This exclusion is not observed in nkd mutants at the same stage. Rather, Wg protein continues to be detected in cells on either side of the wg-expressing row of cells and the levels become substantially higher due to the ectopic stripe of wg expression. These results suggest that nkd gene function may play a role in the posterior restriction of Wg protein that occurs during stage 10. Hence the mutant phenotype is rescued dramatically when this restriction is produced artificially, by expressing shi^D in the en-expressing row of cells. All stage 11 and 12 embryos derived from this cross show posterior restriction of Wg protein, indicating that the nkd homozygotes do not exhibit excess posterior movement of Wg under these conditions. It is suspected that, in wild-type embryos, this restrictive function is not limited to the en-expressing cells. If this were the case, then one would expect to observe excess naked cuticle replacing denticle belts when wg+ is expressed in the en domain. Instead, en-Gal4-driven wg+, either alone or when co-expressed with shi^D, does not produce substantial amounts of ectopic naked cuticle. Thus, it seems likely that some ability to restrict posterior Wg movement during later stages is shared by the rows of cells at the anterior of each segment (Moline, 1999).

It is believed that this analysis of Wg transport by perturbing endocytosis is physiologically relevant because a similar inhibition of transport can be produced by overexpressing Dfz2, the cognate receptor for Wg. It is presumed that these effects result from sequestering ligand, because pattern defects are observed only when Wg levels are limiting. No change from the wild-type cuticle pattern is detected when Dfz2 is driven at ubiquitous high levels of expression with E22C-Gal4. However, in embryos heterozygous for a null mutation of wg, significant pattern defects are observed at a frequency of 60%. Ectopic denticles appear in the domain of cells that normally secrete naked cuticle, similar to what is observed in segments where anterior Wg transport is perturbed by shi^D. These pattern defects caused by Dfz2 overexpression are accompanied by a restricted Wg protein distribution and by a narrowed domain of Arm stabilization. However, it is not possible to directly compare Dfz2 with shi^D in this experiment. E22C-Gal4-driven expression of shi^D , even with UAS lines that express at low levels, results in cell death and failure to secrete cuticle as was the case with the original shi^ts mutation at restrictive temperature (Moline, 1999).

The spread of Wingless within the embryonic epidermis of Drosophila was examined in order to understand the role of Wingless in the segmentation. Using two assays for Wingless activity (specification of naked cuticle and repression of rhomboid transcription), it was found that Wingless acts at a different range in the anterior and posterior directions. This asymmetry follows in part from differential distribution of the Wingless protein. Transport or stability is reduced within engrailed-expressing cells, and farther posteriorward Wingless movement is blocked at the presumptive segment boundary and perhaps beyond. The role of hedgehog in the formation of this barrier is demonstrated (Sanson, 1999).

It is proposed that asymmetric Wingless distribution ensures the establishment of well-differentiated cell fates on either side of the engrailed domain. Anteriorly, at the wingless source, rhomboid expression is repressed. In contrast, reduced Wingless movement and/or stability within the engrailed domain allows nascent posterior rhomboid expression. Around this time (stage 11), a barrier to Wingless that requires hedgehog signaling forms at the posterior of the engrailed domain and ensures that Wingless does not foray across and repress rhomboid. rhomboid then activates the Egfr pathway within its expression domain and in adjacent cells. It may be that rhomboid itself contributes to barrier formation and thus builds a line of defense against invasion by its repressor. In addition, activation of the EGF pathway by rhomboid would antagonize any Wingless leaking through. Denticle formation requires transcription of shavenbaby, which is under positive regulation by the Egfr pathway and negative regulation by the wingless pathway. Activated EGFR and the absence of Wingless posterior to the engrailed domain allow shavenbaby expression and hence denticle formation. At the anterior side, converse conditions exist, since Wingless is present at high levels and the Egfr pathway is inactive. Therefore, it is proposed that polarization of Wingless transport by engrailed and hedgehog guarantees the naked fate anterior to the engrailed domain and the denticle fate posteriorly, and thus establishes the anteroposterior polarity of each segment (Sanson, 1999 and references).

In the ventral abdominal region of the Drosophila embryo, wingless is expressed in single cell-wide stripes. To assess the range of Wingless in specifying the naked fate, these stripes were mapped onto the final cuticle pattern. Unexpectedly, the wingless stripes were found to be are eccentric within each expanse of naked cuticle. Naked cuticle is made over a distance of approximately 3-4 cell diameters anterior to the wingless source. In contrast, posterior to it, only the adjoining cells make naked cuticle; these cells are the most anterior of each engrailed stripe. The denticle fate of more posterior engrailed-expressing cells could be explained if they were unable to respond to wingless. Therefore the responsiveness to Wingless of all epidermal cells was assessed. To ask whether Wingless is required beyond one cell diameter posterior to its source, in individual cells the ability to respond to Wingless was removed and the phenotypic consequences was analyzed at single-cell resolution. Armadillo is a downstream effector of Wingless signal transduction and is also a component of Cadherin-based adhesion complexes. Armadillo is titrated out of its signaling pool when Shotgun (DE-Cadherin) is overexpressed, and in the embryonic epidermis, this leads to a phenocopy of a wingless mutation. Overexpressed Shotgun, therefore, blocks the response to Wingless in a cell-autonomous manner. Shotgun was overexpressed in single cells with the "Flp-on Gal4" system. Clones of cells expressing Gal4, which in turn activates expression of Shotgun and GFP, were induced during early stage 11 and scored at the end of embryogenesis. GFP-positive cells within naked regions make ectopic denticles, confirming that wingless signaling is required everywhere naked cuticle is made. All Shotgun-overexpressing cells located within belts make denticle of the type and size expected for their position. These results show that wingless signaling (via Armadillo) is not required in the presumptive denticle belts. Wingless signaling is only required within the naked domain, and this requirement is asymmetric relative to the wingless source (Sanson, 1999).

Two types of mechanisms could account for the asymmetry of wingless action: (1) the wingless protein itself could be unequally distributed in the anterior and posterior directions, or (2) Wingless could be distributed symmetrically, but downstream signaling would be repressed posterior to the source. Immunocytochemistry has revealed an asymmetric distribution of the wingless-containing vesicles within each segment at stage 11. By colabeling with anti-Engrailed, it has been shown that the posterior transition in Wingless distribution occurs at the interface with engrailed-expressing cells. Although Wingless protein can be detected in the wingless domain and anterior to it, only a small number of Wingless-containing vesicles can be seen in the most anterior row of engrailed cells, and none can be seen more posteriorly. Thus, Wingless distribution is asymmetric, and this could explain the asymmetry of wingless action (Sanson, 1999).

It is conceivable that undetectable yet active Wingless is present in cells posterior to engrailed stripes. To demonstrate the absence of active Wingless there, a functional assay was used, based on the finding that wingless signaling represses rhomboid expression. From stage 11 onward, rhomboid is expressed in stripes just posterior to each engrailed domain, and this expression is abolished by continuous and uniform expression of Wingless. Later ectopic expression, induced at late stage 11, inhibits rhomboid transcription only in the midventral region, and if sibling embryos are left to develop, they make ectopic naked cuticle in the same region. Therefore, Wingless can repress rhomboid transcription in the same time window as it specifies naked cuticle. Wingless is not only sufficient for rhomboid repression, it is also necessary since wingless null mutants have an additional rhomboid stripe in each abdominal segment. The position of these extra stripes relative to landmarks in the CNS suggests that they form at the anterior of the domain of extinct engrailed expression, where wingless would normally be expressed. Thus, in the wild type, the presence of Wingless at the anterior of each engrailed stripe maintains the silence of rhomboid expression there. Significantly, rhomboid is expressed posterior to the engrailed domain of wild-type embryos. Therefore, active Wingless is not present in these cells, at least at late stage 11; if it were, rhomboid would not be expressed. These cells are located only two cell diameters posterior to the Wingless source (Sanson, 1999).

The asymmetric distribution of Wingless could be explained by decreased transport/stability either within the engrailed domain or at its posterior edge, where the segment boundary forms. To explore this, wingless was misexpressed directly in the engrailed domain (posterior to endogenous wingless) and it was determined whether the range of Wingless was shifted posteriorly. Wingless was expressed with the engrailed-Gal4 driver in otherwise wild-type embryos. The only effect on the cuticle pattern is the loss of row 1 denticles. Remarkably, no other denticles are lost. In particular, row 2 denticles are present even though they are adjacent to the Wingless-misexpressing cells. Thus wingless expressed at the anterior side of the presumptive segment boundary does not affect the fate of cells on the posterior side. To confirm this finding, rhomboid expression was used as an early molecular marker for the absence of Wingless. In the wild-type larva, rhomboid is expressed in the cells secreting rows 2-4. In en-Gal4/UAS-wg larvae, this expression is unchanged, indicating that the wingless pathway is not operative in the cells immediately posterior to the wingless-misexpressing cells. Thus, it appears that Wingless cannot cross the posterior edge of the engrailed domain. This was verified by looking directly at the distribution of Wingless protein in en-Gal4/UAS-wg embryos. In these embryos, Wingless is present within the domain of wingless misexpression, as expected. However, it is not detectable posterior to the engrailed-expressing cells. It is concluded that a barrier to Wingless protein movement exists at the presumptive segment boundary (Sanson, 1999).

Among various candidate genes, hedgehog was found to be required for the posterior barrier to Wingless. Assaying the range of Wingless in a hedgehog mutant is not straightforward, since wingless expression requires hedgehog signaling. Therefore, wingless expression was maintained artificially in hedgehog null mutants using en-Gal4. Normally the engrailed promoter also turns off in a hedgehog mutant for lack of wingless, but in en-Gal4/UAS-wg; hh^- embryos, this is remedied by exogenous Wingless. Thus, a hedgehog-independent positive feedback loop is established between engrailed and wingless, and stripes coexpressing Wingless and Engrailed are obtained. The distribution of the Wingless protein in en-Gal4/UAS-wg; hh^- embryos is different from that seen in en-Gal4/UAS-wg control embryos. Wingless spreads posterior to the engrailed domain as if a barrier had been lifted or Wingless movement enhanced. The resulting protein distribution is symmetrical, and this is reflected in the cuticle pattern: in contrast to en-Gal4/UAS-wg embryos, en-Gal4/UAS-wg; hh^- embryos lack rows 2-4 and, instead, have an extra expanse of naked cuticle. At the positions where rows 5 and 6 normally form, lies a thin stripe of small denticles. Naked cuticle is specified equally in the anterior and posterior directions, as shown by marking the wingless-expressing cells with GFP. Thus, in the absence of hedgehog, wingless action is symmetric (Sanson, 1999).

Thus, loss of hedgehog signaling increases the range of Wingless. Now it was asked whether the converse is true. To assay the range of Wingless in the presence of excess hedgehog signaling, endogenous wingless must be removed because hedgehog signaling activates wingless expression and this would confuse the assay. Therefore, the en-Gal4/UAS-wg combination was used again, but this time in a wingless mutant background. The sole source of Wingless in these embryos is in the engrailed domain. The wingless mutant phenotype is significantly rescued: the normal alternation of denticle belts and naked cuticle is restored, and many belts are nearly wild type, except for the loss of row 1. In these embryos, the width of the band of naked cuticle is 4-5 cells, and this provides an assay for the anterior range of Wingless. This assay was validated with a version of Wingless expected to act only at short range. If a membrane-tethered form of Wingless is expressed instead of the wild-type protein, an expanse of naked cuticle only 1-2 cells wide is found. This demonstrates that Wingless has to be physically transported from cell to cell to specify a band of naked cuticle of the normal size, and that there is no relay mechanism. Next, the assay was used to find out the effect of increasing hedgehog signaling on the range of Wingless. Increased hedgehog signaling can be achieved either by overexpressing Hedgehog or by removing patched activity. wg^- en-Gal4/UAS-wg embryos carrying in addition UAS-hedgehog have significantly narrower naked domains. Likewise, wg^- ptc^- en-Gal4/UAS-wg embryos have narrow naked bands as well. This suggests that excess hedgehog signaling reduces the range of Wingless, although excess Hedgehog signaling could also induce ectopic rhomboid, which would in turn antagonize Wingless signaling and bring about the loss of naked cuticle (Sanson, 1999).

It is known that Wingless sustains engrailed expression only in adjoining cells, suggesting that Wingless is not readily transported across the engrailed domain. This is supported by the asymmetric distribution of the protein. Immunostaining reveals the presence of Wingless anterior to its source, whereas very little is detected posteriorly; posterior to wingless-expressing cells, in the engrailed domain, some Wingless staining is found but only in the most anterior cells (nearest the Wingless source). Thus, engrailed-expressing cells appear to restrict Wingless movement. Restricted Wingless transport through the engrailed domain could be explained by the downregulation of a specialized transport receptor in the engrailed cells; the existence of such a receptor has been hypothesized. Alternatively, inefficient transport could follow from selective instability of Wingless or its sequestration within the engrailed domain. In wing imaginal discs, the stability and range of Wingless increase in response to overexpression of its receptor Frizzled2. By analogy, and conversely, Wingless might be particularly unstable within engrailed stripes for lack of a receptor there. Alternatively, the surface or extracellular matrix surrounding engrailed cells might trap Wingless and impede its movement. A receptor of the proteoglycan type could possibly mediate this activity. Indeed, in mutants for the gene encoding UDP-glucose dehydrogenase, that lack HSPGs, embryonic engrailed stripes are temporarily widened, implying an increased range of Wingless. Identification of the relevant receptors and their pattern of expression will be required to discriminate between the above alternatives (Sanson, 1999 and references).

Not only is Wingless movement restricted within the engrailed domain, but a barrier seems to exist at its posterior edge. This is especially evident in embryos that ectopically express Wingless in the engrailed domain. In these embryos, Wingless does not specify naked cuticle nor repress rhomboid posteriorly, even in adjacent cells. The lack of response is unlikely to be due to insufficient expression, since en-Gal4 is a robust driver. Also, uniform wingless expression (even at low levels and up to late stage 11) induces uniform naked cuticle (D) and represses rhomboid transcription. This suggests that all cells, including those posterior to each engrailed stripe, are responsive to Wingless (although it is formally possible that the latter cells are only responsive to autocrine signaling). Thus, the lack of posterior response in en-Gal4/UAS-wg embryos is probably because, in this experimental situation, Wingless does not reach posteriorly. Indeed, in the same embryos, immunostaining fails to detect Wingless protein posterior to the engrailed domain. The best interpretation of the results is that a barrier to Wingless movement exists at the segment boundary, although the possibility that movement is impeded throughout the rhomboid expression domain or that these cells are unable to respond to paracrine Wingless cannot completely excluded (Sanson, 1999).

The notion that Wingless movement is blocked at the forming segment boundary contrasts with an earlier proposal that Wingless spreads symmetrically. According to this view, posterior to its source, Wingless signaling is antagonized by active EGFR. The Egfr pathway is activated within and near the rhomboid stripe, which lies just posterior to the segment boundary. However, it is proposed that this segmental activation, which requires rhomboid, occurs after formation of the restrictions to Wingless movement. If wingless protein were present in the rhomboid cells at late stage 11, rhomboid expression would not be allowed there since wingless has been shown to repress rhomboid transcription. Subsequent establishment of rhomboid expression would further counteract activation of the Wingless pathway in prospective denticle belts (Sanson, 1999).

It is suggested that two mechanisms restrict posterior Wingless movement. The first restriction occurs within the engrailed domain and is unlikely to be under hedgehog control, since engrailed cells are not thought to respond to Hedgehog. Rather, engrailed could implement this restriction by controlling a gene involved in Wingless transport, sequestration, or stability. By contrast, the barrier at the posterior of the engrailed domain requires hedgehog signaling. Wingless produced ectopically in the engrailed domain of hedgehog mutants is allowed to invade posteriorly located cells and induce naked cuticle there. The finding that the same effects are seen in cubitus interruptus mutants indicates that the hedgehog signaling pathway is involved. The role of the hedgehog pathway is confirmed by "gain-of-function" experiments. Loss of patched results in overactivation of the hedgehog pathway and so does excessive hedgehog expression. Both situations reduce the range of Wingless in the anterior direction as if the spread of the protein were reduced. It is presumed that, in the wild type, a downstream Hedgehog target is upregulated at the posterior of each engrailed/hedgehog stripe and this would lead to Wingless destabilization or a block to transport there (Sanson, 1999).

The extracellular matrix, glycosaminoglycan and Wingless

When expressed in S2 cells, the majority (approximately 83%) of secreted Wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparin sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling: (1) treatment of WG-responsive cells with glycosaminoglycan lyases reduces WG activity by 50%; (2) when WG-responsive cells are preincubated with 1 mM chlorate, which blocks sulfation, WG activity is inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells restores WG activity. Based on these results, it is proposed that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules (Reichsman, 1996).

The Drosophila UDP-glucose dehydrogenase gene is involved in wingless signaling. UDP-glucose dehydrogenase is an enzyme responsible for the production of UDP-glucuronate. UDP-glucuronate, a uronic acid, is a precursor in glycosaminoglycan (GAG) biosynthesis. GAG is a complex carbohydrate which is linked to a distinct class of glycoproteins called proteoglycans. A mutation in UDP-glucose dehydrogenase, called kiwi, generates a phenotype identical to that of wingless. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, it has been shown that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Heparin-like GAGs are complex acidic polysaccharides that are important soluble components of the extracellular matrix. They provide the necessary hydration to the ECM scaffold and hence solubilize and modulate the function of such proteins as fibroblast growth factor. This study provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction (Binari, 1997).

In vitro experiments suggest that glycosaminoglycans (GAGs) and the proteins to which they are attached (proteoglycans) are important for modulating growth factor signaling. However, in vivo evidence to support this view has been lacking, in part because mutations that disrupt the production of GAG polymers and the core proteins have not been available. The identification and characterization of Drosophila mutants in the suppenkasper (ska) gene is described. The ska gene encodes UDP-glucose dehydrogenase, which produces glucuronic acid, an essential component for the synthesis of heparan and chondroitin sulfate. ska mutants fail to put heparan side chains on proteoglycans such as Syndecan. Surprisingly, mutant embryos produced by germ-line clones of this general metabolic gene exhibit embryonic cuticle phenotypes strikingly similar to those that result from loss-of-function mutations in genes of the Wingless (Wg) signaling pathway. Zygotic loss of ska leads to reduced growth of imaginal discs and pattern defects similar to wg mutants. In addition, genetic interactions of ska with wg and dishevelled mutants are observed. These data demonstrate the importance of proteoglycans and GAGs in Wg signaling in vivo and suggest that Wnt-like growth factors may be particularly sensitive to perturbations of GAG biosynthesis (Haerry, 1997).

The Drosophila gene sugarless encodes a homolog of vertebrate UDP-glucose dehydrogenase. This enzyme is essential for the biosynthesis of various proteoglycans. In the absence of both maternal and zygotic activities of this gene, mutant embryos develop with segment polarity phenotypes reminiscent of the loss of either Wingless or Hedgehog signaling. To analyze the function of Sugarless in cell-cell interaction processes, attention has been focused on the requirement of Sugarless for Wingless signaling in different tissues. sugarless mutations impair signaling by Wingless, suggesting that proteoglycans contribute to the reception of Wingless. Overexpression of Wingless can bypass the requirement for sugarless, suggesting that proteoglycans modulate signaling by Wingless, possibly by limiting its diffusion and thereby facilitating the binding of Wingless to its receptor. Tissue-specific regulation of proteoglycans may be involved in regulating both Wingless short- or long-range effects. For example, Dally (a Glypican-related heparin sulfate proteoglycan) mutations show wing notching with loss of wing margin structures, an effect seen in wingless and dishevelled mutants, suggesting the potential involvement of Dally in Wg signaling. A Drosophila homolog of vertebrate Syndecans has also been characterized. Syndecan is a transmembrane HSPG and represents the major source of HSPGs in epithelial cells. Syndecan has been demonstrated to function as a coreceptor for FGF signaling. Further genetic and biochemical studies should reveal whether Dally and/or Syndecan play a direct role in Wg signaling (Hacker, 1997).

Wingless is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. The protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. Dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes (Tsuda, 1999).

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Another Drosophila glypican gene, dally-like (dly) has been identified and it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception -- overexpression of DLY leads to an accumulation of extracellular Wg. It is proposed that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. It is concluded that HSPGs play an important role in organizing the extracellular distribution of Wg (Baeg, 2001).

One glypican molecule, Dally, has been implicated in Wg signaling. However, because numerous glypican genes are present in other animals, a search was carried out of the Drosophila database for additional glypican family members. One EST clone showed some sequence similarity to dally and a full length cDNA was cloned. The sequence of the cDNA revealed a potential open reading frame of 765 amino acid residues, with 22% and 35% identity to Dally and mouse K-glypican, respectively. The predicted primary structure of the molecule exhibits the hallmarks of a glypican protein. The hydrophilicity plot of the new molecule is similar to those of the other members of the glypican family, which is characterized by the presence of NH2- and COOH-terminal hydrophobic signal sequences. In addition, this molecule possesses four consensus serine/glycine dipeptide sequences for glycosaminoglycan (GAG) attachment sites, and a signal sequence for a GPI-moiety attachment site at the COOH-terminal region. Moreover, the number and position of cysteine residues, which are a unique feature of glypican family members, are almost completely conserved in the predicted protein. These results led to the identification of Dally-like. Hybridization using a dly-specific probe to polytene chromosomes from salivary glands localized the dly gene to the cytological division 70F on the third chromosome. Finally, Northern blot analysis revealed that dly encodes a single major 3.8 kb transcript (Baeg, 2001).

To discover the function of Dly, its expression in embryos was determined by in situ hybridization. dly transcripts are uniformly expressed at early embryonic stages, but by stage 8 they are enriched in stripes. Double staining for dly mRNA and Wg protein shows that dly transcripts are preferentially expressed in three to four cells anterior to the wg-expressing cells. Interestingly, this expression pattern is similar to that of both dally and frizzled 2, the Wingless receptor (Baeg, 2001).

In an attempt to assess the function of Dly during embryogenesis, the RNA interference (RNAi) method was used to perturb Dly protein synthesis. Embryos were injected with a dly double-stranded RNA (dsRNA). These embryos, referred to as dly dsRNA embryos, show the absence of naked cuticle. This phenotype is reminiscent of loss of either wg or hh gene activities. The segment polarity phenotype is also found when the activity of dally, which is required for Wg signaling in the embryo, is disrupted by RNAi, though the effect is less severe in dally dsRNA embryos than in dly dsRNA embryos. However, when compared with embryos injected with either dly dsRNA or dally dsRNA alone, embryos injected with an equimolar mixture of dly and dally dsRNAs show more severe segment polarity phenotypes. These embryos are smaller, particularly in the tail region. They also show an entire transformation of naked cuticle into cuticle with denticles, which is observed in wg or hh null mutations. Because dally does not appear to play a role in Hh signaling, the interaction between dally and dly observed in the RNAi interference experiment suggests that Dly and Dally function synergistically in Wg signaling. Altogether, these results suggest that dly is a novel segment polarity gene that potentiates Wg signaling (Baeg, 2001).

To further examine the role of Dly in Wg signaling, the function of Dly during wing imaginal disc development was examined. dly transcripts are uniformly expressed in wing discs. In the third instar imaginal disc, wg is expressed at the DV compartment border and acts over short and long ranges to pattern the wing disc. Short range Wg signaling induces the expression of the proneural gene achaete (ac) in a stripe on each side of the DV boundary, while long range Wg signaling controls the expression of Distal-less (Dll) within the wing blade. It was reasoned that overexpression of Dly might activate Wg signaling because dly dsRNA-injected embryos resemble those that have lost Wg activity. Interestingly, overexpression of dly (using the C96-Gal4 driver), which is highly expressed at the DV boundary of the wing disc, results in severe wing margin defects and loss of sensory bristles. These phenotypes are reminiscent of the phenotypes seen when Wg activity is reduced in the wing. Consistent with the adult wing phenotype, Ac expression is dramatically decreased in wing discs overexpressing Dly. Furthermore, when dly is overexpressed using the engrailed-Gal4 (en-Gal4) driver, the expression of Dll is reduced in the posterior compartment (Baeg, 2001).

A test was performed to see whether overexpression of transducers of the Wg signal can rescue the loss-of-function wg-like phenotypes associated with dly overexpression. Ectopic expression of either Wg or a gain-of-function Armadillo (Arm) can rescue the wing margin defects, and induced ectopic bristles that are characteristic of ectopic expression of the Wg pathway. Altogether, these results suggest that overexpression of Dly blocks Wg signaling in the wing disc, and that of Dly acts upstream of Arm (Baeg, 2001).

Since Dly is an extracellular GPI-linked molecule, it was reasoned that patterning defects associated with Dly overexpression might reflect the ability of Dly to sequester Wg, and thus prevent it from accessing and activating Frizzled 2. To visualize the effect of overexpressed Dly on Wg distribution, Dly was overexpressed using various Gal4 lines and the wing discs were stained with anti-Wg monoclonal antibodies. Two different staining protocols were used to detect Wg distribution. The first one, involved fixing the tissue before staining, and thus it detects mostly cytoplasmic Wg present in either secretory or internalized vesicles. In the second protocol, the tissue is incubated with the antibody prior to fixation and mostly detects extracellular Wg (Baeg, 2001).

Using the first protocol, in wild-type discs, Wg protein is found at high levels in a narrow stripe of three to five cells straddling the DV boundary. Following overexpression of Dly using either en-Gal4 or ptc-Gal4 a striking increased accumulation of Wg protein is observed. Using the second protocol, extracellular Wg is organized in a gradient at the basolateral surface of wg-expressing and nearby cells. Following overexpression of Dly, an increased accumulation of extracellular Wg protein is detected. This result indicates that Dly can affect extracellular Wg distribution (Baeg, 2001).

HSPGs are required to increase the local concentration of Wg ligand for its receptors. Considering the roles of Dally and Dly, there are at least two HSPGs at the cell surface of wing disc cells involved in Wg signaling. To generate mutant cells that lack all GAGs and determine the role of the HSPGs in Wg signaling, mutant clones of cells were generated that do not properly synthesize GAGs. sugarless (sgl) mutations cannot be used for this analysis because sgl acts in a cell non-autonomous manner, presumably because the enzyme synthesizes glucuronic acid that diffuses between cells. However, sulfateless (sfl) is involved in GAG modification and in its absence, the GAG chains are not synthesized properly. To determine whether the GAG chains of Dly are modified by sfl, Dly proteins expressed in wild-type or sfl mutant pupae were examined by Western blot analysis using anti-Dly antibodies. The predicted size for Dly is 80 kDa, and in wild-type pupae, Dly protein appears as a broad band that migrates to around 80-110 kDa, which presumably results from addition of GAG chains onto the core protein. In sfl mutant pupae, the modified form of Dly protein is significantly reduced and the sharp band of the core protein is increased. This results indicate that sfl plays a role in Dly modification. Using the staining protocol that detects cytoplasmic Wg, an alteration in the expression of intracellular Wg could not be detected in sfl mutant clones, indicating that sfl mutant cells normally transcribe wg and do not accumulate Wg (Baeg, 2001).

However, using the extracellular staining method, a dramatic decrease of extracellular Wg was detected in sfl mutant cells. Extracellular Wg has been shown to be mainly associated with the basolateral surface of cells, and GPI-anchored protein is thought to be primarily attached to the basal part of the cells. Together, these results suggest that the HSPGs are involved in extracellular Wg accumulation (Baeg, 2001).

Importantly, high accumulation of extracellular Wg can be detected on sfl mutant cells located adjacent to wild-type cells, suggesting that HSPGs act locally in a cell non-autonomous manner. Consistent with this observation, adult wing patterning in sfl mutant clones show local cell non-autonomy as well. Clones of sfl mutant cells are associated with wing margin defects, suggesting that sfl is required for Wg signaling, however some of the sfl mutant cells located near wild-type margin cells have a wild-type morphology (Baeg, 2001).

Taken together, these results suggest that HSPGs are involved in restricting Wg diffusion. Further, the local cell non-autonomy observed in sfl mutant clones may indicate that HSPGs may not be absolutely required for the binding of Wg to its transducing receptor(s) (Baeg, 2001).

The secreted signaling protein Wingless acts as a morphogen to pattern the imaginal discs of Drosophila. A secreted repressor of Wingless activity, called Notum, is described. Loss of Notum function leads to increased Wingless activity by altering the shape of the Wingless protein gradient. When overexpressed, Notum blocks Wingless activity. Notum encodes a member of the alpha/ß-hydrolase superfamily, with similarity to pectin acetylesterases. Evidence is presented that Notum influences Wingless protein distribution by modifying the heparan sulfate proteoglycans Dally-like and Dally. High levels of Wingless signaling induce Notum expression. Thus, Wingless contributes to shaping its own gradient by regulating expression of a protein that modifies its interaction with cell surface proteoglycans (Giráldez, 2002).

The Notum gene was identified in a gain-of-function genetic screen that caused loss of the wing and duplication of the dorsal thorax when expressed under sd^Gal4. Replacement of the wing by a duplicated dorsal thorax resembles the defect caused by the wg¹ mutant, and can also be produced by Gal4-driven overexpression of the GSK3 homolog, shaggy/ZW3, an intracellular repressor of Wg signaling. Expression of Notum throughout the embryo under tubulin^Gal4 control causes expansion of the denticle-producing zone at the expense of naked cuticle. Similar phenotypes can also be obtained by overactivation of the Egf receptor. The balance between Wg and Egfr activity determines the domain in which naked cuticle forms by regulating expression of shavenbaby in the embryonic ectoderm. Likewise, subdivision of the wing disc into wing and thorax territories occurs during the second larval instar and depends on the balance between Wg and Egfr signaling. Ventral expression of either Notum or an activated Egfr under wg^Gal4 control produces similar leg axis duplications. Thus, Notum might act as a repressor of the Wg pathway or as an activator of Egfr signaling when overexpressed. To distinguish between these possibilities, the effects of Notum expression were examined on wing vein formation, where the effects of Wg and Egfr signals differ. During third instar, ectopic Egfr signaling leads to formation of extra vein tissue in the wing blade. In contrast, Notum expression produces scalloping of the wing but has no effect on vein formation. This phenotype resembles late loss of Wg activity. Thus, it is concluded that overexpression of Notum does not work by activation of the Egfr pathway, but rather interferes with Wg activity (Giráldez, 2002).

A number of genes that modulate Wg activity are spatially regulated by Wg signaling in the embryo, including Dfz2, Dally, and naked. It was therefore asked whether Wg regulates Notum expression. In situ hybridization showed that Notum is expressed in a segmentally repeated pattern in two rows of cells anteriorly adjacent to the Engrailed-expressing cells. This corresponds to the Wg stripe in the embryo. Notum expression also mirrors Wg expression in the wing disc. Ectopic activation of the Wg pathway by expression of a constitutively active form of Armadillo induces ectopic Notum expression. Thus, high levels of Wg activity induce Notum expression, which in turn serves as a Wg antagonist (Giráldez, 2002).

To isolate loss-of-function mutations in the Notum gene, a chemical mutagenesis screen was performed for reversion of the thorax duplication caused by expression of EP-Notum. EP-Notum males were treated with EMS and crossed to sd^Gal4 females. Among 15,000 progeny, three flies were recovered that had normal wings despite overexpressing the endogenous Notum gene. These three alleles formed a single complementation group. l(3)72Da and l(3)72CDf were subsequently identified as Notum alleles and have been renamed Notum⁴ and Notum⁵. Sequence analysis revealed an alteration of the splice donor site of exon 1 in Notum¹ and alterations in the coding sequence for the other alleles analyzed (Giráldez, 2002).

Notum zygotic mutant embryos produced a variable naked cuticle phenotype, typical of excess Wg activity. The embryonic phenotype is weak, perhaps due to maternal contribution, wing discs from Notum mutant larvae were used to examine the effects of removing Notum activity. The primordia of the adult wing and thoracic body wall can be visualized in the wing disc by expression of Nubbin and Teashirt proteins . Nubbin is a POU homeodomain protein expressed in the presumptive wing blade and wing hinge. Tsh is a zinc finger protein expressed in the presumptive thorax. Ectopic Wg activity can lead to duplication of wing structures at the expense of thorax. Notum mutant discs showed this phenotype. The severity of these defects ranged from duplication of the wing pouch and hinge associated with a reduced thorax to almost complete loss of thorax associated with a severely abnormal wing duplication. The same range of phenotypes was obtained by activation of the Wg pathway in the early wing disc using Arm^S10 (Giráldez, 2002).

Wg forms a long-range protein gradient and regulates several target genes in different spatial domains in the wing disc. Achaete-Scute is a high-threshold Wg target, expressed in cells close to the DV boundary. Achaete-Scute expression specifies the proneural region in the anterior wing margin in which the single row of sense organ precursor (SOP) cells will form. In wild-type discs, a single row of SOPs forms on each side of the Wg stripe in the anterior compartment. Large clones of cells mutant for Notum³ form additional rows of SOPs and produce extra mechanosensory bristles. Thus, the region in which cells show a high-threshold response to Wg is broadened in the Notum mutant tissue. This defect occurs only when large clones affect both sides of the DV boundary. The defect is rescued when cells on one side of the boundary are able to produce Notum. This suggests that Notum can act nonautonomously. In addition, it was noted that some of the ectopic bristles derived from wild-type cells. This indicates that loss of Notum activity in a mutant clone can lead to increased Wg signaling in nearby wild-type cells. These observations suggest that Notum acts nonautonomously to affect the range of Wg action (Giráldez, 2002).

To examine the effect of large clones of Notum mutant cells on Wg, clones were produced using engrailed^Gal4 to drive Flp recombinase in posterior cells. The mutant posterior compartment was overgrown and the size of the Distal-less (Dll) expression domain increased, suggesting an increased range of Wg action. Antibody labeling showed that the level of Wg protein is elevated in the posterior compartment. The number and brightness of Wg protein vesicles was used to visualize the Wg gradient. Both were increased in the Notum mutant tissue. Thus, reducing Notum activity increases the level of Wg protein and broadens its distribution. The effects on Wg distribution do not make an abrupt transition at the AP compartment boundary. Instead, it increases with distance from the boundary, suggesting that the mutant phenotype may be partially rescued by Notum protein produced by the wild-type anterior cells (Giráldez, 2002).

The results of the clonal analyses suggest that Notum acts nonautonomously. The predicted Notum protein contains a hydrophobic sequence near its amino terminus that might function as a signal peptide. To determine whether Notum is secreted, S2 cells were transfected with expression constructs for wild-type Notum, the Notum² mutant, and a Golgi-tethered form of Notum, in which the signal peptide was replaced with the transmembrane domain from a Golgi-resident enzyme (Notum-GT). Secretion was assayed by immunoprecipitation from cell lysates and from the medium in which the cells were grown. Wild-type Notum protein was recovered from the conditioned medium. Notum-GT was not recovered from conditioned medium. The Notum² mutant protein was recovered at low levels from the medium, suggesting impaired secretion (Giráldez, 2002).

The activities of wild-type and Golgi-tethered Notum were compared in vivo to verify that Notum can act nonautonomously. In wild-type discs, Dll and Hnt are expressed symmetrically in D and V compartments in response to Wg. When overexpressed in the D compartment under apterous^GAL4 control, Notum protein is seen at elevated levels throughout the disc, indicating that it is secreted in vivo. Hnt expression is lost and Dll expression is reduced, symmetrically, in both D and V compartments. This leads to scalloping of the wing. In contrast, expression of Notum-GT affects only the D compartment. The reduced size of the Dll domain can be attributed to a reduced level and range of Wg in the D compartment. This leads to a small D compartment and loss of dorsal wing margin, without any effect on the ventral margin. Thus, overexpression of Notum-GT in the D compartment produces an asymmetric Wg gradient, whereas overexpression of the wild-type protein in the D compartment reduces the Wg gradient symmetrically in both compartments. These observations indicate that wild-type Notum can act as a secreted protein to reduce the effective range of the Wg gradient. However, Notum can also act when retained in the Golgi (Giráldez, 2002).

Pectin acetylesterases from plant pathogens act as secreted enzymes to deacetylate pectins in plant cell walls. Pectins are composed mainly of galacturonic acid residues, some of which are methylated or acetylated. Pectin acetylesterases hydrolyze the ester bond linking acetyl groups to galacturonic acid. Glycosaminoglycans consist of repeated glucuronic acid and GlcNAc disaccharide units. Although GAGs are different in structure from pectins, the similarity to pectin acetylesterases raised the possibility that Notum might act on the GAG side chains of HSPGs. As a first step toward addressing this possibility, it was asked whether Notum could modify Dally and Dlp when coexpressed in S2 cells. Coexpression of Dally-HA with Notum reduced the amount of Dally-HA recovered in S2 cell lysates. In sulfateless mutants, which lack N-deacetylase/N-sulfotransferase (NDST) activity, the level of Dally and Dlp proteins are also strongly reduced, perhaps indicating altered stability of the immature protein. Coexpression of HA-tagged Dlp with Notum altered the electrophoretic mobility of Dlp-HA, without causing substantial loss of the protein. Dlp-HA migrates as a broad band at ~115 kDa, with minor bands at 97 and 105 kDa. Coexpression with wild-type Notum increases the amount of the 97 kDa form (Dlp*), apparently at the expense of the 115 kDa form. The small amount of this band present in S2 cells expressing Dlp-HA may reflect activity of endogenous Notum protein, which was detected on longer exposures (Giráldez, 2002).

The GAG side chains of HSPGs consist mainly of repeated dimers of N-acetylglucosamine (GlcNAc) and glucuronic acid. The first step in modification of the side chains involves replacement of the acetyl moiety on GlcNAc with a sulfate moiety by N-deacetylase/N-sulfotransferase. NDST modifies ~50% of the GlcNAc residues, in blocks along the GAG side chain. The structural similarity between pectins and GAGs raised the possibility that Notum might act by removing acetyl groups from GlcNAc residues of GAGs. If this is the case, GlcNAc residues modified by NDST should not be a good substrate for Notum in cells. To address this possibility, S2 cells were transfected to express a constant amount of Notum and increasing amounts of NDST and the ability of Notum to modify Dlp was assayed. Increasing the ratio of NDST to Notum reduces the amount of Dlp*. These observations support the proposal that NDST and Notum could act on the same substrate. NDST is a Golgi-resident enzyme. In cells where the two proteins are coexpressed, NDST and Notum might compete for modification of GAG side chains, as illustrated by the activity of Notum-GT when expressed in the wing disc. Since there are few cells in the disc where Notum is expressed, it is suggested that secreted Notum may act on HSPGs at the cell surface to deacetylate the blocks of GlcNAc residues that were not modified by NDST during GAG biosynthesis (Giráldez, 2002).

The glypicans Dally and Dlp have been shown to bind and stabilize extracellular Wg, although Dlp is considerably more effective. To ask whether Notum modifies the ability of Dlp to bind Wg, Wg protein was examined in discs expressing Dlp-HA and Notum. Expression of Dlp-HA in a broad band of cells in the center of the wing disc under spalt^Gal4 control causes accumulation of Wg protein, mainly outlining the cell surface. Under these conditions, scalloping of the wing margin was observed, suggesting that Wg is partially sequestered by binding to Dlp and is less available for binding to its receptor. The distribution of Wg differs in discs expressing Dlp-HA and Notum. The total level of Wg accumulation is considerably lower, although Dlp is expressed at a comparable level. In addition, much of the Wg protein appears in intracellular vesicles, instead of outlining the cell surface. These findings support the proposal that Notum modifies Dlp to render it less able to bind and stabilize Wg. Thus, Notum contributes to shaping the Wg gradient by altering the ability of the cell surface glypican Dlp to stabilize extracellular Wg (Giráldez, 2002).

Wg, Dpp, and Hh each regulate the expression of their receptors in ways that can influence the shape of the gradient. These findings indicate that Wg can also influence formation of its own gradient by modulating the activity of cell surface HSPGs. Wg induces expression of Notum, which can act either during GAG biosynthesis or as a secreted protein to modify cell surface HSPGs. Reduced Notum activity allows excess accumulation of Wg protein, resulting in an increased range of Wg activity . Conversely, overexpression of Notum limits the ability of cells to bind and stabilize Wg, thereby limiting the ability of Wg to spread in the disc epithelium and form a long-range gradient. In the embryo, Notum overexpression produces segment polarity defects comparable to those caused by reduced Wg activity. In the wing disc, elevating Notum levels limit Wg movement and cause phenotypes ranging from scalloping of the wing to early failure of wing pouch specification. These changes in the shape of the Wg gradient can be attributed to the effect of Notum on Dally and Dlp. Coexpression of Notum with Dly limit the ability of overexpressed Dlp to accumulate Wg protein. It is suggested that the role of Notum is to limit the ability of Dlp and Dally to bind Wg (Giráldez, 2002).

A novel Drosophila Wingless (Wg) target gene, wingful (wf: termed Notum by FlyBase), encodes a potent extracellular feedback inhibitor of Wg. In contrast to the cytoplasmic protein Naked cuticle (Nkd), the only known Wg feedback antagonist, Wf functions during larval stages, when Nkd function is dispensable. It is proposed that Wf may provide feedback control for the long-range morphogen activities of Wg (Gerlitz, 2002).

A library of 2000 Gal4 enhancer trap P-element insertions was established, each of which reports a gene expression pattern in the wing imaginal disc. This collection was screened with a UAS-GFP reporter for lines that show a wg-like expression pattern. There were 11 insertions identified that reported wg-like gene expression in the embryonic epidermis and all imaginal discs. Four of these lines (S180, ND382, S476, S554) contained an insertion in the wg gene itself; the other seven lines (S141, S145, S163, S330, ND337, ND339, ND634) all carried a P-element insertion at cytological position 72D, only a few base pairs upstream of gene CG13076, referred to as wingful. These enhancer trap insertions indeed report the expression of wf, as revealed by RNA in situ hybridization. wf is ectopically expressed upon wg misexpression, indicating that wf is a Wg target throughout larval development (Gerlitz, 2002).

wf codes for a presumptive protein of 671 amino acids, with an N-terminally situated signal sequence. The wf product is readily secreted from transfected Drosophila cells and has a noticeable propensity to adhere to the surfaces of intact cells. The analysis of the Wf protein sequence reveals a significant structural homology to a subfamily of poorly characterized hydrolases related to plant pectin acetylesterase. Together, these results suggest that the product of the wf gene may catalyze the hydrolytic cleavage of an extracellular substrate (Gerlitz, 2002).

To test the hypothesis that the pan-Wg-target wf encodes an inhibitor of Wg activity, attempts were made to abolish wf function by genetic means. From a collection of six EMS-induced lethal mutations, located between the distal breakpoint of Df(3L)st-f13 and the proximal breakpoint of Df(3L)brm11, a putative null allele of wf was identified with a stop codon at amino acid position 141, encoding a severely truncated protein. Animals homozygous for wf¹⁴¹ or animals of the genotype wf¹⁴¹/Df3(3L)st-f13 die during pupal stages, and show various phenotypes. The most prominent of these are patterning defects in the wing imaginal disc. Wg signaling plays at least two distinct roles during wing development. Early reduction of wg activity results in a wing-to-notum transformation, indicating a requirement for Wg in defining the wing blade primordium, but later reductions cause the loss of wing margin and adjacent tissue, indicating its subsequent role in specifying the wing margin and organizing wing blade development. wf mutants show phenotypes opposite to both classes. Wing discs mutant for wf are enlarged with an extended wing blade region (hence the name wingful). Often these discs contain two wing pouches at the expense of notal structures. Although no apparent expansion of Distalless-lacZ (Dll-lacZ) expression was detected along the dorsoventral axis, there was a significant increase in the number of cells expressing neuralized, a high-threshold Wg target expressed in neural wing margin cells. Consistent with this observation, rare adult escapers mutant for wf show a dramatically increased number of mechanosensory bristles in the wing. Wg signaling also distinguishes between sternite and ventral pleura development in the adult abdomen. wf adult escapers show extra sternite bristles, an effect that was also observed with ectopic expression of wg. Finally, wf adults show extra dorsocentral bristles, sensory organs on the notum whose specification has been shown to depend on wg activity. Taken together, these results show that the absence of wf function causes a gain of Wg activity in developing adult tissues. Therefore, the function of the wild-type wf product is to limit Wg signaling activity (Gerlitz, 2002).

A further prediction of the assumption that Wf functions as a Wg feedback inhibitor is that wf overexpression should lead to wg loss-of-function phenotypes. Three lines of evidence are presented to show that this is, indeed, the case. (1) One of the wf enhancer trap P-element insertions was replaced with an EP element positioning 10 UAS sites upstream of the wf gene, rendering it transcriptionally responsive to Gal4 expression. Alterations of wf expression have unusually potent effects, since all commonly used Gal4 drivers caused lethality in combination with UAS-wf. The only exceptions were S168-Gal4 and scalloped-Gal4, which are expressed in the wing pouch and represent Wg targets, providing a self-regulating circuit in combination with UAS-wf. Adult animals carrying the S168-Gal4 and UAS-wf transgenes have severely reduced wings that lack all wing margin structures. (2) The expression of two target genes were analyzed in this context. S168-Gal4 expression was virtually abolished by UAS-wf expression, whereas the expression domain of wg itself is expanded. Wg is known to narrow its own domain of expression, because a reduction in Wg signal transduction causes ectopic wg transcription. (3) Finally, and perhaps most strikingly, driving expression of wf with scalloped-Gal4 results in a wing-to-notum transformation, the founding loss-of-function phenotype of the wg gene (Gerlitz, 2002).

Based on its structural features as a secreted protein with homologies to pectin acetylesterases, Wf could exert its function by modifying polysaccharide-based properties of cell surface proteins and thereby impeding the intercellular movement of the Wg protein. Alternatively, Wf could counteract Wg signaling by modifying the transducing properties of Wg or one of its receptors. To distinguish between these two possibilities, tests were performed to see whether Wf also antagonizes a derivative of Wg, Nrt-Wg, that is tethered to the cell surface and does not move through tissue. Expression of Wg or Nrt-Wg driven by dpp-Gal4 results in a robust activation of ectopic Dll-lacZ expression. Surprisingly, wf expression extinguishes Dll-lacZ expression induced by tethered Wg, as well as that induced by free Wg. From this experiment it can be ruled out that the primary function of Wf is to impede the extracellular transport of Wg. Therefore, Wf must interfere with the signaling activities of either Wg or its receptor components.Because no physical interaction between Wf and Wg, or between Wf and Frizzled-2 (Dfz2), or between Wf and Frizzled-2's LRP-like partner Arrow could be detected in tissue culture systems, the view is favored that Wf inhibits the activity of a coreceptor component, such as Dally or Dally-like (Dly), proteoglycans that appear to participate in Wg reception (Gerlitz, 2002). Wf may inhibit such receptor components via its presumptive esterase activity, for example, by modifying Dally or Dly glycosaminoglycan chains. A definitive proof for this mode of action could be achieved by the genetic demonstration that in larval dally;dly double-mutant situations, loss of wf function has no antagonistic effect (Gerlitz, 2002).

The discovery of Wingful as an essential Wg feedback antagonist may provide an explanation of why Nkd has no apparent role in imaginal tissues of Drosophila. The function of Nkd may be superseded by that of Wf, which functions in a powerful negative-feedback loop in adult development. Conversely, when both maternal and zygotic components of wf are removed, no obvious requirement was observed for Wf in embryonic development, possibly because the nkd system is operative at this stage of development. Both Naked and Wf can, however, inhibit Wg signaling throughout development if they are overexpressed, but each of them is operating more effectively at only one of the two stages. It may not be coincidence that Nkd, as the intracellular feedback antagonist, is used during embryonic patterning, (where Wg functions at short range), whereas Wf, as a secreted extracellular antagonist, primarily regulates patterning processes that depend on long-range Wg signaling. Wf functions nonautonomously and, like Argos (a secreted feedback antagonist of the Drosophila EGF system), may have a different range of action compared with the primary signal, providing an intricate means to shape the range and slope of the cellular responses to a morphogen gradient (Gerlitz, 2002).

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. A gene has been identified that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which has been called slalom, display defects in Wg and Hh signaling; these defects are likely due to the lack of sulfation of glycosaminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant Pipe (Lüders, 2003).

Secreted signaling molecules of the FGF, Hh, TGF-ß and WNT families rely on proteoglycans (PGs) for efficient activation of their respective signaling pathways. PGs consist of secreted or transmembrane core proteins to which glycosaminoglycan (GAG) side chains are attached at specific consensus sites. In Drosophila, the secreted PG perlecan, the transmembrane PG syndecan and two members of the glypican family of glycosylphosphatidylinositol (GPI) anchored PGs have been identified. Phenotypes associated with loss of function mutations of the glypican-encoding genes dally and dally-like (dlp) have revealed the requirement of these PGs for efficient activation of several signal transduction pathways (Lüders, 2003).

The function of PGs is critically dependent on the integrity of the attached GAGs. GAGs are unbranched polysaccharide chains, which are synthesized on proteoglycan core proteins in the Golgi and undergo complex modification reactions before the PG to which they are attached is transported to the cell surface. In the case of glypican, heparan sulfate (HS) chains, consisting of a sugar backbone of alternating units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA), are synthesized on the core protein. The nucleotide sugar substrates for this reaction are synthesized in the cytoplasm and must be transported into the Golgi. In Drosophila, the activated precursor UDP-GlcA is synthesized from UDP-glucose by the enzymatic activity of the sugarless (sgl) gene product, a homolog of mammalian UDP-glucose dehydrogenases. The gene product of fringe connection (frc), a predicted ER/Golgi multipass transmembrane protein, has been shown to transport UDP-GlcA and UDP-GlcNAc from the cytosol into the Golgi. Mutations in either gene severely affect the Wg and FGF signaling pathways. Elongation of the HS chains requires the activity of HS polymerases. tout-velu (ttv) encodes a protein with homology to the mammalian HS co-polymerase EXT1 and has been demonstrated to be required specifically for Hh signaling. Subsequent to their synthesis, GAGs undergo multiple modifications such as epimerization and sulfation. Mutations in sulfateless (sfl), a homolog of vertebrate N-deacetylase/N-sulfotransferases, lead to a severe reduction in the activity of the Wg, Hh and FGF signaling pathways. A characteristic feature of all mutations in genes involved in GAG biosynthesis is that their segment polarity phenotypes can be rescued by ectopic expression of wg or hh, suggesting that GAGs are not essential components of the respective signaling cascades but accessory factors most likely required for the proper distribution of extracellular signaling molecules throughout morphogenetically active tissues in vivo (Lüders, 2003).

GAGs have also been proposed to play a role in the determination of the dorsal-ventral (D/V) axis of the Drosophila embryo. The D/V polarity of the embryo is established during oogenesis by asymmetric expression of the key D/V determinant pipe (pip) in the follicle cell epithelium. pip expression in the ventral follicle cell layer is necessary and sufficient to trigger a serine-protease cascade in the perivitelline space; this leads to the generation of an active ligand for the transmembrane receptor Toll (Tl). Activation of Tl on the ventral side of the embryo results in a gradient of nuclear localization of the transcription factor Dorsal, which patterns the D/V axis. Based on sequence similarity to a family of vertebrate enzymes and its localization in the Golgi apparatus, pip has been hypothesized to encode a heparan sulfate 2-O-sulfotransferase. However, neither the enzymatic activity nor the substrate specificity of pip have been demonstrated directly (Lüders, 2003).

Sulfation of secreted molecules occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to be present within that organelle. In Drosophila, PAPS is synthesized in the cytoplasm by PAPS-synthetase, which incorporates both ATP-sulfurylase and adenosine 5'-phosphosulfate-kinase (APS-kinase) activity. PAPS must be transported into the Golgi thereafter to serve as a substrate for sulfotransferases. This study reports the molecular identification and functional characterization of a PAPS transporter. Mutations in this gene, called slalom (sll), are associated with defects in multiple signaling pathways, including Wg and Hh signaling. A phenotypic analysis suggests that the effects of sll on signal transduction are caused by its requirement for GAG modification. Evidence is presented that sll is also required to supply PAPS to the machinery initiating the establishment of embryonic D/V polarity, supporting the view that Pipe protein is a sulfotransferase (Lüders, 2003).

A hydrophobicity analysis of the putative Sll protein predicts a hydrophobic polypeptide with at least 10 transmembrane regions, a structural characteristic of many nucleotide-sugar transporters. A BLAST search of non-redundant protein databases with the Sll sequence revealed that Sll is conserved throughout the animal kingdom, as well as in plants, and shares nearly 40% of amino acid sequence identity with predicted mouse and human proteins of unknown function. Sll is also similar to mammalian proteins that have been classified as nucleotide-sugar transporters on the basis of their homology to the human UDP-galactose transporter hUGT. However, Sll itself has no significant sequence similarity to hUGT. While these data suggest that Sll encodes a transmembrane transporter, they leave open what substrate Sll may be transporting (Lüders, 2003).

In order to demonstrate directly that Sll has transporter activity, and to determine its substrate specificity, an in vitro vesicle transport assay was used to examine the ability of Sll to facilitate the transport of defined nucleotide substrates into Saccharomyces cerevisae microsomes. Heterologous expression of sll in yeast was favored over other systems since procedures in this organism have been successfully used for transfection of nucleotide-sugar genes, high-level preparation of sealed microsomal vesicles and nucleotide-sugar assays. In vesicles derived from cells transfected with a sll-expression construct, a 5-fold increase in the uptake of PAPS was detected. Import of several other nucleotide-sugars did not increase after sll expression. Importantly, the sll-dependent PAPS transport activity could be efficiently competed by structurally similar substrate analogs but not by more distantly related nucleotide monophosphates, confirming that the transport was specific (Lüders, 2003).

PAPS is synthesized in the cytoplasm and serves as a substrate for sulfotransferases during the post-translational modification of macromolecules in the Golgi. Therefore, the result that sll encodes a PAPS transporter suggests that Sll should be localized to the Golgi membranes. To address this question, peptide antibodies were raised against Sll. Localization of Sll in salivary glands of wild-type embryos, using Sll antiserum, showed a punctate staining pattern in the perinuclear region of cells. Co-staining of salivary glands with antibodies against an endogenous Golgi protein revealed extensive co-localization of the two proteins, indicating that Sll is a resident Golgi protein. Together, these data suggest that Sll encodes a nucleotide transporter required to translocate the high-energy sulfate donor PAPS into the lumen of the Golgi (Lüders, 2003).

The phenotypical analysis of sll mutants shows that sll function is required for Wg and Hh signaling, and suggests that sll may also play a role in the activation of other signaling pathways, such as the TGF-ß pathway and possibly others. The broad requirement for sll raises the question of how sulfation influences the function of multiple secreted signaling factors. Proteins entering the secretory pathway are ubiquitously sulfated on specific tyrosine residues. However, according to sulfation consensus prediction algorithms, neither Wg nor Hh is predicted to be a substrate for tyrosine sulfation (see 'Sulfinator' at http://www.expasy.org/tools/sulfinator/). In addition, overexpression experiments show that wg and hh have the ability to at least partially activate their respective signaling cascades in sll mutants, which would be unlikely if the signaling factors themselves were non-functional due to lack of sulfate modification. Therefore, it is unlikely that lack of tyrosine sulfation of either Wg or Hh proteins is responsible for the defects in Wg or Hh signaling pathways in sll mutants (Lüders, 2003).

The Wg and Hh signaling pathways are activated through interaction of secreted ligands with their respective cell surface receptors. This activation relies on the action of GAGs attached to proteoglycan core proteins of the glypican family. In the case of Wg, the proteoglycans are thought to act as coreceptors, retaining the ligand at the cell surface in the vicinity of its receptor. Hh has been proposed to depend on GAGs for efficient transport to its target cells. In the absence of functional GAGs, the signaling molecules are not present in sufficiently high concentrations at the receptor to activate the signal transduction pathway. This defect can be compensated by overexpression of the signaling molecule. Therefore, a characteristic feature of mutations in genes involved in GAG metabolism is that their segment polarity defects can be rescued by overexpression of the ligand. These data show that Wg protein levels and Hh signaling activity are reduced in sll mutant clones. The segment polarity defects of sll mutants can be rescued by overexpression of wg or hh. In addition sll and dally mutants affect Wg signaling synergistically. The data also suggest that the ability of cells to sulfate GAGs is dramatically reduced in sll mutants because the sulfate donor necessary for sfl to modify the polysaccharide chains cannot be transported into the Golgi in sufficient amounts. Taken together, these observations strongly suggest that lack of functional GAGs is responsible for the segment polarity defects in sll mutants (Lüders, 2003).

Interestingly, the overall size of sugar chains attached to the glypican Dally does not appear to be affected in sll mutants. Consistent with this result, it has been observed that the overall level of HS, which is reduced to trace amounts in the sgl mutant that affects GAG biosynthesis, is not markedly changed in sfl mutants, which should affect sulfate modification but not synthesis of GAG chains. However, it cannot be excluded that residual sulfation of GAGs occurs in the cell culture assay owing to incomplete inhibition of the PAPS transport activity of sll by dsRNA interference. However, the phenotypic analysis of sll suggests that sll function is essential for at least two sulfotransferases, sfl and pip, and that the GAG chains present in sll mutants are not able to fulfill their normal function owing to altered sulfation patterns (Lüders, 2003).

The central position of sll in sulfate metabolism along the secretory pathway makes it an interesting tool for the identification of developmental pathways sensitive to sulfate modification. The results demonstrate that several cell-cell communication pathways are critically dependent on the sulfation of macromolecules, and highlight the importance of sulfation during pattern formation and development (Lüders, 2003).

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of toutvelu (ttv), were identified. The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, ttv mutations were also found to affect Dpp and Wg. These data lead to the conclusion that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. It is proposed that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients (Takei, 2004).

In addition to monitoring signaling in EXT mutant cells, antibodies that recognize Hh, Dpp and Wg, and a GFP-tagged version of Dpp were used to analyze whether the levels or distribution of these morphogens had been affected. Levels of each of these proteins were significantly reduced in the mutant, both in the morphogen-expressing region and in the receiving region. For Hh, Dpp and Wg, similar results were observed in cells mutant singly for any of the EXT genes. Single mutation was not tested for the distribution of Dpp-GFP. In the morphogen-expressing region, hh expression was not downregulated, however levels of Hh protein were significantly decreased. This may indicate that Hh protein is destabilized and/or not retained efficiently on the cell surface in the absence of HSPGs. In contrast to hh, expression of the wg and dpp and levels of Wg and Dpp were decreased in the EXT clones. The decrease in dpp expression is easily accountable because Hh signaling is impaired in the absence of HSPGs. In contrast, the decrease in wg expression is not as readily explainable: cut and wg are both targets of Notch signaling, however the protein level of Cut was not altered in EXT clones. This suggests that wg is also regulated by an unknown mechanism dependent on HSPGs (Takei, 2004).

In the morphogen-receiving region, each of these proteins was significantly decreased in the clones of cells mutant for EXT genes, although a little leakage of morphogen molecules was seen even in the clones doubly mutant for ttv and botv. This suggests two possible mechanisms that do not exclude each other: in the absence of HSPGs these three morphogens are (1) destabilized and/or are not retained efficiently on the cell surface, like Hh in morphogen-expressing region, or (2) prevented from diffusing efficiently into the region consisting of EXT mutant cells. Intriguingly, close observation of the distribution of Hh strongly suggested a function for HSPGs in morphogen movement. In the wild-type discs, Hh protein synthesized in the posterior compartment appears to flow into the anterior compartment, with a moderate concentration gradient starting from the middle of the posterior compartment. However, Hh abnormally accumulates in the posterior compartment when the EXT mutant clone is in the anterior compartment along the A/P boundary. This effect is seen both in the ventral region and in the dorsal region. This suggests that Hh fails to move into the mutant cells and as a consequence accumulates in posterior cells instead. Dpp-GFP and Wg accumulation in front of the mutant clones was also apparent, however less pronounced compared with the case of Hh. Therefore it is concluded that the HSPG-dependent diffusion is the common mechanism for the movement of these three morphogens (Takei, 2004).

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. Mutations have been isolated in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. These results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv (Bornemann, 2004).

A role for HSPGs in Wg signaling in Drosophila is well established. Addition of heparin sulfate (HS) or chondroitin sulfate (CS) GAGs to culture medium causes S2 cells to release secreted Wg protein, while heparinase treatment reduces their ability to respond to exogenous Wg. Furthermore, both Wg levels and signaling efficiency are sensitive to HSPG concentration; Wg activity is reduced when either HS synthesis or the core proteins for the glypicans Dally and Dlp are compromised. Severe disruption of extracellular Wg distribution and the altered expression of the downstream target Ac, in ttv and sotv mutant clones, demonstrates that HS chains are essential for establishing and/or maintaining the Wg gradient (Bornemann, 2004).

The effects of HSPGs on signaling are further modulated by secondary modifications to the GAG chains. For example in Drosophila, loss of either sfl or slalom (sll) compromises both Wg and Hh signaling. Sfl is an N-deacetyl/N-sulfotransferase, and Sll is required to transport a high-energy sulfate donor molecule to the golgi, where HSPGs such as dally and dlp are sulfate modified. Loss of GAG sulfation results in signaling defects, despite the fact that unsulfated HS-GAGs are still present in sfl mutants. Consequently, not only are HS-GAG chains required for signaling, but the extent of sulfation is also crucial. In vertebrates, Qsulf1 promotes Wg signaling by catalyzing removal of 6-O sulfate groups from HS chains of HSPGs, including the Dally homolog glypican 1. It has been proposed that in the absence of Qsulf1, Wg stays tightly bound to HSPGs, which prevents the ligand from interacting with receptors. However, in Qsulf1-expressing cells, selective 6-O desulfation reduces the binding affinity sufficiently to permit ligand-receptor interaction. In this context, the altered HS disaccharide distribution and abnormal sulfation patterns encountered in hypomorphic sotv alleles is intriguing. If partial loss of function EXT2 mutations in individuals with HME causes similar disruption of GAG modifications, they could also have complex and allele-specific effects on signaling (Bornemann, 2004).

Opposing activities of Dally-like Glypican at high and low levels of Wingless morphogen activity

The glypican family of heparan sulfate proteoglycans has been implicated in formation of morphogen gradients. The role of the glypican Dally-like protein (Dlp) in shaping the Wingless gradient was studied in the Drosophila wing disc. Surprisingly, Dlp has opposite effects at high and low levels of Wingless. Dlp promotes low-level Wingless activity but reduces high-level Wingless activity. Evidence is presented that the Wg antagonist Notum acts to induce cleavage of the Dlp glypican at the level of its GPI anchor, which leads to shedding of Dlp. Thus, spatially regulated modification of Dlp by Notum employs the ligand binding activity of Dlp to promote or inhibit signaling in a context-dependent manner. Notum-induced shedding of Dlp could convert Dlp from a membrane-tethered coreceptor to a secreted antagonist (Kreuger, 2004).

Notum encodes a secreted member of the alpha-hydrolase superfamily, which includes serine proteases, lipases, and other enzymes in which a Ser-Asp-His catalytic triad comprises the active site. Mutation of Ser237 to Ala was shown to remove Notum activity in vivo and in vitro (Giraldez, 2002). On the basis of its similarity to pectin acetylesterase enzymes from plants and plant pathogens and on the basis of its ability to compete with the N-deacetylase-N-sulfotransferase enzyme for use of Dlp as a substrate, it has been proposed that Notum might function as a GAG deacetylase. If this were the case, it would be expected that Notum would be unable to modify a form of Dlp lacking GAG side chains (Kreuger, 2004).

Dally and Dlp each contain five putative GAG addition sites and have been shown to carry predominantly heparan sulfate (HS, as shown for vertebrate glypicans). GAG biosynthesis is initiated by addition of a xylose residue to the hydroxyl group of a serine in a serine-glycine (SG) motif in the stem region of the glypican (Lindahl, 1998). Mutant forms of Dally and Dlp proteins were prepared in which all the putative GAG-addition sites were mutated. For Dally, the serine residues in the five SG motifs were mutated to alanine. For Dlp, four SG motifs were deleted by removing a block of 25 residues, and the fifth was mutated to AG (Kreuger, 2004).

The glycosylation states of epitope-tagged Dally and Dlp and the GAG addition site mutants were compared by anion exchange chromatography. Glypicans isolated from mammalian cells in culture are highly negatively charged and are typically retained on a strong anion exchange matrix up to 1 M NaCl. When expressed in Schneider S2 cells, a considerable fraction of Dally bound to Q-Sepharose in 0.3 M salt and some was able to bind in up to 1 M salt. Dally migrated as a broad band in SDS-PAGE. The more negatively charged forms that were bound to Q-Sepharose at higher salt exhibited a higher apparent molecular weight. This may reflect the presence of more and longer HS side chains as well as differences in charge due to sulfation. Dally behaves like a conventional glypican (Tsuda, 1999). In contrast, most of the Dlp protein bound to Q-Sepharose in 0.15 M salt, but none bound at higher salt concentrations. Dlp migrated as a more tightly resolved band in SDS-PAGE. These observations suggest that Dally and Dlp differ in the extent or quality of GAG modification. Removal of the GAG addition sites considerably reduced retention of both mutant proteins on Q-Sepharose. The proportion of the mutant form of Dally that bound was reduced at all salt levels and none was bound above 0.3 M salt. Very little of the mutant form of Dlp was able to bind to Q-Sepharose, even in physiological salt (Kreuger, 2004).

To further evaluate their GAG content, lysates of S2 cells transfected to express wild-type or mutant Dally or Dlp were digested with heparitinase, and the remaining glycan stub was detected with 3G10 antibody. Dally-expressing cells showed elevated GAG labeling in a broad band that comigrated with the major form of endogenous glypican in untransfected cells. Although the level of expression of the mutant form of Dally was comparable, GAG labeling was not detectable above background. The level of GAG modification on overexpressed Dlp was lower than that seen on the endogenous glypicans. This indicates that Dlp has a lower GAG content than Dally, consistent with its poor retention on Q sepharose. The mutant form of Dlp showed much reduced GAG labeling, barely distinguishable from background levels (Kreuger, 2004).

Dally resembles a conventional glypican, being extensively modified by GAG side chains. Dlp appears to have less heterogeneous and less extensive GAG modification than Dally. The mutant form of Dlp has low levels of residual GAG and would be expected to be a poor substrate for Notum, if Notum acts directly on the GAG side chains. Coexpression of Notum and HA-tagged Dlp in S2 cells caused a large shift in the electrophoretic mobility of Dlp in SDS-PAGE to a faster migrating species, and the Notum S237A mutant had no effect. S2 cells contain endogenous Notum, so there is always a background level of Dlp processing. Interestingly, Notum caused a shift in the mobility of the low-GAG form Dlp comparable in magnitude to the shift in the wild-type protein, though the processing efficiency was lower. This would be difficult to explain if the shift were due to alteration in the amount or in the negative charge of the remaining GAG due to deacetylation. Further, no increase in deacetylase activity was detected in lysates of S2 cells overexpressing Notum, nor was there any detectable decrease in the degree of HS acetylation in larvae overexpressing Notum. These observations suggest that Notum does not act as a GAG-modifying enzyme (Kreuger, 2004).

The mobility shift of Dlp caused by Notum is ~15 kDa. Proteolytic cleavage at the N or C termini of Dlp could cause the apparent size reduction, although other modifications such as removal or attachment of lipids, sugars, or phosphate groups also could cause anomalous migration in SDS-PAGE. To test the possibility of proteolytic processing, forms of Dlp were prepared with epitope tags close to the two ends. For Dlp-HA-C, an HA epitope tag was inserted at serine 732, just before the putative cleavage site for addition of the GPI anchor (S733). To produce GFP-Dlp-HA-C, the HA tag was placed at the corresponding position in GFP-Dlp, which contains a GFP moiety inserted in place of residue G68, close to the N terminus of the protein following removal of the signal peptide at residue 41. S2 cells were transfected with Dlp-HA, Dlp-HA-C, or GFP-Dlp-HA-C together with normal or mutant forms of Notum. Notum induced comparable mobility shifts in all three forms of Dlp. The C-terminally HA-tagged forms of Dlp were processed more efficiently than Dlp-HA, so that more of the faster migrating from was observed without addition of wild-type Notum (Notum is expressed in S2 cells). These observations are difficult to reconcile with Notum acting as a protease (Kreuger, 2004).

The possibility was considered that the shift in Dlp mobility could be due to cleavage of the GPI anchor. Lysates were prepared from S2 cells transfected with Dlp-HA with or without Notum, and half of each was treated with phospholipase-C (PI-PLC) to cleave the GPI anchor of Dlp. Interestingly, PI-PLC cleavage caused a mobility shift in Dlp comparable to that caused by Notum. Notum had no additional effect on PI-PLC-treated Dlp. Although the magnitude of this shift in apparent molecular weight is larger than would be expected from the mass of the GPI anchor alone, anomalous migration of proteins in SDS-PAGE has been observed following removal of GPI anchors (Kreuger, 2004).

To further evaluate the possibility that the mobility shift caused by Notum might be due to cleavage of the GPI anchor, a Triton X-114 phase separation analysis was performed. Following phase separation, integral membrane proteins and GPI-anchored proteins are recovered mainly in the detergent phase, while other proteins are mainly in the aqueous phase. In cells cotransfected to express Dlp and Notum, processing of Dlp was efficient and the faster migrating form of Dlp was mainly recovered in the aqueous phase. In cells transfected to express Dlp, the slower migrating form was mainly recovered in the detergent phase, consistent with it having a GPI anchor. The faster migrating form partitioned between aqueous and detergent phases. These observations are consistent with the suggestion that the faster migrating form of Dlp produced by Notum lacks a GPI anchor (Kreuger, 2004).

It was next asked if Notum could act on forms of Dlp that lack a GPI anchor. The predicted GPI anchor site of Dlp consists of the sequence SDA (at S733) followed by a characteristic hydrophobic motif. Mutation of any of the three residues of the GPI anchor site to proline has been shown to reduce GPI addition in other glypicans. The SDA motif was mutated to PDA in GFP-Dlp-S988P (residue numbers are for GFP-Dlp). A truncated version of GFP-Dlp was also prepared in which a stop codon was inserted after S987 to mimic the effects of cleavage of the protein chain at this position without addition of GPI. Coexpression with Notum had little or no effect on these forms of GFP-Dlp. GFP-Dlp-S988P migrated predominantly at an intermediate position, faster than the GPI-anchored form but slower than the Notum-processed form. This presumably reflects the full-length protein that has not been cleaved for GPI addition (this form appears as a minor band in most preparations). The major band of GFP-Dlp-S988P is unaffected by Notum, though a small amount of the protein does appear to have had GPI added (perhaps at S987) and was subject to Notum activity. The truncated GFP-Dlp-S987Z protein did not produce any of the slower migrating GPI anchored form and was not affected by Notum. GFP-Dlp-S987Z migrated slightly faster than the Notum-modified form of GFP-Dlp. In Triton X-114 phase separation analysis, GFP-Dlp-S987Z partitioned into the aqueous phase, also consistent with the lack of a GPI anchor. The form of Dlp cleaved by PI-PLC comigrated more closely with the Notum-processed form, consistent with the possibility that Notum cleavage was in the GPI moiety (Kreuger, 2004).

Next a version of Dlp was prepared that was not GPI anchored, in order to ask if it would be a substrate for Notum. GFP-Dlp was fused at residue S975 to residue R23 of the transmembrane protein CD2 and expressed in S2 cells. This protein contains the entire ectodomain (CRD and stem) of Dlp, but lacks the GPI addition signals (the fusion is at the equivalent position in Dlp to the S987Z construct; numbering differs because the HA tag was removed). GFP-Dlp-CD2 was not cleaved by PI-PLC, verifying that it does not contain a GPI anchor. GFP-Dlp-CD2 was also not a substrate for Notum. If Notum acted as a protease to cleave in the Dlp protein, it would be expected that GFP-Dlp-CD2 fusion would be a substrate for Notum. Likewise, if Notum acted on the GAG side chains or on another posttranslational modification, it would be expected that GFP-Dlp-CD2 would be affected. Taken together, these observations indicate that Notum can induce modification of Dlp in a manner that resembles cleavage of the GPI anchor. Given that the alpha-hydrolases includes lipases, it is possible that Notum might act directly to cleave within the GPI anchor. Alternatively, Notum could induce the activity of an endogenous phospholipase (Kreuger, 2004).

Can cleavage at the GPI anchor cause Dlp to be shed from the cell surface? The faster and slower migrating forms of Dlp-HA were efficiently recovered from the cell lysates. In addition, the faster migrating cleaved form of Dlp-HA was recovered from the culture medium of cells overexpressing Notum. The slower migrating GPI-anchored form of Dlp-HA was not recovered from the medium. Very little Dlp-HA was recovered from medium conditioned by cells transfected with Dlp alone or with the Notum mutant. This indicates that Notum can release Dlp from the cell surface, causing it to be shed into the medium. Notum does not induce detectable shedding of HA-tagged Dally (Kreuger, 2004).

Dlp-HA or Dally-HA were expressed under en-Gal4 control in the wing disc to compare the effects of the endogenous Notum protein on Dlp and Dally. Notum is expressed at the dorsoventral boundary under control of Wg. En-Gal4 produced a uniform level of Dally-HA across the wing pouch. In contrast, Dlp-HA protein levels were lower at the dorsoventral boundary. Notum caused loss of Dlp, but had little effect on Dally. This presumably reflects shedding of Dlp protein from cells in the disc in the region where Wg levels are highest (Kreuger, 2004).

The finding that Notum can cleave and release Dlp from cells raised the possibility that Dlp might be released together with bound Wg. This could explain how Dlp acts to reduce Wg activity where Wg and Notum levels are high. Therefore the possibility was tested of synergistic action of Notum and Dlp on Wg function. Overexpressed Dlp binds and sequesters Wg at the cell surface, leading to reduced Wg activity. Coexpression of Notum with Dlp reduced the ability of Dlp to bind and retain Wg (Giraldez, 2002). To examine the effects of Notum on Dlp activity, transgenes expressing Notum or Dlp were selected at levels that were on the threshold for altering wing morphology. When expressed under en-Gal4 control, each transgene caused loss of some of the posterior wing margin bristles, indicating mild reduction of Wg activity. Expression of the two transgenes together caused scalloping of the wing, a typical defect caused by more severe reduction of Wg activity. This observation suggests that expression of Notum enhanced the ability of Dlp to reduce Wg activity. In view of the finding that Notum can cause Dlp to be released from the cell surface, it is suggested that coexpression of Notum causes Dlp to be released together with bound Wg (Notum reduced the level of Wg bound in the disc; Giraldez, 2002). Release of Wg bound to Dlp could reduce the level of Wg available for signaling and cause the observed wing scalloping phenotype. Notum causes release of Dlp from cells at the DV boundary, where Wg levels are highest (Kreuger, 2004).

Therefore, Notum can induce cleavage of Dlp in a manner that resembles PI-PLC cleavage of the GPI anchor. Notum and PI-PLC produce comparable shifts in the electrophoretic mobility of Dlp. Although the magnitude of this shift is larger than would be expected from the mass of the GPI anchor, other studies have shown that the effects of removing GPI anchors are not predictable. Examples of increased or decreased mobility have been reported, and the magnitude of the shifts can be large. Notum-induced processing also renders Dlp soluble in the aqueous phase following detergent phase separation of soluble and membrane-associated proteins, consistent with removal of the GPI anchor. Also, a Dlp-CD2 fusion protein that is not GPI anchored, and is therefore not a substrate for PI-PLC, is insensitive to Notum. This would not be expected if Notum acted on the GAG side chains or if Notum was a protease cutting within the Dlp core protein. These observations are consistent with two modes of Notum action. Notum might act directly to cleave the GPI anchor or it might modify Dlp in some way that makes Dlp a substrate for an endogenous phospholipase. The exact nature of the Notum-induced cleavage remains to be determined; however, one intriguing possibility is that Notum might cleave within the glycan linker of the GPI anchor (Kreuger, 2004).

Can the effects of Notum on Dlp provide an explanation for the apparently opposing activities of Dlp at high and low levels of Wg? How could Notum-induced cleavage of Dlp lead to reduced Wg activity, whereas removal of Dlp by RNAi increases Wg activity? If Wg remains bound to Dlp when Dlp is cleaved and shed from the cell, bound Wg would also be shed and so become unavailable for signaling. Shedding of Dlp as a consequence of Notum-induced cleavage could reduce peak levels of available Wg. Notum is expressed at the source of Wg and so would be expected to shed Dlp and reduce Wg activity where Wg levels are highest. In this way, removal of Dlp protein as a consequence of Notum-induced cleavage could have a different effect than failure to express Dlp. In the absence of Dlp, Wg would not bind Dlp and be shed with it, so that more Wg might be available to interact with Dally and/or the Wg receptor complex. This could increase the effective concentration of Wg locally near the site of Wg production (Kreuger, 2004).

This model is consistent with the observed synergy between low-level expression of Dlp and Notum. Overexpression of Dlp is thought to increase the number of Wg binding sites and shift the equilibrium toward more Wg bound to Dlp. At high levels of Dlp, this can reduce the amount of Wg available for signaling and produce a Wg loss-of-function phenotype. A level of Dlp overexpression was chosen that produces a mild defect due to reduced availability of Wg. Coexpression of Notum would lead to shedding of the Dlp bound Wg and thus remove this fraction of Wg from the pool on the cell surface so that it would no longer be able to contribute to the pool of Wg in equilibrium with the receptor. This would be expected to further reduce Wg activity and increase the severity of the defect, as observed (Kreuger, 2004).

Localized cleavage of Dlp induced by regulated expression of Notum may provide a unifying explanation for the opposing effects of the Dlp glypican in different regions of the tissue. A mathematical model of Wg gradient formation has invoked a need for an elevated level of Wg turnover in cells close to the source of Wg. The mechanism described here would be sufficient to provide the reduction of Wg activity in cells closest to the source of the secreted ligand that is needed for formation of a robust morphogen gradient (Kreuger, 2004).

Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein

Wingless is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix (Kirkpatrick, 2004).

By a number of cellular and molecular markers, compromising dlp function produced ectopic activation of high-threshold Wg target genes in the wing imaginal disc. dlp adult escapers have ectopic mechanosensory bristles and dlp third instar larvae show expanded expression of Ac, a marker for sensory organ precursor cells. Wg signaling was abnormally elevated along the DV boundary of dlp mutant discs by a number of other measures, including expansion of the zone of nonproliferating cells (ZNC) and ectopic expression of Cyclin A. These findings are consistent with the expansion of sensory organ precursor cells produced by RNAi inhibition of dlp function. These phenotypes show that Dlp serves to limit activation of high-threshold target genes near the DV boundary (Kirkpatrick, 2004).

The expansion of Wg signaling found in dlp mutants is accompanied by locally elevated levels of extracellular Wg. The increase in Wg in the matrix of dlp mutant wing discs is not associated with increases in Wg production or altered expression of D-fz2, a known regulator of Wg levels. These findings argue against the model that dlp controls Wg levels indirectly via control of Wg expression or other regulatory genes. It is well established that Wg binds heparan sulfate, and therefore it is likely that Dlp controls Wg levels through a direct interaction (Kirkpatrick, 2004).

The expansion of Wg-directed patterning in the wing disc of dlp mutants was a surprising finding on a number of counts: (1) mutations in genes encoding heparan sulfate biosynthetic enzymes compromise rather than increase the levels of Wg in the matrix; (2) ectopic expression of Dlp results in higher levels of Wg in the matrix. These findings suggest that loss of Dlp would destabilize Wg on cell surfaces and in the matrix, thereby reducing Wg signaling. However, exactly the opposite result was obtained in dlp mutants: loss of Dlp increases extracellular Wg and signaling. These findings emphasize that while mutations compromising synthesis of all HSPGs might destabilize Wg in the matrix, loss of any one proteoglycan can have very different effects (Kirkpatrick, 2004).

The phenotypes of dlp mutants suggest that Dlp serves to limit Wg distribution and/or alter Wg stability, thus affecting the Wg gradient in the wing disc. Genetic mosaic analysis of dlp clearly demonstrates that the ectopic activation of Wg signaling, as measured by Arm, Cyclin A, and Ac expression, occurs non-cell autonomously, providing support for this model. Recent mathematical modeling of morphogen gradients demonstrates that binding of morphogens to receptors or coreceptors on cell surfaces or in the matrix can have profound effects on diffusion and subsequently limit morphogen activity. Consistent with such modeling, it has been demonstrated experimentally that a nonheparin binding isoform of the secreted growth factor VEGF adopts a broader, shallower distribution than heparin binding forms and the differential localization of VEGF-A isoforms in the matrix controls the vascular branching pattern. It is also well established that heparan sulfate proteoglycans mediate endocytosis of extracellular ligands, and the findings suggest the possibility that Dlp might mediate endocytic control of Wg levels in the matrix. dlp mutants display locally elevated levels of extracellular Wg without an increase in production, and therefore Wg turnover must be affected in some way (Kirkpatrick, 2004).

In contrast to the higher levels of extracellular Wg found near the DV boundary in dlp mutant wing discs, there is no apparent change in the low level of Wg at the edges of the gradient. However, genetic mosaic analysis of dlp in wing development shows that loss of dlp can modestly reduce the activation of a low-threshold Wg target gene in those regions where Wg levels are low. Since this effect on Wg signaling is not a consequence of reduced levels of Wg in the matrix, it must result from an alteration of cell responsiveness or the bioactivity of the Wg that is present. The findings are consistent with studies showing that RNAi of dlp in the wing disc also inhibits transcriptional activation of Wg target genes in regions where Wg is low. It is interesting to note that dlp expression, like that of the Wg receptor D-fz2, is highest outside the presumptive wing margin. Perhaps the high level of dlp expression in this region serves, in part, to enhance cellular responses to Wg. The ability of Dlp to enhance Wg signaling as well as limit Wg distribution is not unprecedented: the other glypican in Drosophila, Dally, enhances Dpp signaling in regions of the wing disc where Dpp levels are modest in addition to regulating the distribution of Dpp protein (Kirkpatrick, 2004).

Mutations in Notum produce ectopic activation of Wg signaling and alter the Wg gradient, similar to what was observed in dlp mutants. Notum encodes a protein with homology to plant hydrolases and has been proposed to alter the structure of Dlp, thus affecting its affinity for Wg. Coexpression of Notum with Dlp decreases Dlp-mediated stabilization of Wg protein in the wing pouch. dlp and Notum mutants fail to complement: this is strong genetic evidence that Dlp and Notum cooperate to control Wg-mediated patterning. Biochemical studies show that Notum can cleave Dlp from the cell surface. The finding that heterozygosity for dlp ameliorates patterning abnormalities produced by ectopic Notum suggests that Dlp is a principal substrate for Notum activity. The results suggest that Notum-mediated release of Dlp from the cell surface is required for Dlp to limit Wg signaling (Kirkpatrick, 2004).

Notum and dlp are expressed in complementary patterns in the wing disc, with Notum levels highest near the DV boundary and dlp levels highest elsewhere. Thus, a gradient of Dlp cleavage may be established across the disc, with some Dlp remaining on cell surfaces in regions distant from the DV boundary, enhancing cellular responses to Wg. Cleavage of Dlp would remove this coreceptor activity, but shed Dlp-Wg complexes also could limit Wg signaling by promoting Wg clearance or could serve as dominant-negative inhibitors of signaling. Coexpression of Notum and Dlp in the wing disc produced more intracellular vesicles compared to expression of Dlp alone: this is consistent with a role for shed Dlp in promoting endocytosis and clearance of Wg from the matrix (Giraldez, 2002). Such a role is also consistent with the non-cell-autonomous effects of dlp somatic cell clones on extracellular Wg distribution. Loss of dlp in clones might locally reduce Wg turnover as well as reducing cell surface binding sites for Wg, permitting increased diffusion of Wg and accumulation on wild-type cells at clone boundaries. Spatial regulation of Dlp activity by Notum can explain how Dlp primarily limits Wg signaling near the DV boundary and enhances signaling away from the boundary, but other Wg-dependent factors may also influence the ability of Dlp and Notum to downregulate Wg levels. Notably, recent mathematical modeling suggests that elevated degradation of Wg close to its source is necessary to enhance the robustness of the morphogen gradient. Together, Dlp and Notum may provide this locally increased turnover and hence stabilize the Wg gradient. Enzymatic modification of a proteoglycan to influence its cell surface localization may enable it to play both positive and negative roles in signaling and provides another potential mechanism for regulating morphogen distribution in tissues (Kirkpatrick, 2004).

In humans, mutations in glypican-3 (GPC3) were identified as the genetic basis of a human overgrowth and tumor susceptibility syndrome, Simpson Golabi Behmel Dysmorphia (SGB). SGB patients display both prenatal and postnatal overgrowth, a number of morphological abnormalities including renal dysplasia and skeletal defects, as well as a high incidence of tumors. Subsequently, loss of GPC3 expression has been found associated with a number of cancers, including breast, mesothelioma, and ovarian neoplasias. Given the established role of proteoglycans as molecules facilitating growth factor signaling at the cell surface, the effects of loss of GPC3 on growth and tumor development are a bit puzzling. The current findings provide a molecular mechanism for understanding the capacity of glypicans to serve as tumor suppressors. Dlp restricts the cellular domain of Wg signaling during wing development, and loss of dlp results in ectopic growth factor signaling. Clearly, ectopic signaling produced by loss of GPC3 could readily contribute to tumor development and growth in humans. Recently, analysis of a gene-trap mutant in mouse Ext1 has provided further evidence for the capacity of heparan sulfate proteoglycans to limit the range of morphogen activity during chondrocyte differentiation. Mice partially defective for heparan sulfate biosynthesis as a consequence of hypomorphic mutations in Ext1 show ectopic Indian Hedgehog signaling and altered Hedgehog distribution at the growth plate (Kirkpatrick, 2004 and references therein).

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

The respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient has been examined. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, it was found that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. These studies reveal that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, this analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient (Baeg, 2004).

Sfl encodes a homolog of the Golgi enzyme HS N-deacetylase/N-sulfotransferase that is required for the modification of HS. It was of interest to determine whether the retention of Wg at the cell surface involves HSPGs in receiving cells, since it has been proposed that HSPGs are unlikely to be required in Wg-receiving cells. In the wing blade, Wg, originating from the D/V border, is detected in an irregular pattern of puncta in receiving cells; these puncta correspond to the internalized Wg protein. The intensity and number of puncta decreases from the source of Wg. Furthermore, using an extracellular labeling method, a gradient of Wg protein that appears broader, shallower, and with less puncta is observed. Both in sfl mutant wing discs and in large sfl mutant clones, a striking decrease in the number of Wg puncta was observed. This decrease is not due to a change in wg transcription because it is not affected in sfl mutant cells. Further, lack of Sfl activity does not appear to disrupt the overall amount of Wg produced by wg-expressing cells but is associated with a dramatic decrease in extracellular Wg. These results suggest that HSPGs are required for sequestering extracellular Wg in receiving cells (Baeg, 2004).

To gain further insights into the role of HSPGs in receiving cells to shape the Wg gradient, an examination has been made of the distribution of Wg in patches of WT cells located within a large sfl mutant territory. In such cases, bright spots of Wg were be detected within the patch of WT cells, indicating that sfl-expressing cells are able to sequester extracellular Wg, unlike neighboring cells that lack sfl. This result is consistent through an analysis of more than 10 clones. Further, clones of cells that overexpress Notum-GT (Golgi-tethered), which acts cell autonomously in receiving cells, were generated. Notum, which encodes a member of the α/β-hydrolase superfamily, antagonizes Wg signaling and it has been proposed that Notum acts by altering the ability of the cell surface glypican molecules Dally and Dlp to stabilize extracellular Wg (Giraldez, 2002). Consistent with the conclusion that HSPGs are required in receiving cells to capture extracellular Wg, a decrease in the formation of Wg puncta was observed in cells overexpressing Notum-GT. These results are consistent with at least two nonexclusive models: (1) HSPGs could be required for Wg stability and/or trapping of Wg at the cell surface such that it does not diffuse away; (2) HSPGs could be involved in promoting Wg movement throughout tissues. The role of HSPGs in sequestering and/or stabilizing the ligand is supported by previous observations that overexpression of either Dlp or Dally results in the accumulation of extracellular Wg (Baeg, 2004).

Because HSPGs have been implicated in endocytosis of ligands such as FGF, possibly HSPGs also play a role in Wg internalization. A role for HSPG in Wg endocytosis would be consistent with the absence of puncta in sfl clones, and also the observed accumulation of extracellular Wg following overexpression of Dlp-HA (Giraldez, 2002). However, much of Wg proteins appeared in intracellular vesicles, instead of outlining the cell surface, in discs overexpressing both Dlp-HA and Notum. If HSPGs were directly involved in Wg internalization, fewer intracellular vesicles would be detected in discs overexpressing Dlp-HA and Notum since Notum acts to decease the affinity of Dlp for Wg. Furthermore, if the primary function of HSPGs were to internalize Wg, then extracellular Wg accumulation in cells lacking HSPGs activity would be expected, which is not the case. However, the data does not rule out the possibility that HSPGs play a direct role in Wg endocytosis, and, thus, further analysis will be required to clarify this issue. Taken together, these results suggest that the primary role of HSPGs is to trap and/or stabilize extracellular Wg in receiving cells where it is then able to interact with its signaling receptor as well as other factors that are responsible for its internalization, and thus contributes to shaping the Wg gradient (Baeg, 2004).

Previous ectopic expression studies have shown that Dlp can trap extracellular Wg and prevent activation of the Wg signaling pathway. Because Dlp appears to be a major HSPG required to regulate Wg signaling, its endogenous distribution was examined in the wing imaginal disc using a polyclonal antibody against Dlp and a staining method that primarily detects extracellular proteins. The specificity of the Dlp antibody was confirmed by misexpressing dlp using the ap-Gal4 driver. In the third instar wing imaginal disc, Dlp was detected throughout the disc; however, a significant decrease in the level of Dlp was detectable at the D/V border. This domain of low Dlp expression correlates with the region where high level of Wg signaling is required to induce the expression of short-range target genes. It is noted that since dlp mRNA expression is uniform throughout the disc, the down-regulation of Dlp at the D/V border must occur post-translationally. Interestingly, an optical cross section of the disc has revealed that endogenous Dlp localizes mostly on the basolateral surface of the cell where extracellular Wg is detected. The subcellular localization of Dlp protein was also examined using a GFP-dlp expressed under the control of a Gal4 driver. Consistent with the Dlp antibody result, it was found that GFP-Dlp localizes predominantly to the basolateral membrane. Altogether, these observations suggest that Dlp can bind to extracellular Wg and that Dlp levels need to be reduced for high-level Wg activity in cells near the D/V boundary (Baeg, 2004).

In a genome-wide RNAi screen in S2R+ cells to identify genes that either up- or down-regulate Wg signaling, Dlp was identified as both a negative and positive regulator of Wg signaling under stimulated and nonstimulated conditions, respectively. The cell-based assay devised consists of the activation of a Tcf/Arm-dependent Wg-reporter gene upon induction of S2R+ cells by expressing a wg cDNA by transient transfection. The activity of the Wg pathway and the effect of the addition of various dsRNAs on the pathway were assayed by monitoring Luciferase-reporter activity using a luminescence-based plate reader. Using this assay, the addition of dsRNAs of positive transducers of Wg signaling, such as arm, decrease the Top12X-HS-Luciferase reporter activity, while dsRNAs to negative Wg regulators, such as Daxin, increase its activity. Interestingly, under condition of Wg induction, dlp was found to act as a negative regulator of Wg signaling, since dlp dsRNA led to a twofold increase in luciferase activity. This increase is significant since it is comparable to that of daxin dsRNA. In the absence of Wg induction, dlp was found to positively regulate Wg signaling, since dlp dsRNA leads to a fivefold decrease in luciferase activity, a decrease that is similar to that observed by the addition of arm dsRNA. These results suggest Dlp acts as a positive regulator of the Wg pathway when Wg level is low and negatively influences signaling when Wg is abundant. These results are consistent with in vivo results that demonstrate that Dlp has both a positive and negative role in Wg signaling (Kirkpatrick, 2004). The observations in S2R+ are consistent with the hypothesis that low Dlp levels at the D/V boundary supports high-level Wg signaling, while further away from the D/V boundary where the Wg concentration is lower, Dlp positively influences Wg signaling. Overall, the result from the S2R+ RNAi experiments indicates that (1) Dlp is not an essential component of the Wg signal transduction pathway; and (2) Dlp can either have a negative or positive impact on Wg signaling depending on the level of Wg available. The negative effect of Dlp is consistent with previous in vivo studies that have shown ectopic Dlp expression can trap extracellular Wg and prevent activation of the Wg signaling pathway. Therefore, a reduction of Dlp levels at the D/V border would be expected to contribute to high-level Wg signaling. The positive effect of Dlp in Wg signaling needs to be understood in the context of the previous findings that loss of HSPG activity results in wg loss-of-function phenotypes, as shown by a decrease in dll expression in clones mutant for enzymes involved in GAG biosynthesis. One attractive model is that Dlp would act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with the signal transducing Fz receptors in cells located at a distance from the D/V boundary where low level of Wg is available. Finally, these observations are consistent with a recent study (Kirkpatrick, 2004) that showed that (1) ectopic activation of Wg signaling at the wing margin occurs in dlp mutant tissues, and (2) a cell autonomous reduction in Wg signaling in dlp clones located distal to the Wg-producing cell