Transcriptional regulation

In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. High levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation (Cadigan, 1998).

Dfz2 binds and transduces the Wg signal in cell culture. To examine Dfz2's role in vivo, its expression pattern was examined in the developing wing. In the wing pouch, the region of the disc destined to become wing blade, Dfz2 is expressed in an inverse pattern to that of Wg, with the lowest levels found at the D/V boundary. This pattern is Wg-dependent, since Dfz2 expression near the D/V stripe is derepressed when Wg activity is blocked for 24 hr in wgts discs , as compared to wgts discs at the permissive temperature. To extend these findings, the Gal4/UAS system was used to express deleted versions of two Wg signaling components, Armadillo and dTCF (Pangolin), which constitutively activate (armact) or inhibit (dTCFDN) Wg signaling. Expression of armact throughout the wing pouch represses Dfz2 expression, while expression of dTCFDN in a Patched (Ptc) pattern (i.e., a stripe that runs perpendicular to the D/V wg stripe at the anterior/posterior boundary) leads to derepression of Dfz2 within the Ptc domain. Thus, Wg signaling is responsible for the lower expression of Dfz2 near the D/V boundary (Cadigan, 1998).

To test whether wg-dependent repression of Dfz2 expression is important for normal wing development, Dfz2 was misexpressed using UAS-Dfz2 lines crossed to various Gal4 drivers. This Gal4 driver is expressed in every cell in the wing pouch except those at the D/V boundary, and 1J3-Gal4/UAS-Dfz2 (1J3/Dfz2) expression overwhelms the endogenous graded Dfz2 pattern. Ectopic expression of Dfz2 throughout the wing disc causes an expansion of Wg target gene expression, resulting in "Hairy" wings. All surviving animals have ectopic bristles on their wing blades. These sensory organs are normally only found at the wing margin, the adult structure corresponding to the D/V boundary (bristle formation depends on Wg activity). The ectopic bristles in the anterior compartment are almost always of the slender or chemosensory type, though an occasional stout bristle is also observed. In the posterior compartment, the extra bristles are similar to the noninnervated ones found at the posterior margin (Cadigan, 1998).

The slender and chemosensory bristle cell fates are determined during the third larval instar by proneural genes such as ac, whose expression is wg-dependent. ac is initially expressed at mid-third instar in the anterior compartment in a stripe on each side of the D/V boundary. The cells destined to become bristle precursors gradually accumulate Ac to higher levels than their neighbors. Consistent with the hairy wing phenotype, 1J3/Dfz2 discs have a dramatic increase in cells expressing high levels of Ac. These cells are found at a greater distance from the D/V stripe than in controls and presumably cause the ectopic bristles seen in adult wings (Cadigan, 1998).

In the morphogen model of Wg action in the wing blade, ac is an example of a short-range target, requiring high levels of Wg signaling to be expressed. As was found for ac, the expression domain of another short-range target, Delta (Dl), normally expressed in a narrow stripe on either side of the wg stripe, is much broader in 1J3/Dfz2 wing discs. The model also states that Wg acts directly on long-range targets, such as Dll, which require less Wg signaling for activation and thus are normally expressed in wider domains centered on the D/V boundary. Dll is expressed at highest levels close to the Wg stripe and then at progressively lower levels at further distances. In 1J3/Dfz2 discs, the higher expression levels of Dll are seen much further from the stripe. Thus, misexpression of Dfz2 at high levels throughout the wing pouch expands the domains of both short- and long-range Wg targets (Cadigan, 1998).

The increased activation of Wg targets by misexpression of Dfz2 could be due to a heightened response of the cells to the Wg signal, or a constitutive activation of the signaling pathway. To address this, the effect of Dfz2 misexpression was examined in discs from wgts mutants reared at the restrictive temperature. Both Ac and Dll expression are dramatically reduced in these discs to levels seen in wgts discs grown under the same conditions. This indicates that the primary effect of Dfz2 misexpression is to potentiate the ability of Wg to signal to target cells (Cadigan, 1998).

The 1J3/Dfz2 experiments suggest that Dfz2 can transduce the Wg signal in the wing. Presumably, as has been shown in cell culture, this occurs through direct binding. To examine this in more detail, an altered Dfz2 cDNA was expressed in flies. The cDNA encodes the extracellular domain anchored to the cell surface via a glycerol-phosphatidyl inositol linkage. This is a truncated protein and consequently should not be able to transduce the signal to intracellular targets, since it lacks the seven transmembrane and intracellular domains. Therefore, if Dfz2 and Wg can interact in vivo, GPI-Dfz2 should block Wg signaling by binding the protein nonproductively. Expression of GPI-Dfz2 in the wing pouch does abolish the expression of the Wg targets ac and Dll and causes severe notching of the wings in adults. Experiments in the embryo and eye indicate that GPI-Dfz2 efficiently blocks Wg signaling in these tissues as well. These data are consistent with the hypothesis that Dfz2 is a physiologically relevant Wg receptor (Cadigan, 1998).

Misexpression of Dfz2 alters Wg distribution by increasing its stability. Wg is normally found at high levels in the cells expressing wg RNA but drops off sharply moving away from the stripe. Previously, it has been reported that Wg is undetectable more than 10 cell diameters from the D/V boundary. Using an affinity-purified Wg antibody, it is found that low levels of Wg are still detected up to 25 cell diameters away from the site of secretion. This Wg signal is punctate and favors the apical portion of the epithelium. It is not seen in wgts discs grown at the restrictive temperature, indicating that it is due to Wg and not a cross-reaction artifact. Thus, the physical distribution of Wg is consistent with the genetic evidence that it can directly affect gene expression over long distances (Cadigan, 1998).

Misexpression of Dfz2 or GPI-Dfz2 causes a dramatic posttranscriptional spread of Wg, with 1J3/Dfz2 discs, which overexpress Dfz2, having high levels of Wg several cell diameters away from the RNA stripe. A greater accumulation of Wg is seen with GPI-Dfz2, when compared to Dfz2. This could simply be due to a higher amount of truncated receptor present or caused by the inability of GPI-Dfz2 to internalize Wg after binding. While Dfz2 does not appear to facilitate the diffusion of Wg directly, its ability to protect Wg from degradation can indirectly promote the movement of Wg. Endogenous Dfz2 levels are modified by activating or inhibiting Wg signaling and determined the effect on Wg levels. Expression of dTCFDN in the posterior compartment of wing discs (which blocks Wg signaling) results in derepressed levels of Dfz2 transcripts and accumulation of Wg outside the RNA expression domain. Clones mutant for dishevelled (dsh) activity, which lack Wg signaling, also have a similar accumulation of Wg. Conversely, clones lacking zeste white 3 (zw3), also known as shaggy, which constitutively activate Wg signaling and are predicted to have repressed Dfz2 levels, have less Wg inside them, when compared to surrounding tissue. These results show that Wg signaling has a negative effect on the accumulation of Wg, which can be explained by the ability of Wg to inhibit Dfz2 expression (Cadigan, 1998).

It is proposed that the ability of Dfz2 to stabilize Wg, combined with the Dfz2 expression pattern, plays a major role in shaping the Wg morphogen gradient. Wg concentration initially decreases rapidly moving away from the D/V boundary but then plateaus at a low level. The data support a model where Wg is normally able to travel up to 25 cell diameters away from its source, consistent with genetic data on the range of action of Wg. This diffusion/transport of Wg does not appear to be enhanced by increased Dfz2 levels. However, the distribution of Dfz2 creates a situation where Wg is unstable near the D/V boundary and more stable at further distances. High levels of Dfz2 near the boundary, through expression of a transgene or derepression of the endogenous Dfz2 genes, stabilizes Wg so that elevated levels are observed. Repression of Dfz2 expression away from the boundary, via activation of the Wg signaling pathway, destabilizes Wg, resulting in lower levels found in these cells. Thus, Dfz2-mediated stabilization of Wg can, in large part, explain the biphasic nature of the Wg morphogen gradient. This work raises additional questions regarding Wg function. What is the biochemical basis for the enhanced Wg instability near the site of its production, and what is the basis for the enhanced Wg stability from its site of secretion? (Cadigan, 1998).

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Frizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression, and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during Drosophila development is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch (Wesley, 1999).

A variety of factors could influence how far developmental signals spread. For example, the Patched receptor limits the range of its ligand Hedgehog. Somehow, the Frizzled2 receptor has the opposite effect on its ligand. Increasing the level of Frizzled2 stabilizes Wingless and thus extends the Wingless gradient in Drosophila wing imaginal disks. Here it is asked whether Frizzled or Frizzled2 affects the spread of Wingless in Drosophila embryos. In the embryonic epidermis, the combined expression of both receptors is lowest in the engrailed domain. This is because expression of Frizzled is repressed by the Engrailed transcription factor, whereas that of Frizzled2 is repressed by Wingless signaling. Receptor downregulation correlates with an early asymmetry in Wingless distribution, characterized by the loss of Wingless staining in the engrailed domain. Raising the expression of either Frizzled or Frizzled2 in this domain prevents the early disappearance of Wingless-containing vesicles. Apparently, Wingless is captured, stabilized, and quickly internalized by either receptor. As far as is possible to tell, captured Wingless is not passed on to further cells and does not contribute to the spread of Wingless. Receptor downregulation in the posterior compartment may contribute to dampening the signal at the time when cuticular fates are specified (Lecourtois, 2001).

Both Frizzled and Frizzled2 proteins are expressed in a dynamic fashion during the first 12 h of development. In particular, the level of Frizzled is down in the engrailed domain and Frizzled2 is relatively less abundant in the apparent domain of Wingless action. The patterns of transcription around Stages 8 and 11 (3.5-7 h AEL) were studied. Although frizzled expression is initially uniform during gastrulation, it begins to resolve into a periodic pattern by Stage 9 (4 h AEL). Double staining shows that, at Stage 10 (4.5-5 h AEL), frizzled transcripts are abundant in all cells except those that express engrailed. Expression of frizzled2 also becomes segmental around Stage 9, a pattern that is clearly marked at Stage 10: broad stripes of frizzled2 expression are detected at the posterior of each engrailed stripe. Thus, at Stage 10 (4.5-5 h AEL), combined expression of frizzled and frizzled2 is lowest in engrailed-expressing cells, especially those nearest to the source of Wingless. Note, however, that residual mRNA remains, possibly as a result of maternal contribution or low-level zygotic transcription. In fact, intensive studies support the view that Engrailed directly represses frizzled (Lecourtois, 2001).

At Stage 10 of Drosophila embryogenesis, the amount of detectable Wingless decreases within the engrailed domain. This corresponds to the time when both frizzled and frizzled2 are transcriptionally downregulated there. Artificially increasing the expression of frizzled or frizzled2 prevents the early loss of Wingless staining; binding of Wingless to its receptors may render it inaccessible to extracellular proteases. This suggests that, in the wild type, transcriptional downregulation of the receptors causes the early loss of Wingless immunostaining. Two distinct mechanisms repress the transcription of frizzled and frizzled2: Engrailed itself appears to repress frizzled, whereas Wingless signaling represses frizzled2. Repression of frizzled expression by Engrailed is not seen in imaginal disks where, presumably, a cofactor is missing. In contrast, repression of frizzled2 by Wingless signaling appears to be a general feature. As a result of two distinct repression mechanisms, the combined expression of frizzled and frizzled2 is lowest in the engrailed cells, especially those nearest to the source of Wingless. Nevertheless, residual activity must remain because engrailed-expressing cells respond to Wingless as late as 8.5 h AEL, whereas the complete absence of frizzled and frizzled2 activity phenocopies a wingless null mutation (Lecourtois, 2001).

The results suggest that downregulation of the Frizzled receptors reduce the spread of Wingless into the posterior compartment, not by affecting its transport but rather by reducing its stability. This would lead to a reduced number of effective receptor-ligand complexes and hence dampened signaling. This is thought to commence during Stage 10. Transcriptional repression of receptor expression has been shown to contribute to dampening of signaling in other instances. Additional strategies such as desensitization are also at work. Likewise, additional mechanisms for dampening Wingless signaling are likely to exist. Indeed, after Stage 11, residual Wingless/receptor complexes are rapidly degraded (and hence rendered ineffective) in prospective denticle-secreting cells. This targeted degradation of Wingless can account for the fact that row 1 denticles still form in embryos that massively express frizzled or frizzled2. Both mechanisms of signal downregulation (repression of receptor transcription and degradation of receptor/ligand complexes) dampen the action of Wingless toward the posterior, although more work is needed to assess their relative importance. Another outstanding issue is whether Frizzled and Frizzled2 are equivalent with respect to signal downregulation. Clearly, these receptors differ in terms of affinity for the ligand. It may also be that differences in intracellular trafficking lead to distinct effects on Wingless signal downregulation (Lecourtois, 2001).

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophila embryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. This approach was validated by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Strong evidence is presented showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo (Solano, 2003).

the expression of 14 genes was studied that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability. The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved. frizzled 2 expression is activated in the presence of (VP16-En) and repressed in the presence of En. This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type. This expression is slightly activated in the presence of (VP16-En), but strongly repressed when En is overexpressed, suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type. In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch, whereas no activation of bnl is observed after wild type En overexpression. As expected, because MS1096 drives Gal4 expression only in the wing pouch, endogenous bnl expression outside the wing pouch is not affected, showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc. This expression is activated when (VP16-En) is overexpressed, and repressed upon En overexpression (Solano, 2003).

In conclusion, these data demonstrate that the expression of several potential target genes identified via UV-X-ChIP is modulated when engrailed is misexpressed. This test has been successfully performed on 12 genes of the 14 that were tested (Solano, 2003).

Interactions between engrailed and the wingless signaling pathway have been extensively described. A direct regulation of frizzled receptor expression by engrailed has been documented. The other wingless receptor gene, frizzled2 (fz2), might also be directly regulated by engrailed. A high-affinity Engrailed-binding fragment (1A4) was detected in the close vicinity of the fz2 transcription unit. In wild-type embryos, fz2 expression becomes segmentally repeated around stage 9, in two or three rows of cells just anterior to engrailed. In stage 9 engrailed mutant embryos, fz2 expression is extended posteriorly, being detected in 4 rows of cells. This shows that Engrailed acts as a repressor of fz2 expression in embryos, as has been suggested with the previous test in the wing disc. Whether the 1A4 Engrailed-binding fragment was able to drive the expression of a reporter gene was verified in vivo and whether it responds to engrailed regulation was examined. For this purpose, this 170 bp fragment, either as a monomer or a trimer, was cloned upstream of a GFP reporter gene and hsp70 minimal promoter and introduced into the Drosophila genome by P element-mediated transposition. In these transgenic lines, GFP expression was essentially detected in the embryonic hindgut and in half of the larval hindgut. GFP is expressed in the ventral cells of the larval hindgut that do not express engrailed, which mimics endogenous fz2 expression. This demonstrates that the 1A4 DNA fragment might be a part of endogenous fz2 regulatory sequences. Overexpression of (VP16-En) fusion protein driven by hs-Gal4 leads to ectopic GFP expression in the entire hindgut, but also in tissues that do not express the transgene in wild type, such as the midgut, the salivary glands, and the wing disc. Overexpression of (VP16-En) fusion protein driven by en-Gal4 in embryos leads to ectopic GFP expression in a striped pattern. Such activation does not occur with overexpression of wild-type Engrailed, confirming a repressor role of Engrailed on fz2 expression through this 1A4 fragment. These results show that 1A4 is able to respond to engrailed regulation in vivo. Altogether, these data show that the 1A4 fragment that was isolated by UV-X-ChIP is a part of the fz2 regulatory regions and is able to directly respond to engrailed regulation in vivo (Solano, 2003).

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. A chromatin immunoprecipitation (ChIP)-chip strategy has been developed for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. Use of ChEST in Drosophila is described to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. The approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome (Junion, 2005).

To predict Dmef2-dependent CRMs, the Drosophila genome was scanned for modules containing one of the three previously described in vivo-acting Dmef2 binding sites. Because muscle differentiation events are controlled by the synergistic action of MADS-box (Mef2 family) and E-box (Twist and MyoD bHLH family) factors modules were sought containing Mef2- and E-box-binding sites. This scanning procedure led to the identification of ~1,243 potential Dmef2-binding CRMs, from which 99 modules were selected, amplified, and spotted to produce a computed Dmef2-CRM array. Three of four previously described Dmef2-CRMs, located in the vicinity of Paramyosin, Act57B, and Dmef2, were identified during genome scanning and selected for spotting as positive control. The fourth in vivo-acting Dmef2-CRM located close to b3-tubulin did not come out from the screen because of the high number of E-box sites used as the scanning criterion. The CRMs located in the vicinity of genes that are not expressed in the mesoderm or genes of unknown expression and function (some of CGs) were rejected. In parallel, X-ChIP was used to isolate DNA fragments to which Dmef2 binds in vivo. ChIP-DNA immunoprecipitated either with Dmef2 antibody or with nonimmune serum was then labeled and used to probe the Dmef2-CRM array. The three previously described in vivo-acting CRMs were found enriched in ChIP-DNA immunoprecipitated with Dmef2 antibody. Importantly, numerous other in silico-identified Dmef2-CRMs were enriched in ChIP-DNA, thus demonstrating the efficacy of the ChEST method (Junion, 2005).

Of the 99 in silico-predicted Dmef2-CRMs, 62 were enriched in the DNA immunoprecipitated with anti-Dmef2 antibody. The CRM-associated Dmef2 targets included genes expressed in all muscle cell types, in which Dmef2 has previously been reported to function. In addition to expected candidates encoding fusion (Lmd, Hibris) and structural (Ket, Pod1) muscle proteins, a large number of CRM-associated Dmef2 target genes coded for TFs and signal transduction proteins. For example, CRMs upstream of Fz2 and within the introns of Ci and Pan indicate a potential role of Dmef2 in transcriptional regulation of genes transducing to the mesodermal cells ectodermal Wg and Hh signals, whereas CRMs within the introns of If and Pka-C3 suggest that by regulating transcription of these genes Dmef2 is involved in the attachment of muscle fibers and in fiber contraction, respectively. In some cases (e.g., Kettin, NetB, N-cad), several Dmef2-dependent CRMs were mapped in the vicinity of the same gene, highlighting the complexity of transcriptional regulation in which Dmef2 is involved. Analysis of the position of CRMs in relation to adjacent genes revealed that the majority of ChIP-enriched modules are located upstream (42%) or within the introns (39%) of target genes. In these two categories, the most frequent positions of Dmef2-CRMs appear between 1 and 5 kb upstream of the gene and within the first intron (Junion, 2005).

To determine whether the ChEST-identified DNA fragments are able to act as regulatory modules in vivo, ten Dmef2-CRMs by reporter gene transgenesis were tested. Nine of 10 CRMs were found to drive reporter gene expression in Dmef2-positive muscle cells. In all transgenic Drosophila lines, lacZ reporter expression at least partially reproduced endogenous gene expression, indicating that the identified CRMs were bona fide enhancers of adjacent genes. For example, the Ket-1, Ket-2, and Ket-3 CRMs laying in the vicinity of the kettin gene, which encodes a giant muscle protein required for the formation and maintenance of normal sarcomere structure, were found to drive lacZ expression in distinct subsets of differentiating body wall muscles. These data indicate that the pan-muscular expression of kettin is regulated in a muscle-type-specific manner, and by multiple Dmef2-binding enhancers. Interestingly, four other analyzed CRMs located within the introns of N-cad and acon and upstream of fz2, sfl, and Meso18E also drive lacZ expression in discrete subsets of somatic muscle precursors. The muscle-type-restricted activity of these modules suggests that both CRM regulators (Dmef2, E-box factors) and their target genes are involved in different aspects of muscle precursors diversification, including muscle fiber shape and axial positioning. Alternatively, the observed muscle-type-specific expression of lacZ may result from the limited size (250-300 bp) of genomic sequences tested. In embryos carrying a Dmef2-dependent CRM found in the intron of If, lacZ expression is detected in a group of ventrolateral muscles. This lacZ pattern correlates with distribution of endogenous If, which accumulates at the extremities of ventrolateral muscle fibers and is required for their correct epidermal insertion. The reduced level of target gene expression in Dmef2 mutant embryos provides an additional support for Dmef2-dependent in vivo regulation of ChEST-identified CRMs (Junion, 2005).

A regulatory receptor network directs the range and output of the Wingless signa>

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg [Arrow (Arr) and Frizzled 2 (Fz2)] are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. This study analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. The experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by an in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, a 200 bp regulatory element in the arr locus was identified that can account for the Arr gradient, and it was shown that this is indirectly negatively controlled by Wg activity. Finally, the role of Frizzled 3 (Fz3) in was analyzed this system, and its expression, which is induced by Wg, was found to contribute to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, these results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system (Schilling, 2014).

During the development of a metazoan organism, signaling events are precisely regulated. One frequently employed mode of regulation is feedback loops. This study analyzed a network of feedback loops in the Drosophila wing pouch that regulate receptor abundance, and thus the range of distribution and signaling output of Wg (Schilling, 2014).

Receptors sequester their ligands and, thereby, impact upon the range of the signal. A transcriptional regulatory link between receptor expression and signaling activity, causing up- or downregulation of receptor levels in cells in response to the signal, can thus restrict or extend the signaling range. For example, the Hedgehog (Hh) signal induces the expression of its receptor Patched (Ptc), a regulatory link which severely narrows the Hh activity. In the case of Wg, this study observed the opposite. Wg signaling appeared to extend the range of Wg distribution by transcriptionally downregulating expression of arr and fz2; downregulation of the receptors hinders the formation of Wg-Arr-Fz2 complexes. This allows superfluous Wg to diffuse further away from the source without being immobilized by its receptors. In agreement with this notion, a slightly narrower distribution was observed of extracellular Wg in discs that expressed Fz2 or Arr under the tubulinα1 promoter. Quantifying these observations in discs that compartmentally overexpressed the receptor, a subtle reduction of the decay length (corresponding to a slightly steeper Wg distribution) was observed in compartments that overexpressed the receptor compared with that in wild-type compartments (Schilling, 2014).

In apparent contradiction, a previous study has shown that high levels of Fz2 can stabilize Wg and promote Wg spreading; accordingly, this study observed an accumulation of Wg when repeating this experiment by overexpressing Fz2 using the GAL4-UAS system. These contradictory findings can be reconciled by taking into account the different strength of Fz2 upregulation in the two experimental setups -- Fz2 expression that is driven by the tubulinα1 promoter leads to a relatively mild upregulation of the receptor by, approximately, a factor of 2, whereas overexpression by using the GAL4-UAS system causes a much stronger overexpression. Presumably, Arr becomes the limiting factor in UAS-Fz2-overexpressing cells, a situation that might prevent the surplus Wg-Fz2 complexes from being internalized, thus causing an extracellular accumulation of Wg. If Fz2 is only moderately overexpressed, sufficient Arr protein might be available to allow this extra Fz2 to form Wg-Arr-Fz2 complexes, which are subsequently internalized, leading to a slight narrowing of the gradient because there is less free and diffusible Wg. Consistent with this notion, simultaneous strong overexpression of both of the receptors Fz2 and Arr, by means of Gal4, leads to a reduction of extracellular Wg levels (Schilling, 2014).

Although Wg signaling transcriptionally represses both Arr and Fz2, ubiquitous overexpression of Arr, or Fz2, had no phenotypic consequences. Unexpectedly, however, severe phenotypes arose upon mosaic expression of the tub>arr or tub>fz2 transgenes. Theoretical modeling and reporter gene analysis indicated that cells that had elevated receptor levels ectopically activated the pathway when situated close to wild-type cells. Apparently, the 'high-receptor-level state' allows tub-fz2 or tub-arr cells to engage in ligand-receptor interactions that depend on the 'low-receptor-level state' of their neighbors. One plausible explanation might be that tub-fz2, or tub-arr, cells bind to Wg that diffuses in from neighboring wild-type cells (Schilling, 2014).

The different outcome of clonal versus uniform alteration of the Wg pathway is reminiscent of observations that have been reported by Piddini and Vincent (2009), where loss of Wg signaling in the entire P compartment had no impact on the expression of low-threshold target genes but resulted in their repression, and in patterning defects, when Wg signaling was only clonally abolished. Piddini and Vincent also used different patterns of Wg receptor expression for their experiments, and they explained their findings by postulating that there is a Wg-induced, still to be identified, inhibitory signal that negatively regulates target gene expression in surrounding cells (Schilling, 2014).

In an additional layer of negative-feedback regulation in the wing pouch, Wg signaling activates the expression of the Frizzled family member Fz3. Fz3 seems to act as a negative regulator of Wg signaling by repressing Wg signaling readouts and downregulating Wg receptor levels. Various models could be envisaged of how Fz3 acts as an inhibitor of Wg signaling. As it has been demonstrated that Fz3 is able to bind Wg, Fz3 could work as a decoy receptor that acts as a molecular trap by binding to Wg without eliciting a signal. Decoy receptors are often part of negative-feedback mechanisms. In the Drosophila epidermal growth factor (EGF) system, the pathway inhibitor Argos is a target of EGF signaling and functions as a decoy receptor. In vertebrates, decoy Frizzled receptors have been identified that modulate Wnt signaling - secreted Frizzled-related molecules (sFRPs) have strong homology to the Frizzled extracellular domains. sFRPs inhibit signaling by directly binding to the Wnt ligands. No sFRP gene has been identified in the Drosophila genome (Schilling, 2014).

In another scenario, Fz3 could work as a negative regulator of Wg receptors. Its function could be analogous to that of ZNRF3 and RNF43 in crypt base columnar intestinal stem cells. These related E3 ubiquitin ligases have been shown to regulate the stability and levels of cell-surface Fz and LRP5/6, through internalization and lysosomal degradation of the receptor components in the presence of Wnt signaling. Several of the current experimental findings indicate that Fz3 might work as an inhibitor of Wg feedback at the receptor level - firstly, decreased Arr and Fz2 levels were observed in compartments that overexpressed Fz3, and secondly, Arr and Fz2 levels were increased in fz3 mutant wing discs. Most probably, Fz3 acts by more than one mechanism - cells that overexpressed Fz3 in the Wg stripe lead to Arr downregulation, whereas cells that overexpressed Fz3 outside of the Wg stripe lead to Arr upregulation. Furthermore, extracellular Wg was stabilized upon Fz3 overexpression. In a wild-type situation, this stabilization of Wg might contribute to a broader Wg gradient and promote signaling in the outskirts of the wing pouch. Taken together, these findings suggest that only Wg-bound Fz3 causes inhibition of the pathway (Schilling, 2014).

The post-translational regulation of Wg receptor levels was not the focus of this study, but substantial efforts were undertaken to further characterize the transcriptional regulation of the receptor genes. In particular, attempts were mead to identify the regulatory elements of these genes that mediated the feedback loops. The isolation of a 200 bp fragment of the arr locus and a 300 bp fragment of the fz3 gene (each of which was responsive to Wg signaling and drove reporter gene expression in a pattern that was reminiscent of the endogenous expression pattern) allowing an investigation of whether the Wg pathway controls these genes directly or indirectly; fz3 appeared to be a direct target of canonical Wg signaling, whereas arr did not. Pan-binding sites were dispensable in the minimal arr enhancer, indicating that either Arm regulates the transcriptional activity of arr through another DNA-binding protein, or that Arm and/or Pan transcriptionally induce one (or more) negative regulators that, in turn, regulate arr expression. Hence, although the Wg pathway has been reported to possess the capacity to directly negatively regulate transcription, it apparently does not use this mechanism to attenuate arr expression (Schilling, 2014).

Including transcriptional Wg receptor downregulation in the model led to a broader distribution of Wg - receptor downregulation by ligand-induced endocytosis consumes the ligand, this was not the case for transcriptional repression. The broadening of the Wg distribution area under a mechanism of transcriptional receptor repression might facilitate a robust signaling readout for high-threshold Wg target genes (Schilling, 2014).

A recent study indicates that the long-range Wg gradient might be less important for imaginal disc patterning than assumed previously. Hence, it is also conceivable that the receptor gradients are not essential, a notion supported by the finding that uniform misexpression of Arr or Fz2 in the wing imaginal disc had no phenotypic consequences. Nevertheless, it remains to be determined whether the Arr and Fz2 gradients are dispensable; the tubArr transgene is not able to rescue arr loss-of-function mutants (Schilling, 2014).

So far, most quantitative models of the Wnt-Wg pathway have focused on intracellular events, and only a few models have taken into account the spatial aspects of this signaling system. The model in this paper is the first to systematically study the roles of Wg-receptor complexes -- Wg-ArrFz2 and Wg-Fz3 -- in the spatial profile of Wg signaling, as well as being the first to be challenged experimentally by manipulations of the receptor levels. The cell-based modeling approach of ligand receptor interactions allowed varying of all parameters in a cell-autonomous manner, which has not been done in previous studies. This technique is, thus, an ideal tool to predict the impact of clonal conditions with cellular precision, which have historically formed the basis of experimental approaches in Drosophila but have also become increasingly available in vertebrates (Schilling, 2014)

Protein Interactions

Dishevelled acts in both wingless and frizzled signaling. It is interesting to note that many frizzled proteins, but not Frizzled itself contain an S/T-X-V motif at their C-terminal ends: this motif has been shown to interact with PDZ (or DHR) domains in a variety of proteins (Gomperts, 1996). DSH contains a PDZ domain, suggesting a direct interaction of DSH with FZ2 (Klingensmith, 1994).

Frizzled homologs have an extracellular cysteine-rich domain (CRD). This is joined to the transmembrane domain by a variable linker. The CRD may constitute part or all of the ligand-binding domain. Strong surface staining is detected when a truncated form of FZ2 carrying an isolated CRD segment and part of the linker region is displayed on the surface of cultured cells and incubated with Wingless and anti-WG antibodies. This provides evidence that the CRD constitutes a significant part of the WG binding domain (Bhanot, 1996).

To examine the question of ligand-receptor specificity in the Wingless-frizzled system, the ability of Wingless to bind six mammalian frizzled sequences was examined. Cells transfected with fz2, human fz5 or mouse fz4, fz7 and fz8 bind wingless, while transfection with mouse fz3 and fz6 do not confer WG binding. CRD-deleted mouse fz4 also does not confer WG binding (Bhanot, 1996).

Wingless (Wg) treatment of the Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Frizzled2. However, a line of S2 cells obtained from another source express both Frizzled-2 and Frizzled. Thus, this cell line was designated as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and Shotgun protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induces the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells is phosphorylated as a consequence of expression of Frizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells, but theycan grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling (Yanagawa, 1998).

Thus expression of Dfz2 or Mfz6 induces phosphorylation of Dsh in S2 cells and a small proportion of Dsh protein is phosphorylated in S2R+ and clone 8 cells. These results suggest that expression of frizzled family proteins induces the basal phosphorylation of Dsh. In this regard, Casein kinase 2 (CK2), which binds to the PDZ domain of Dsh, is known to be the major kinase responsible for phosphorylation of Dsh upon Dfz2 overexpression in S2 cells. Therefore, CK2 may take part in the basal phosphorylation of Dsh in Dfz2/S2, Mfz6/S2, clone 8 and S2R+ cells not stimulated with soluble Wg. In addition, Frizzled overexpression leads to translocation of Dsh from cytoplasm to plasma membrane. Overexpression of rat frizzled-1 has been shown to result in recruitment of Xwnt-8 and XDsh to the plasma membrane in Xenopus embryos. Thus, it is possible that Dfz2 or Mfz6 expression induces translocation of at least a part of Dsh to the plasma membrane in S2 cells and that this Dsh translocation in some way stimulates Dsh phosphorylation by CK2. However, it is not clear whether CK2 also participates in Wg-induced hyperphosphorylation of Dsh or whether other kinase(s) are activated by the binding of Wg to Dfz2 in clone 8, S2R+, and Dfz2/S2 cells and that these other kinases induce the hyperphosphorylation. In view of the reports indicating association (probably indirect) between frizzled family proteins and Dsh and the binding of Dsh to CK2, it is attractive to speculate that Wg binding induces aggregation of Fz2 receptors, which, in turn, brings the Dsh-CK2 or other kinase complexes close together, and this aggregation stimulates the Dsh phosphorylation by CK2 or other kinases in these Dsh-kinase complexes. This could explain how Wg induces Dsh hyperphosphorylation in clone 8, S2R+, and Dfz2/S2 cells. However, it is noteworthy that Dfz2 overexpression leads to marked phosphorylation of Dsh, but not to elevation of Arm, in S2 cells, indicating that phosphorylation of Dsh, at least by Dfz2 overexpression, cannot activate the Wg signaling pathway by itself. Clearly, further detailed experiments are necessary to evaluate the function of Dsh phosphorylation in Wg signaling (Yanagawa, 1998 and references).

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, biologically active Xenopus Wnt8 (XWnt8) production by Drosophila S2 cells is reported. Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 mg/liter and 0.5 mg/liter, respectively. The frizzled family of Wnt receptors all have at the amino terminus a conserved extracellular cysteine-rich domain, referred to as the cysteine-rich domain (CRD), that spans 120 amino acids and contains 10 invariant cysteines, followed by seven putative membrane spanning domains. Conditions also are described for the production in 293 cells of an IgG fusion of the CRD of mouse Frizzled 8 with a yield of 20 mg/liter. The use of these proteins is described for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is ~9 nM (Hsieh, 1999).

To begin to define the regions of the CRD that mediate Wnt binding, the binding of XWnt8-AP to live cells that had been transfected with a series of 23 insertion mutants in the CRD of Dfz2 was measured. Insertional mutagenesis was performed with a three-codon cassette that codes for gly-ser-gly, an insertion that would be predicted to sterically disrupt binding if located at any point on the ligand-binding surface. Failure to bind XWnt8-AP also could reflect a defect in the folding or stability of the CRD. To minimize the probability that the tripeptide insertion might cause a structural defect, the 23 insertions were placed at least one amino acid from any cysteine and within those regions that are most hydrophilic. Given the predicted flexibility of the gly-ser-gly tripeptide, it is presumed that most insertions within surface loops would be compatible with a correctly folded CRD. Each of the 23 insertion mutants was studied in the context of the full-length Dfz2 protein and in a CRD-myc-GPI construct. Of interest, all 23 mutants resemble wild-type Dfz2-CRD-GPI: they show robust cell surface immunostaining and significant release of the low mobility electrophoretic species on PI-PLC treatment (corresponding to 10%-30% of the total Dfz2-CRD protein), indicative of a high degree of plasma membrane localization. On the assumption that passage through the endoplasmic reticulum-Golgi-plasma membrane pathway reflects correct folding, this result implies that the native CRD structure can tolerate insertions at each of these positions. This observation raises the possibility that the CRD may be a relatively extended structure with a high surface to volume ratio (Hsieh, 1999).

Different Dfz2 insertion mutants differ markedly in XWnt8-AP binding when assayed on the surface of live transfected cells. Mutants 15 and 18 (130GSG131 and 144GSG145) bind XWnt8-AP when they are present in the context of the full-length Dfz2 sequence but fail to bind as GPI-anchored CRDs, suggesting the possibility that stabilizing interactions may occur between the membrane-embedded domain of the Frizzled protein and the CRD or the CRD-Wnt complex. Among the Dfz2CRD-GPI mutants, 5 bind with a strength indistinguishable from that of the wild type, 4 bind weakly, and 14 fail to bind. Binding and nonbinding mutants are distributed throughout the length of the CRD with nonbinding mutants showing significant clustering (Hsieh, 1999).

In Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and Frizzled2 (Fz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately, suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Overexpression of Fz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz is able to substitute for Dfz2 only under conditions where the Wg ligand is present in excess. The wgPE2 mutant phenotype is also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. It is concluded that wild-type Wg signaling requires efficient interaction between ligand and the Fz2-proteoglycan receptor complex to promote the naked cuticle cell fate (Moline, 2000).

The wgPE2 allele contains a single amino-acid substitution in the carboxyl terminus of the molecule, changing Val453 to Glu. Unlike wgCX4 loss-of-function mutants, which produce a cuticle pattern lacking both naked cuticle and denticle diversification, wgPE2 mutants lack only naked cuticle and secrete an essentially wild-type array of denticle types in each segment. This pattern also differs from that of reduced wg expression levels. Df(2)DE disrupts the wg promoter and results in low-level expression of wild-type wg RNA. These hypomorphic mutants produce small patches of naked cuticle in addition to a diverse array of denticles. Since this pattern is distinct from that of the wgPE2 mutants, the wgPE2 pattern defect appears to represent a qualitative rather than a quantitative change in Wg activity levels (Moline, 2000).

The receptors Fz and Dfz2 are thought to function redundantly in embryonic Wg signaling because neither mutation alone produces a pattern defect, but double mutant embryos phenocopy wg loss of function. Nevertheless, it was found that they do not function equivalently, with respect to the wgPE2 mutant phenotype. Overexpression of wild-type Dfz2, but not fz, rescues naked cuticle specification in wgPE2 mutant embryos. Expression of a UAS-Dfz2 transgene under the control of a prd-Gal4 driver promotes proper naked cuticle secretion in odd-numbered segments, where the transgene is expressed, whereas unaffected even-numbered segments remain mutant. These effects are not an indiscriminate consequence of raising the activity level of Wg. Driving ubiquitous Fz2 overexpression with E22C-Gal4 or arm-VP16-Gal4 has no effect on epidermal patterning in wg null mutant embryos. Furthermore, the pattern produced by the hypomorphic allele Df(2)DE is not rescued by overexpression of Fz2. Overexpression of Fz does not rescue the wgPE2 phenotype, even though roughly equivalent levels of protein product are produced by both transgenes. This suggests that the wgPE2 mutant phenotype reflects a specific problem in activation of the endogenous Fz2 receptor. Furthermore, Fz function can not account for the denticle diversity that is present in wgPE2 mutants. No effect on denticle diversity was seen when maternal and zygotic fz gene product was removed from wgPE2 mutants (Moline, 2000).

Wg signaling results in stabilization of Armadillo (Arm) protein, which activates Wg target genes, such as engrailed (en). Wild-type embryos show broad stripes of intense Arm staining centered over the wg-expressing cells. No striped increase in Arm staining is detected in wgPE2 mutant embryos; only membrane-associated Arm is detected in these embryos, as in wg null mutants. Nevertheless, wgPE2 mutants retain almost wild-type levels of en expression throughout development, whereas wg null mutants lose all epidermal en expression by stage 10. Thus, the wgPE2-encoded ligand is able to maintain en expression and promote denticle patterning, but it does so without stabilizing detectable amounts of Arm. This suggests either that amounts of Arm below the level of detection suffice for some Wg functions, or that Arm is not directly required for those functions (Moline, 2000).

Restoration of naked cuticle in wgPE2 mutant embryos, by prd-Gal4-driven expression of Fz2, correlates with stabilization of Arm in odd-numbered segments. No Arm elevation is observed when fz is overexpressed, nor when Fz2 is overexpressed in Df(2)DE mutant embryos, consistent with the lack of naked cuticle specification in such embryos. Furthermore, prd-Gal4-driven Fz2 expression restores a normal width to en expression domains and corrects defective tracheal pit morphogenesis in odd-numbered segments of wgPE2 mutant embryos, suggesting that all aspects of the wgPE2 mutant phenotype are rescued by Fz2 overexpression (Moline, 2000).

When ubiquitously expressed in a wild-type embryo, wgPE2 subtly changes the denticle pattern and shows a slight dominant-negative effect on naked cuticle formation. This contrasts with ubiquitous expression of wild-type wg, which produces uniform naked cuticle. Ubiquitous expression of wgPE2 in a wg null mutant embryo rescues denticle diversity, but does not significantly rescue naked-cuticle formation. However, coexpression of Fz2 and wgPE2 in wg null mutants produces uniform naked cuticle, as does ubiquitous expression of wild-type wg alone. Thus, the ability of wgPE2 to generate the naked-cuticle cell fate depends on overexpression of Fz2. A slight interaction was also detected with Fz under conditions of high-level coexpression, suggesting that amounts of Wg in excess of physiological concentrations permit interaction with Fz receptor (Moline, 2000).

Indeed, this observation offers an explanation for the apparent genetic redundancy of Fz and Fz2 in embryonic Wg signaling. In the absence of zygotic Fz2 receptor, Wg protein may accumulate to a level sufficient to activate Fz receptor, which then promotes normal epidermal patterning. An increased accumulation of Wg protein was detected in embryos zygotically deficient for Fz2, compared either with wild-type embryos or embryos maternally and zygotically deficient for fz. This suggests that Wg ligand is not internalized and degraded as efficiently when Fz2 is absent from the cell surface, thereby permitting interactions with Fz that are not relevant under wild-type conditions. Abnormal accumulation of Wg protein has also been observed in wgPE2 mutant embryos, which similarly show a broader and less punctate pattern of Wg antibody staining. This staining pattern is restored to a more wild-type appearance by overexpressing Fz2 in wgPE2 mutant embryos, further supporting the idea that the wgPE2 lesion compromises interaction with the Fz2 receptor (Moline, 2000).

It is curious that ectopic Fz2 restores the interaction with wgPE2 ligand, whereas ectopic wgPE2 alone does not. This may indicate either that endogenous levels of Fz2 are limiting for naked-cuticle specification or that overproduction of Fz2 saturates a modification system that regulates its interaction with Wg, and with which the wgPE2 mutant molecule has a defective interaction. For example, glycosaminoglycans have been shown to be required for efficient Wg signal transduction, and the Drosophila glypican encoded by dally appears to act as a co-receptor in the Fz receptor complex. Therefore, the possible involvement of proteoglycans in the wgPE2-Fz2 interaction was examined (Moline, 2000).

In wgPE2 mutant embryos that are zygotically mutant for either dally or sugarless (which encodes an enzyme involved in polysaccharide synthesis), a substantial expanse of naked cuticle is produced. Both mutations are hypomorphic, semi-lethal P-element insertions that do not affect embryonic patterning in the context of wild-type Wg. Therefore, mild reductions in sugar modification suffice to restore functionality to the wgPE2 mutant ligand. Moreover, ectopic expression of dally, using a hs-dally transgene, worsens the wgPE2 mutant phenotype. These effects are specific for the wgPE2 phenotype: the hypomorphic Df(2)DE phenotype is not affected by zygotic loss of sugarless or dally and is partially suppressed, rather than enhanced, by providing ectopic dally. Thus, excess Dally improves signaling efficiency for low levels of wild-type Wg, as has been demonstrated for other hypomorphic wg phenotypes, but has the opposite effect on the partial signaling activity of wgPE2 (Moline, 2000).

Finally, overexpression of dally reverses the rescuing effect of overexpressing Fz2 in wgPE2 mutants. This suggests that ectopic Fz2 expression allows interaction with the mutant ligand because it shifts the ratio of Fz2 to Dally molecules at the cell surface, presumably increasing the number of Fz2 receptor complexes that lack Dally co-receptor and that are therefore, as free receptors, able to bind the mutant Wg ligand. As the wgPE2 genetic lesion changes an uncharged valine to a negatively charged glutamic acid, it is conceivable that introduction of a negative charge in the carboxyl terminus prevents proper binding between Wg ligand and negatively charged sulfated sugar groups (Moline, 2000).

In conclusion, it is proposed that interactions between Wg and proteoglycans are required for promoting naked-cuticle specification, but not denticle diversification, and that wgPE2 cannot promote this high-level response because of abnormal interactions with proteoglycans. It is further concluded that the Fz receptor is able to substitute for Fz2 under conditions of excess Wg ligand, but under normal circumstances, does not appear to have a major role in transducing the naked-cuticle cell fate (Moline, 2000).

Wg/Wnt signal can be transmitted through Arrow/LRP5,6 and Axin independently of Zw3/Gsk3ß activity

Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).

The prevailing view on Wnt reception states that Arrow/LRP5,6 function as coreceptors together with Frizzled proteins. It is well established that Frizzled proteins bind Wnt ligands and that this interaction is essential for Wnt signal transduction. Initial work on LRP6 extended this model in suggesting that Wnt provides a bridging function in assembling a complex of Frizzleds and Arrow, at least for the particular combination mFz8/mWnt-1/mLRP6. However, biochemically, such complex formation has also not always been confirmed. In addition, the functional significance for signaling of the observed ternary complex has not been demonstrated in vivo. Therefore an experiment was designed that tested whether, in vivo, physical proximity of Arrow and Frizzled-2 is sufficient for signaling. In fact, it was found that Frizzled-2 can initiate ligand-independent signal transduction. The constitutive activity of the Fz2-Arr[intra]chimeric protein is significant, since only expression of the fusion protein but not expression of the individual components (Fz2 and Arr[intra]) activate signal transduction. It is inferred that association of Frizzled2 with the Arrow C terminus is indeed a key step in signal initiation in vivo, and that the proximity afforded here by protein fusion also occurs during normal signaling. The Fz2-Arr[intra] chimera is uncoupled from the need for ligand to trigger the intracellular signal transduction cascade. Therefore, whether the Arrow extracellular domain participates in a true 'reception' complex with Fz2 in Wg binding cannot be addressed. Nevertheless, Arrow, or at least its C terminus, likely interacts intimately with Fz2 during signal initiation at the membrane. In addition, activation of the pathway by the Fz2-Arr[intra] chimera proceeds through canonical pathway components, most notably requiring Disheveled, a result consistent with the finding that Dsh functions downstream of Arrow. In cultured vertebrate cells, one report has suggested that in some circumstances, LRP6 can induce Wnt signal transduction independently of Disheveled. In contrast, the experiment of overexpressing biologically active Arrow cytoplasmic sequences in the form of the Fz2-Arr[intra] chimera revealed a strict Dsh dependence, suggesting instead an obligate role for the Dsh protein at signal initiation by Arrow, and by extension, presumably by vertebrate LRP5,6 (Tolwinski, 2003).

Though the Fz2-Arr[intra] chimera clearly signals, it is not as active as a Wg-stimulated endogenous receptor complex. Presumably, and not surprisingly, the protein fusion will present a distorted topology to cofactors required in the signal initiation complex, and therefore is not optimally configured for initiating signal transduction. This may explain why the chimera retains some measure of reliance on endogenous Arrow, as is apparent from a reduced level of signaling in its absence (Tolwinski, 2003).

In summary, Arrow and the Frizzled family of Wnt receptors function in a protein complex that triggers the intracellular signaling cascade. By binding to and causing a reduction in steady-state levels of Axin, Arrow provides a pivotal link between the receptor complex on the cell surface and the downstream events that control Arm activity. One consequence of Axin degradation may reflect its role as a scaffold for Zw3-mediated degradation of Arm. However, because zw3- embryos still respond to Wg input though they fail to degrade Arm, regulation of the degradation complex cannot be the only target of Wg signaling. A Zw3-independent branch in the Wg pathway is proposed, one that might regulate the release of Armadillo from Axin, resulting in nuclear accumulation and signaling (Tolwinski, 2003).

Self-enhanced ligand degradation underlies robustness of morphogen gradients

Morphogen gradients provide long-range positional information by extending across a developing field. To ensure reproducible patterning, their profile is invariable despite genetic or environmental fluctuations. Common models assume a morphogen profile that decays exponentially. Exponential profiles cannot, at the same time, buffer fluctuations in morphogen production rate and define long-range gradients. To comply with both requirements, morphogens should decay rapidly close to their source but at a significantly slower rate over most of the field. Numerical search has revealed two network designs that support robustness to fluctuations in morphogen production rate. In both cases, morphogens enhance their own degradation, leading to a higher degradation rate close to their source. This is achieved through reciprocal interactions between the morphogen and its receptor. The two robust networks are consistent with properties of the Wg and Hh morphogens in the Drosophila wing disc and provide novel insights into their function (Eldar, 2003).

Wg is produced by two cell rows in the dorsoventral boundary of the wing disc. The shape of the Wg gradient is affected by interactions with its principle receptor, Fz2. Overexpression of Fz2 increases the net levels of Wg, as judged by whole-disc Western blots, and leads to accumulation of Wg on the surface of the overexpressing cells. It was also reported that Fz2 expression is repressed at regions of high Wg signaling. Indeed, those two features, Wg stabilization by its receptor and downregulation of receptor expression by Wg signaling, characterize the Wg-like class of robust networks identified in a numerical screen (Eldar, 2003).

Stabilization of Wg by Fz2 could be due to passive protection, namely by sequestration of the receptor-bound Wg from degradation. Alternatively, it could also stem from active interference of Fz2 with the degradation of free Wg, e.g., by sequestering or inhibiting a putative protease. This analysis makes a clear distinction between those two alternative mechanisms: in all networks assigned to the Wg-like class, the receptor reduces free Wg degradation through active stabilization, which is required for achieving robustness. Previous reports, however, attributed Wg stabilization to its passive protection by Fz2 (Eldar, 2003).

A central issue is how to examine experimentally the involvement of active ligand stabilization, since a graded morphogen profile is obtained in the presence of either active stabilization or passive protection. Computer simulation was used to examine the expected Wg accumulation upon ectopic expression of Fz2 in a stripe that is perpendicular to the rows of Wg-expressing cells. This setup allows for a direct comparison of the levels of free Wg within the stripe to those in the adjacent cells. In the case of passive protection, the distribution of free ligand outside the ectopic stripe is the same as that within the stripe. Indeed, under steady-state conditions, the flux of dissociated ligand is precisely balanced by the flux of associated ligand. In contrast, in the case of active stabilization, the diffusion length of the free ligand is enhanced by the presence of receptor, allowing it to move further inside the stripe. Moreover, an asymmetry in free ligand level is generated between the stripe and the adjacent regions, leading to a net flow of ligand from the stripe, resulting in a wedge-like distribution of free ligand that peaks at the center of the ectopic stripe. Note also that conversely, if receptor-mediated endocytosis is a major factor in ligand degradation, the diffusion length of free ligand is in fact reduced in the presence of receptors, leading to an inward flow of ligand from the external regions. Importantly, since free and receptor-bound Wg are at equilibrium, altered diffusion length of free Wg within the stripe is reflected also in the distribution of receptor-bound Wg, which is significantly easier to detect experimentally. It has also been verified that the wedge-like shape in Wg distribution is unique to the model of active stabilization and does not appear as a transient state in the other cases (Eldar, 2003).

The distribution of receptor-bound Wg within the stripe of ectopic receptor expression can thus be used to test if Wg is actively stabilized by its receptor. Previous experiments examined situations of high receptor levels, by ectopically expressing the extracellular domain of Fz2, anchored to the cell surface via a glycerol-phosphatidylinositol linkage (GPI-Fz2). A non-cell-autonomous increase in free Wg was observed, reflected by an elevation in endocytotic Wg vesicles in cells adjacent to the GPI-Fz2-expressing cells. Those experiments, however, led to the stabilization of Wg throughout the pouch and were thus not sufficient for elucidating the pattern of Wg distribution. To generate lower levels of Wg stabilization, ectopic receptor expression was induced in a stripe perpendicular to normal Wg expression using the intermediate-level driver dpp-Gal4. An accumulation of Wg within the GPI-Fz2 stripe which displayed a clear wedge-like pattern was observed. Neither the wedge-like shape of Wg accumulation within the stripe nor the non-cell-autonomous increase in Wg seen previously is consistent with the receptor passively protecting the bound Wg from degradation but all results point to the involvement of active stabilization of the free Wg. These results are thus consistent with the theoretical proposal that Fz2 plays an active role in stabilizing free Wg (Eldar, 2003 and references therein).

It should be noted that, since GPI-Fz2 functions as a dominant-negative receptor (Rulifson et al., 2000), the current results are also consistent with an alternative interpretation whereby Wg signaling enhances the degradation of free Wg, e.g., by a transcriptional induction of a protease. Such a mechanism would also increase Wg degradation close to its source, thus enhancing the system robustness through the same mechanism of self-enhanced ligand degradation (Eldar, 2003).

Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors

Wnt/Wingless (Wg) signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single-transmembrane LDL-receptor-related proteins 5 or 6 (LRP5/6) or Arrow. The aminotermini of LRP and Fz were reported to associate only in the presence of Wnt, implying that Wnt ligands form a trimeric complex with two different receptors. However, it was recently reported that LRPs activate the Wnt/beta-catenin pathway by binding to Axin in a Dishevelled-independent manner, while Fzs transduce Wnt signals through Dishevelled to stabilize beta-catenin. Thus, it is possible that Wnt proteins form separate complexes with Fzs and LRPs, transducing Wnt signals separately, but converging downstream in the Wnt/beta-catenin pathway. The question then arises whether both receptors are absolutely required to transduce Wnt signals. A sensitive luciferase reporter assay in Drosophila S2 cells was established to determine the level of Wg-stimulated signaling. Wg can synergize with DFz2 and function cooperatively with LRP to activate the beta-catenin/Armadillo signaling pathway. Double-strand RNA interference that disrupts the synthesis of either receptor type dramatically impairs Wg signaling activity. Importantly, the pronounced synergistic effect of adding Wg and DFz2 is dependent on Arrow and Dishevelled. The synergy requires the cysteine-rich extracellular domain of DFz2, but not its carboxyterminus. Finally, mammalian LRP6 and its activated forms, which lack most of the extracellular domain of the protein, can activate the Wg signaling pathway and cooperate with Wg and DFz2 in S2 cells. The aminoterminus of LRP/Arr is required for the synergy between Wg and DFz2. This study indicates that Wg signal transduction in S2 cells depends on the function of both LRPs and DFz2, and the results are consistent with the proposal that Wnt/Wg signals through the aminoterminal domains of its dual receptors, activating target genes through Dishevelled (Schweizer, 2003).

Trimeric G protein-dependent Frizzled signaling in Drosophila

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galphao subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).

The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).

The inherent subviability of Go clones prevented a frank assessment of their loss-of-function effects on Wg transduction: surviving cells likely carried perduring wild-type transcripts or protein. This offers a simple explanation for why not all Go cells showed effects on Wg targets -- many cells still carried enough Go function to transduce Wg. However, even given the lack of penetrance of the clones, there was a striking correspondence between Go mutant clones and the loss of expression of Wg targets, thereby arguing that Go gene function is critically required for Wg signal transduction (Katanaev, 2005).

Further evidence for the role of Go in transducing Wg comes from the overexpression experiments. When Go is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If Go achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of Go should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, Go overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).

In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-Go wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of Go overexpression lose Dll expression to a level comparable with clones in which Go is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of Go requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).

Upon activation of serpentine receptors, GDP is exchanged for GTP on Galpha, and the complex dissociates, leaving Galpha-GTP and ßγ free to signal to downstream components. To test whether Go-GTP is able to activate the transduction pathway, a form of Go containing an inactive GTPase was overexpressed. Overexpression of Go-GTP induces Wg targets, indicating that Go-GTP is a positive transducer of the pathway and that one function of Fz activation is to catalyze the release of Go-GTP. Any signaling role of the ßγ moiety remains to be investigated. Overexpression of the Go-GDP mutant form did not produce any effect. This form has a low affinity for GTP and could be expected to have dominant-negative effects. However, this form may not be sufficiently inactive to allow any effects on Wg transduction (and the PCP pathway) to be detected (Katanaev, 2005).

The epistasis experiments provide two key indications that Go represents an immediate transducer of Fz signaling. (1) Dsh (previously the highest element of the transduction cascade identified downstream of the receptors) is necessary for the effects of Go overexpression. (2) Since serpentine receptors act as exchange factors for trimeric G proteins, the effects of overexpression of a wild-type form should require the presence of the exchange factor to load and subsequently reload GTP. Conversely, once loaded with GTP, the form lacking GTPase activity (Go-GTP) will be a long-lived activated subunit. Thus, if Fz acts as the exchange factor for Go, then it would be expected that wild-type Go would require Fz for its overexpression effects but that the activated form would be significantly less dependent. This is what was observed: Wg signaling is significantly rescued in fz, fz2 cells concomitantly expressing Go-GTP as compared to those expressing wild-type Go (Katanaev, 2005).

Given that Go functions in the Wg transduction pathway, given that its overexpression effects require Dsh, and given that its activated form is receptor independent, the simplest explanation is that Go functions in a trimeric G protein complex that relays signals from Fz receptors. These data do not necessarily suggest that Go is the exclusive transducer of Wg signals: other trimeric complexes may be involved, and non-G protein-mediated signaling may also occur (Katanaev, 2005).

In the wing, the key molecular events associated with PCP occur by 30 hr APF, when Fz becomes specifically localized to the distal membrane of the cell. The localization of Fz appears to require its own signaling, since, in dsh mutants, Fz localization does not occur. A similar effect occurs when Fz is overexpressed: Fz is no longer restricted to the distal membrane. Given this complexity, the following feature of Go can be predicted if it indeed acts as a transducer of Fz signaling. (1) Loss of Go activity should induce PCP phenotypes; (2) Fz localization should not occur correctly when Go signaling is compromised. In regard to these two predictions, it has been shown that (1) reduction of Go function or Go overexpression induces clear PCP defects and (2) Fz localization is aberrant when Go function is down- or up-regulated. Furthermore, it has been shown that Go itself undergoes a striking asymmetric redistribution in a fz-dependent manner (Katanaev, 2005).

Go clones can show nonautonomous polarity defects on their proximal side, whereas fz clones show effects on their distal sides. This may indicate that Go relays a negative signal in PCP transduction. Go localizes proximally in polarizing cells, as does Strabismus/van Gogh, which also shows proximal nonautonomous effects. Hence, the proximal nonautonomous effects of Go may result from it functioning negatively in the PCP pathway, from it becoming localized proximally, or from some combination of the two. A further aspect of Go clones is the inappropriate localization of Fz at the interface of mutant and wild-type cells. It is not clear if this protein is derived from the wild-type cells, the mutant cells, or both. But it implies that the cells are in communication, and again a similar phenomenon has been described for Strabismus/van Gogh clones that may relate to the nonautonomous effects (Katanaev, 2005).

Overexpression of either Go or Go-GTP causes PCP defects, suggesting that one function of Fz signaling in the PCP pathway is the generation of free Go-GTP. However, given the difficulty in distinguishing gain-of-function from loss-of-function effects, it is not possible to say whether Go-GTP acts positively (as in the Wg pathway) or negatively. Any role for the ß/gamma dimer in transducing PCP signals remains to be established. The secreted multiple wing hairs produced by overexpression of wild-type Go or Go-GTP show a marked difference: the effects of wild-type Go require the presence of the receptor (Fz), whereas the activated form does not. As for the Wg pathway described above, the most likely explanation of this observation is that Fz functions as an exchange factor for Go (Katanaev, 2005).

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).

The distribution of Dlp protein is reminiscent of the down-regulation of Dfz2 transcription near the D/V border. Wg-mediated repression of DFz2 expression has been shown to affect the shape of the Wg gradient, resulting in a gradual decrease in Wg concentration. Because these results indicate that HSPGs affect Wg distribution, the functions of the two seven transmembrane Wg receptors, Fz and DFz2, were examined to evaluate how the signal transducing receptors cooperate with HSPGs in shaping the Wg gradient. To determine the role of Fz and DFz2 in Wg movement, the distribution of Wg in fz DFz2 double-mutant clones was examined. In these clones, an expansion of wg expression was observed, which is consistent with the previously described Wg “self-refinement” process, by which Wg signaling represses wg expression in cells adjacent to wg-expressing cells. Unexpectedly, within these clones, Wg puncta are still present, indicating that Fz/DFz2 receptor activities are not required for Wg spreading (Baeg, 2004).

To determine whether Wg is present in endosomes in the absence of Fz/DFz2 activities, wing discs were labeled with the endosomal marker Texas-red dextran. More than 50% of Wg puncta co-localize with red dextran, indicating that Wg is internalized in the absence of Fz/DFz2 activities. These observations are consistent with results in the embryo, and altogether suggest that internalization of Wg can be accomplished by proteins other than Fz/DFz2. Interestingly, this observation contrasts with the role of HSPGs in Wg distribution, since wing discs lacking GAGs show alteration in Wg puncta in receiving cells. The extracellular distribution of Wg was examined in fz DFz2 mutant clones. Interestingly, accumulation of extracellular Wg was detected throughout these clones, thus revealing that Wg can bind to the cell surface and that Fz/DFz2 receptors are required somehow for Wg degradation. To exclude the possibility that accumulation of extracellular Wg results from increased wg expression or secretion in fz DFz2 clones that cross D/V boundary, small clones that do not include the D/V boundary were generated. Accumulation of extracellular Wg was detected in these clones, which is reminiscent of the finding that overexpression of a dominant-negative form of DFz2 (ΔDFz2-GPI) driven by en-Gal4 in embryonic tissue prevents Wg decay within the en domain. It has been proposed that endocytosis of a Wg/receptor complex is responsible for down-regulating Wg levels. Further, because Wg is still organized in a graded manner in these clones, as shown by the distribution of the Wg puncta, it indicates that Wg movement can occur in the absence of Fz/DFz2 (Baeg, 2004).

There is a third member of the Frizzled family encoded by DFz3 that could influence the distribution of Wg in tissues. DFz3 expression is similar to that of wg, and a constitutively activated form of Arm up-regulates its expression in the wing disc, suggesting that DFz3 is transcriptionally regulated by Wg signaling. Based on these observations, little or no DFz3 protein would be expected to be present in cells that lack Fz/DFz2 activity, suggesting that internalization of Wg in Fz/DFz2 mutant cells is unlikely to be mediated by DFz3. Another candidate that could affect Wg distribution is Arrow, which is a Drosophila homolog of a low-density lipoprotein (LDL)-receptor-related protein (LRP) and has been shown to be essential in cells receiving the Wg signal. However, because a soluble form of the Arrow fails to bind Wg and Fz receptors in vitro, and because Arrow functions after DFz2 engages Wg, it is unlikely that Wg internalization in Fz/DFz2 mutant cells is mediated by Arrow. Finally, as is case for FGF endocytosis, HSPGs themselves possibly play a role in Wg internalization (Baeg, 2004).

In summary, there is a Fz/DFz2 receptor-independent mechanism that organizes Wg distribution, and Fz/DFz2 proteins play a role in Wg gradient formation by decreasing the level of extracellular Wg. Regulation of extracellular Wg levels by Fz/DFz2 may occur through receptor-mediated endocytosis, or by some other mechanisms. If Wg degradation occurs by receptor-mediated endocytosis, it indicates that there may exist more than one way to generate Wg puncta since these are still present in the absence of Fz/DFz2 receptor activity. These findings also emphasize that the amount of Fz/DFz2 receptors at the cell surface must be precisely regulated to achieve the proper spreading of Wg, an observation that is underscored by the transcriptional down-regulation of DFz2 expression near the source of Wg (Baeg, 2004).

To further examine the role of Fz/DFz2 receptors in Wg gradient formation, DFz2 was overexpressed at the D/V boundary using the C96-Gal4 driver, and the effect on Wg distribution and wing patterning was examined. Analysis was focused on DFz2 since DFz2 has been shown to bind Wg with high affinity and to stabilize it. Further, DFz2 expression is down-regulated by Wg signaling, and this regulation has been shown to play a critical role in the overall shape of the Wg gradient. Interestingly, ectopic expression of DFz2 results in wing notching and ectopic bristles at the wing margin of adult wing. Previous studies have shown that wing nick phenotypes result from an inhibition in Wg signaling activity while the presence of ectopic bristles on the wing blade corresponds to an increase in Wg signaling. Thus, based on the wing phenotypes, it appears that overexpression of DFz2 paradoxically both increases and decreases Wg signaling (Baeg, 2004).

Overexpression of the DFz2 could interfere with Wg signaling and its distribution in a number of ways. For example, an increase in DFz2 could increase the efficiency of Wg signaling, if the amount of receptor is limiting. Further, since wg expression in the wing disc is restricted to the D/V margin, and Wg diffuses from it, trapping of Wg near these cells most likely will have an effect on Wg short- and long-range activity since the shape of the Wg gradient will be disrupted. To distinguish between these possibilities, Wg distribution was examined in discs with clones of cells that overexpress DFz2 at the D/V boundary. These clones were associated with two effects on Wg distribution. (1) The level of Wg was increased in the clones of cells where DFz2 was overexpressed, indicating that an increase in the level of DFz2 in receiving cells leads to an increase in trapping extracellular Wg. This observation is consistent with the occurrence of extra bristles on the wing blade since they reflect high levels of Wg signaling activity. (2) A dramatic reduction in Wg puncta was detected in WT cells located adjacent to the cells overexpressing DFz2, suggesting that Wg movement from the D/V margin into the wing blade is impaired as a result of the excess trapping of Wg by cells that overexpress DFz2. To demonstrate that Wg accumulation correlates with an increase in Wg signaling and that the absence of Wg puncta correlate with an absence of Wg signaling, the effect of DFz2 overexpression on the expression of senseless was examined. sen expression is expanded in cells overexpressing DFz2, yet sen is not expressed in WT cells near a clone of cells overexpressing DFz2. This is consistent with the observation that more Wg can be detected in cells overexpressing DFz2 and that less Wg puncta are present in WT cells near a clone of cells overexpressing DFz2 (Baeg, 2004).

It has been proposed that Fz proteins contribute to Wg turnover. Thus, it is intriguing to note that overexpression of DFz2 leads to an accumulation of extracellular Wg. This may reflect saturation of the endocytotic pathway when DFz2 is overexpressed or an inability of the regulatory pathways that normally control Dfz2 endocytosis in the wing disc to appropriately respond under this overexpressed condition. Another possibility is HSPGs themselves might play an important role. Wg endocytosis and the stoichiometry of Fz to HSPGs is essential to promote proper Wg internalization. Detailed biochemical and cell biological studies are now required to clarify the role(s) of these receptors in Wg movement (Baeg, 2004).

Finally, whether overexpression of DFz2 at the D/V boundary could affect long-range activity of Wg was examined, using wing disc overexpressing DFz2 driven by C96-Gal4 driver. Interestingly, Dll expression is dramatically shortened in wing disc overexpressing DFz2 at the D/V boundary when compared to that of WT disc. It is concluded that DFz2 has multiple roles in Wg signaling: First, it transduces Wg signaling and its level is limiting in amount; and second, it affects Wg short- and long-range activity by modulating the availability of extracellular ligand (Baeg, 2004).

In this study, the respective roles of HSPGs and Fz/DFz2 receptors in Wg distribution and gradient formation were examined. Interestingly, it was found that loss of Dlp activity significantly increases the level of Wg activity in S2R+ cells upon Wg induction, indicating that Dlp acts as a negative regulator in Wg signaling and that it is not required for transducing the Wg signal. Interestingly, the in vivo results show that Dlp protein levels are low near the D/V boundary. Thus, low levels of Dlp near the source of Wg production may allow for activation of high threshold Wg target gene. It is of interest to note that Notum is highly expressed along the D/V boundary (Giralez, 2002), which would be predicted to further diminish HSPGs activity. In addition, it was found that Dlp positively influences Wg signaling in S2R+ cells when Wg is not induced, suggesting that it is required for Wg signaling in cells where Wg level is low. A possible explanation for this result is that Dlp may act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with signal transducing Fz receptors. In the wing imaginal disc, given that Dlp is required for Wg signaling in cells where Wg levels are low, HSPG activity is possibly required for Wg signaling by somehow facilitating Wg movement. The binding of extracellular Wg to the low-affinity HSPG receptors in receiving cells may result in the association of Wg to cell membranes. Ligand movement could then occur by a mechanism that directly involves HPSGs where subsequent cycles of Wg dissociation/reassociation with HSPGs might promote the movement or require other yet to be identified extracellular molecules. To distinguish these possibilities, the role of HSPGs in Wg movement will require further detailed analysis. Regardless, these results clearly indicate that the primary role of HSPGs is to sequester and/or stabilize extracellular Wg in receiving cells. The imposition of the HSPG-mediated Wg accumulation and the Fz-dependent degradation mechanism would thus contribute to the Wg morphogen gradient. It is important to note that the expression levels of some of the critical components of each systems (i.e., dally, DFz2) are also regulated by the Wg pathway itself, indicating that the slope of the Wg gradient is established by the delicate balance between these two systems (Baeg, 2004).

Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs

Lysosome-mediated ligand degradation is known to shape morphogen gradients and modulate the activity of various signalling pathways. The degradation of Wingless, a Drosophila member of the Wnt family of secreted growth factors, was investigated. One of its signalling receptors, Frizzled2, stimulates Wingless internalization both in wing imaginal discs and cultured cells. However, this is not sufficient for degradation. Indeed, as shown previously, overexpression of Frizzled2 leads to Wingless stabilization in wing imaginal discs. Arrow (the Drosophila homologue of LRP5/6), another receptor involved in signal transduction, abrogates such stabilization. Evidence is provided that Arrow stimulates the targeting of Frizzled2-Wingless (but not Dally-like-Wingless) complexes to a degradative compartment. Thus, Frizzled2 alone cannot lead Wingless all the way from the plasma membrane to a degradative compartment. Overall, Frizzled2 achieves ligand capture and internalization, whereas Arrow, and perhaps downstream signalling, are essential for lysosomal targeting (Piddini, 2005).

The main conclusion of this work is that two receptors contribute distinct though overlapping trafficking activities that together lead to degradation of Wingless. Binding data support the earlier suggestion that normally Wingless is primarily captured by a Frizzled family member and that this facilitates subsequent binding to Arrow. Wingless is internalized by Frizzled2 in the absence of Arrow. This result extends and complements recent evidence that mammalian Frizzled4 is endocytosed upon stimulation by Wnt5a. Moreover, Wingless internalization in the absence of Arrow also shows that Wingless signalling is not required for endocytosis. However, in the absence of further targeting to a lysosomal compartment, endocytosis would clearly be insufficient for degradation (Piddini, 2005).

Using gain-of-function experiments, Arrow is shown to contributes to the targeting of Wingless, maybe as a complex with Frizzled2, to a degradative compartment. As expected, loss of either Arrow or Frizzled and Frizzled2 leads to extracellular accumulation of Wingless. Frizzled and Frizzled2 are clearly redundant in this respect (as in signalling) because removal of either receptor has no noticeable effect on Wingless distribution. Interestingly, large intracellular vesicles are lost in the absence of Frizzled;Frizzled 2 but not in the absence of Arrow. It is suggested that Frizzled-mediated endocytosis is sufficient to generate these large vesicles in the absence of Arrow. The fine-grained Wingless staining seen in the absence of Frizzled;Frizzled 2 could be internalized by Arrow or by another receptor, such as Dally or Dally-like. The distinct intracellular distribution of Wingless in the absence of Frizzled;Frizzled 2 when compared with that in Arrow-deficient cells is consistent with the suggestion that the two receptor classes have distinct trafficking activities (Piddini, 2005).

It is unclear at this point whether the degrading activity of Arrow is regulated by post-translational modification or by the recruitment of other factors. Either process could be impaired in ArrowDeltaC. Work in Xenopus has identified negative regulators of Wnt signalling, Kremens, which operate by triggering LRP6 endocytosis and possibly degradation. It remains to be seen whether this leads to degradation of a Wnt during frog embryogenesis. Moreover, there is no Kremen homologue (a negative regulator of Wnt signalling identified in Xenopus that operates by triggering LRP6 endocytosis and possibly degradation) encoded by the fly genome. Clearly further work will be needed to understand the genetic control of Wnt/Wingless degradation both in flies and other systems. The data provide a simple explanation of why overexpression of Frizzled2, a receptor that mediates Wingless internalization, causes Wingless stabilization. Under such experimental conditions, Arrow becomes limiting and in the absence of an effective degradation signal, Wingless accumulates (Piddini, 2005).

Because the receptors involved in Wingless degradation are those required for signalling, Wingless degradation cannot be initiated before a signalling-competent complex is assembled. Even though signalling downstream of Armadillo is not sufficient to activate the degradation of Frizzled2-Wingless complexes, it is not known yet whether downstream signalling is necessary for degradation. In the case of EGF receptor signalling, ubiquitination (the first step towards degradation of the ligand) is contingent on the tyrosine phosphorylation that accompanies receptor activation. However, in this case, a single receptor type is involved. In the case of TGFß signalling, two receptor types are required for signal transduction. Type 2 receptor is believed to capture the ligand and this is followed by the formation of a tripartite complex with type 1 receptor. Interestingly, like Arrow, type 1 receptor brings a degradation signal such that the two types of receptor cooperate to direct the ligand towards degradation and signalling pathways appropriately. Sharing of trafficking duties by distinct receptors may provide cells with increased flexibility as expression or turnover of the two receptors could be independently modulated. It may not be a coincidence that both Dpp (the fly TGF-ß) and Wingless, which can act over a relatively long distance, use two receptors for signalling and degradation. Maybe separation of capture and degradation is a feature required for long-range signalling, perhaps by allowing modulation of local relative receptor levels (Piddini, 2005).

Further work will be needed to identify the relevant trafficking signals in Arrow and Frizzled2, as well as the mechanisms that control relative receptor levels in order to obtain a full understanding of how degradation of Wingless is tuned to generate a reliable concentration gradient (Piddini, 2005).

Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2

Wingless secretion provides pivotal signals during development by activating transcription of target genes. At Drosophila synapses, Wingless is secreted from presynaptic terminals and is required for synaptic growth and differentiation. Wingless binds the seven-pass transmembrane Frizzled2 receptor, but the ensuing events at synapses are not known. Frizzled2 is shown to be endocytosed from the postsynaptic membrane and transported to the nucleus. The C terminus of Frizzled2 is cleaved and translocated into the nucleus; the N-terminal region remains just outside the nucleus. Translocation of Frizzled2-C into the nucleus, but not its cleavage and transport, depends on Wingless signaling. It is concluded that, at synapses, Wingless signal transduction occurs through the nuclear localization of Frizzled2-C for potential transcriptional regulation of synapse development (Mathew, 2005).

Members of the WNT signaling family function in synapse formation and maturation. In Drosophila, the WNT homolog Wingless (Wg) is secreted from presynaptic cells at glutamatergic larval neuromuscular junction (NMJs). The Wg receptor Frizzled2 (Fz2) is present in both pre- and post-synaptic cells and is required for synaptic Wg function. Wg secretion from the presynaptic cell is crucial for both the formation of active zones (regions where synaptic vesicles accumulate adjacent to the presynaptic membrane) and postsynaptic specializations that are assembled during proliferation of synaptic boutons in larval development. How these Wg-dependent signaling events coordinate synapse differentiation remains unknown. To investigate the effect of Wg signaling on the distribution of its receptor and subsequent signal transduction, antibodies to the extracellular amino acids 1 to 114 (Fz2-N) and to the intracellular amino acids 600 to 694 (Fz2-C) were used. Staining of body wall muscles from third instar larvae show that Fz2-C antibodies label the same NMJs as Fz2-N (Mathew, 2005).

Antibodies against Fz2-C also label spotlike structures within each of the multiple nuclei in each muscle cell. Quantification of the number of Fz2-C spots in each nucleus has revealed that spots are more numerous in nuclei close to the NMJ than in those more distal. Fz2-N immunoreactive puncta are observed near the nucleus, but unlike those seen from Fz2-C immunoreactivity, these puncta are much smaller, are localized outside the nuclear boundary, and are never observed inside the nucleus. Smaller Fz2-C immunoreactive puncta are also observed at the perinuclear area, but their abundance is low (Mathew, 2005).

Intranuclear localization of Fz2-C was confirmed by double-labeling with propidium iodide (PI) and antibodies against the chromatin remodeling protein Osa, which associates with chromosomal DNA. Preparations were also labeled with antibody against HP-1, which labels heterochromatin. Regions of the nuclei containing HP-1 had either no Fz2-C spots or only showed marginal coincidence, which suggests that Fz2-C spots are mostly excluded from regions of transcriptionally inactive DNA. Nuclear localization of Fz2-C appears to be cell-type-specific. Although Fz2-C spots are always observed in the nuclei of larval muscles, Fz2-C is not observed in epithelial cells or neurons (Mathew, 2005).

To test whether Fz2 might be cleaved, as are some other membrane receptors such as Notch and ß-amyloid precursor protein (APP), Drosophila Schneider-2 (S2) cells were transfected with full-length Fz2 or with Fz2 fragments containing the N-terminal region (amino acids 1 to 605) or C-terminal region (amino acids 606 to 694). On Western blots of lysate from S2 cells transfected with Fz2, two protein bands are detected, an 83-kD band (full-length Fz2) and an 8-kD band. The 83-kD band is recognized by both the Fz2-N and the Fz2-C antibodies, but only the Fz2-C antibody recognizes the 8-kD band, which suggests that full-length Fz2 may be cleaved to produce a C-terminal fragment. In extracts of wild-type body wall muscle, full-length Fz2 is detected at very low levels by Western blots, but the 8-kD band is not detected. However, if full-length Fz2 is overexpressed in muscle cells, an 8-kD fragment is detected (Mathew, 2005).

The putative amino acid sequence of Fz2 was compared with those of its most related Frizzled counterparts from different species, because regions of functional significance are highly conserved across phylogenies. A sequence in the cytoplasmic domain proximal to the transmembrane domain (VWIWSGKTLESW) is virtually identical in all species, from flies to humans, and contains a glutamyl-endopeptidase cleavage site. In eukaryotes, glutamyl-endopeptidase activity is observed in peptidases of the ADAM (a disintegrin and metalloprotease) family, and ADAM members have also been implicated in APP and Notch receptor cleavage. Although, in the case of APP and Notch, ADAM proteases cleave the extracellular domain of the proteins, ADAM proteases are also observed intracellularly (Mathew, 2005).

Site-directed mutagenesis was used to construct three mutants: two deleting the coding sequences for KTLES, which contains the glutamyl endopeptidase cleavage site (DeltaKTLES and DeltaSGKTLESW), and another mutating the adjacent upstream sequence VWIWSG (Fz2-DeltaVWIWSG). The amount of cleavage product was reduced in Fz2-DeltaKTLES-expressing S2 cells, and no cleavage product was detected in Fz2-DeltaSGKTLESW cells, but Fz2-DeltaVWIWSG cells had normal amounts. Thus, KTLES is apparently contained in the cleavage site or required for cleavage (Mathew, 2005).

Localization of Fz2-C and Fz2-N fragments into different compartments within and around the nucleus may occur immediately after Fz2 biosynthesis, or Fz2 fragments may translocate to the nucleus through a retrograde pathway after integration into the plasma membrane. To distinguish between these possibilities, whether cell surface Fz2 was internalized and transported to the nucleus was tested. Larvae were dissected, and body wall muscles were incubated in situ in physiological saline containing antibody against Fz2-N. Under these conditions, the antibody was expected to label only surface Fz2. Then, unbound antibody was washed away, the preparations were fixed, and a secondary antibody conjugated to a blue fluorescent marker (Alexa-647) was added under nonpermeabilizing conditions to detect surface Fz2. To determine whether any cell surface Fz2 had been internalized during the initial incubation, the preparation was permeabilized and then incubated with secondary antibody conjugated to a green fluorescent marker (FITC). A prerequisite for such an experiment is that the antibody should label the extracellular region of Fz2 in situ, and indeed, it was found that anti-Fz2-N could label NMJs in situ. A fraction of surface-labeled Fz2 was internalized into the muscle and appeared as puncta at the NMJ (Mathew, 2005).

To determine whether nuclear Fz2 is derived from receptors that are internalized at the postsynaptic membrane, an antibody pulse-chase experiment was conducted in living preparations. The primary antibody-binding step was done at 4°C to inhibit internalization during antibody incubation. Unbound antibody was washed away, and samples were shifted to room temperature for various time intervals before fixation. In samples that were fixed after a 5-min shift at room temperature, most of the internalized Fz2 was observed close to the NMJ, but after 60 min, little internalized Fz2 was observed at the NMJ. There was a comparatively small decrease in surface Fz2 over time at the NMJ, suggesting that only a fraction of labeled Fz2 was internalized. Parallel with the changes in Fz2 internalization at the NMJ, at 5 min, minimal internalized Fz2 was observed at the periphery of nuclei, whereas at 60 min, the amount of internalized Fz2 at the nuclear periphery was increased. Thus, cell surface Fz2 appears to be transported from the plasma membrane to the nucleus (Mathew, 2005).

If cell surface Fz2 is endocytosed and transported to the nucleus, then blocking endocytosis or retrograde vesicle transport should block the nuclear localization of Fz2. Therefore, in a subset of muscle cells, dominant-negative transgenes were expressed that block endocytosis [dominant-negative form of the Drosophila Dynamin, Shibire (Shi-DN)] or retrograde transport (dominant-negative form of Glued, a component of the dynein-dynactin complex). In both cases, the number of Fz2-C spots per nucleus was reduced. These results, together with the in vivo internalization assays, indicate that Fz2 is internalized from the plasma membrane and is carried by retrograde transport to the nucleus (Mathew, 2005).

Whether Wg signaling is required for Fz2 transport to the nucleus was tested. To decrease Wg signaling, a temperature-sensitive wgts mutant was used, as well as two conditions that disrupt Wg-dependent Fz2 signaling: overexpression of full-length Fz2 in muscles and expressing a Fz2 dominant-negative Fz2 construct (Fz2-DN). Wg was also overexpressed in the presynaptic cells, causing those cells to increase Wg secretion. Disrupting Wg signaling caused a decrease in the number of Fz2-C spots inside muscle nuclei. In contrast, when presynaptic secretion of Wg was increased, there was an increase in the number of nuclear spots (Mathew, 2005).

Transgenic Fz2 variants were also expressed in muscles; full-length Fz2, Fz2DeltaSGKTLESW, and Myc-NLS-Fz2-C [consisting of a Myc-tagged Fz2-C fragment alone or fused to a nuclear localization sequence]. When Fz2 was overexpressed in muscle, bright Fz2-C immunoreactivity accumulated just outside the nucleus, which suggests that overexpression of Fz2 does not disrupt retrograde transport of Fz2, but rather, the nuclear import of Fz2-C. To further test the model that the Fz2 pool transported to the nucleus is derived by endocytosis from the plasma membrane, and not from an internal pool, Fz2 and the Shi-DN were simultaneously expressed in muscle cells. In the presence of Shi-DN, no accumulation of Fz2-C at the perinuclear area was observed. Mutations in the Fz2 cleavage site did not alter the endocytosis of Fz2; expression of transgenic Fz2DeltaSGKTLESW in muscles did not suppress the accumulation of perinuclear Fz2-C spots (although it did not enter the nucleus), and the perinuclear spots had a distribution that was indistinguishable from that of cells expressing transgenic wild-type Fz2. Muscle cells expressing the Fz2-C transgenes showed diffuse Myc immunoreactivity in the cytoplasm and nuclei (Mathew, 2005).

Whether expressing Fz2, Fz2DeltaSGKTLESW, or Fz2-C could rescue the synaptic phenotypes of a mutant of the Fz2 gene (Fz2C1/DfFz2) was tested. Interfering with Fz2 function prevents the proliferation of synaptic boutons and the formation of pre- and post-synaptic specializations in many boutons. Like wgts mutants, Fz2C1/DfFz2 NMJs had irregular and tightly spaced boutons and a reduced number of boutons (Mathew, 2005).

Expression of Fz2 in a Fz2c1/DfFz2 mutant background completely rescued the decrease in bouton number and partially restored the abnormal morphology of the boutons. It also restored the presence of nuclear spots in the mutant larvae. In contrast, only a slight rescue was observed when Fz2DeltaSGKTLESW was expressed, and no rescue was detected when Myc-NLS-Fz2-C was expressed. Thus, cleavage of Fz2 appears to be needed for Fz2 signaling at the NMJ, and Fz2-C is necessary but not sufficient for Fz2 function. The slight rescuing activity observed in Fz2c1/DfFz2 mutants expressing Fz2DeltaSGKTLESW may indicate that not all of Fz2's function at the NMJ is accomplished through Fz2 cleavage and nuclear import (Mathew, 2005).

To test whether Wg is required for nuclear import of Fz2, Fz2-transfected S2 cells were treated with conditioned medium containing soluble Wg. In the presence of Wg, prominent immunoreactive spots were detected inside the nucleus of Fz2-transfected cells, but not in Fz2-DeltaSGKTLESW-transfected cells or in transfected cells not exposed to Wg-conditioned medium (Mathew, 2005).

These results indicate that, at the Drosophila NMJ, Wg secretion initiates a signaling mechanism, whereby Fz2 receptors at the postsynaptic muscle membrane are endocytosed and undergo retrograde transport to the nucleus. The C-terminal fragment is cleaved during this process and is ultimately transported into the nucleus. It is propose that Wg binding to Fz2 may initiate an event that marks the Fz2 C-terminal region. Endocytosed vesicles containing the entire Fz2 receptor travel toward the nucleus. Once at the periphery of muscle nuclei, the C terminus is cleaved, and only marked C-terminal fragments are imported into the muscle nuclei, where they may regulate gene transcription. These studies help unravel a mechanism by which pre- and postsynaptic cells communicate during the coordinated growth and maturation of synaptic specializations (Mathew, 2005).

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. This study characterizes the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigates whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. A role for Arrow and DFz2 in Wg internalization has been demonstrated. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, it is suggested that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event (Rives, 2006).

During patterning and growth of the wing imaginal disc, cells along the D/V axis interpret positional information and, hence, their fate, from the concentration of Wg ligand. The graded distribution of Wg, with high levels near the source at the D/V boundary and low levels toward the edges of the wing pouch, is therefore crucial for normal wing development. Lysosomal targeting of Wg and its receptors has been proposed as a mechanism for shaping the Wg gradient and attenuating signal transduction. To address this model, both trafficking to the lysosome and lysosome function was interfered with using genetic and pharmacological means (Rives, 2006).

In Drosophila, the hrs loss of function allele is a valuable tool for interrupting vesicular traffic to the lysosome. Hrs functions in late endosome invagination, a process that separates endocytic cargo to be recycled from cargo destined for the lysosome. Trafficking of the EGFR and Torso RTKs into the late endosome/MVB is an important step in signal attenuation; hrs mutant embryos experience elevated tyrosine kinase signaling due to the persistence of active receptors. Likewise, in the wing disc and the ovarian follicle cell, Hrs is required for downregulation of Tkv levels and dampening of the Dpp signal. Thus, Hrs activity is required to attenuate multiple developmental signals (Rives, 2006).

The fact that RTK and Dpp signaling levels are elevated in hrs mutant cells implies that active receptor complexes continue to signal inside the cell from an endocytic compartment. Although receptor internalization may turn off signaling by preventing ligand-receptor interaction, it is clear that many receptors remain active on endosomal membranes. For instance, activated EGFR can be detected in association with downstream signaling effectors on early endosomes, suggesting that signaling persists after endocytosis. This study reports dramatic intracellular accumulation of Wg, Arrow, and DFz2 in hrs or deep orange (dor) mutant wing discs; dor encodes a yeast VPS homologue required for delivery of vesicular cargo to lysosomes. Similar observations have been made for Wg and for Wg and Arrow. Given this dramatic intracellular accumulation of ligand, receptors, and a signal transducer, Wg signaling levels are expected to be elevated in hrs mutant cells. However, based on antibody stains for three Wg targets, no altered Wg signaling was detected in mutant cells. This was true for large null mutant clones, induced early in development, as well as in discs from larvae bearing a null hrs allele. The attenuation of Wg signaling, therefore, appears to be regulated differently from the attenuation of RTK and Dpp signaling (Rives, 2006).

The data suggest that Wg signaling is attenuated prior to Hrs-mediated lysosomal targeting of the receptor complex. In this case, removal of hrs prevents receptor and ligand degradation but has no bearing on signal output. Following endocytosis, internalized receptor-ligand complexes may be deactivated by physical dissociation in the increasingly acidic environment as they move through the endocytic compartment or by targeting to the lysosome for degradation. A model is favored in which the active Wg receptor complex is attenuated by dissociation earlier in the endocytic pathway; perhaps, this complex is more sensitive to pH levels in early endosomes, whereas, for example, a Dpp receptor complex is only uncoupled at the lower pH of later endosomal compartments (Rives, 2006).

Alternatively, there may be residual lysosomal degradation in hrs cells sufficient to effectively terminate signaling despite the accumulation of Wg, Arrow, and DFz2. It is not certain that Hrs is obligatory in targeting endocytic cargo to the lysosome. Internalized avidin, an endocytic tracer, still localizes to a low pH compartment in hrs mutant garland cells, suggesting that some trafficking to the lysosome continues in the absence of Hrs. Perhaps, this residual trafficking is sufficient to dampen Wg signaling levels but not RTK or Dpp levels (Rives, 2006).

In contrast to the genetic removal of hrs, treatment of wing discs with the lysosomal protease inhibitors chloroquine or NH4Cl leads to expansion of SOPs, a Wg gain-of-function phenotype. While this result agrees with the previous finding that chloroquine-treated embryos generate excess smooth cuticle, indicative of enhanced Wg signaling, it is surprising that disruption of lysosome function can affect signaling. Once internalized, receptors are sorted into inner MVB vesicles, they are presumably sequestered from intracellular effectors and thereby deactivated. If mild bases, such as chloroquine, solely affect lysosomal protease function, a step subsequent to MVB sorting, this should not affect Wg signaling output in embryos or in imaginal discs. As all endocytic compartments maintain an acidic environment that is crucial to their function, it is unlikely that alkalizing agents solely inhibit the lysosome. In a caution to their use, pharmacological reagents such as chloroquine and NH4Cl almost certainly disturb earlier pH-dependent trafficking steps as well, resulting in the accumulation of active receptor complexes. It is hypothesized that chloroquine- and NH4Cl-mediated alkalization prevents the dissociation of Wg from its receptor(s), thereby resulting in prolonged signaling (Rives, 2006).

Consistent with the excess SOP specification in chloroquine- and NH4Cl-treated discs, RNAi knockdown of Rab5 in cultured cells causes an increase in Wg-dependent reporter activation. These findings suggest that Wg signaling is normally attenuated at a trafficking step after internalization from the plasma membrane, but prior to Hrs-mediated lysosomal targeting. Such findings should be interpreted cautiously, however, as S2 cells are reported to be macrophage-like, and, thus, any effects on signaling output in these cells might not compare to that in wing disc cells in vivo. Nevertheless, attempts were made to define more precisely the trafficking step involved by treating cultured S2 cells with Shi dsRNA. So far the results have been ambiguous, since two trials demonstrated increased reporter activation while two other trials exhibited no such increase. Unfortunately, due to the compromised viability of endocytosis-defective cells in the wing disc, the DRab5 or Shi cell culture results could not be varified in vivo. However, in agreement with the data, a recent report shows enhanced Wg signaling, as evidenced by accumulation of the signal transducer Armadillo, in cells expressing a temperature-sensitive dominant negative variant of Shi. The viability issue was circumvented by transiently expressing dominant negative Shi with a 3-h upshift to the non-permissive temperature. Interestingly, no change was observed in Wg target gene expression under these conditions, suggesting that cell viability becomes compromised before such changes can occur (Rives, 2006).

While no evidence was found that lysosomal targeting modulates Wg signal output in the developing wing, it is clear that Wg, Arrow, and DFz2 are trafficked to the lysosome by Hrs. Hrs contains a conserved ubiquitin-interacting motif (UIM) and binds ubiquitin in vitro, suggesting that it regulates MVB sorting via direct interaction with ubiquitinated receptors. Monoubiquitination of cell surface receptors is emerging as an important signal for internalization and lysosomal sorting. It will be of interest to determine whether Arrow, Fz, and DFz2 undergo signaling-dependent monoubiquitination, and whether this has a consequence for Wg signaling output (Rives, 2006).

Signaling ligands are commonly internalized by receptor-mediated endocytosis, during which a ligand–receptor complex accumulates in coated pits on the plasma membrane and enters the cell in clathrin-coated vesicles. In the embryonic epidermis, endocytosis of Wg is thought to be receptor-mediated; expression of DFz2-GPI, which presumably lacks an endocytic signal, binds Wg but does not cause internalization. A similar model is predicted in the wing imaginal disc, where expression of DFz2-GPI stabilizes Wg to a greater extent than full length DFz2, most likely due to an inability to internalize Wg. Consistent with these views, it was found that extracellular Wg accumulates on the surfaces of arrow and fzdfz2 mutant cells. This striking accumulation cannot be explained by ectopic wg gene expression and likely results from impaired Wg internalization. In support of this conclusion, Wg and Arrow can colocalize in endosomes. It was still possible to detect residual Wg internalization into arrow mutant cells and fzdfz2 mutant cells. Yet, given the striking excess of extracellular Wg on receptor-deficient cells, a large increase was expected in the number of intracellular Wg puncta if Wg is internalized at a normal rate. This was not observed and led to a suggestion that Arrow, Fz, and DFz2 function as endocytic receptors for Wg. Since Fz does not contain an obvious endocytic signal, it is presumed that Arrow and DFz2 play more prominent roles. The residual intracellular Wg in receptor-deficient cells might be explained by a functional redundancy of Arrow and DFz2 in ligand internalization, in which case an absolute defect could only be observed by producing arrow-dfz2 doubly mutant cells. While this manuscript was in preparation, Piddini (2005) also reported that both DFz2 and Arrow contribute to Wg trafficking and degradation. A model was proposed in which DFz2 is important for Wg binding and internalization, while Arrow targets the Wg-DFz2 complex for degradation in the lysosome (Rives, 2006).

Contrary to hypothesis, recent evidence suggests that the accumulation of extracellular Wg on arrow and fzdfz2 mutant clones is due to upregulation of the glypican Dally-like protein (Dlp) (Han, 2005). That study also observed an increase in the level of extracellular Wg on arrow and fzdfz2 mutant clones. However, Wg accumulation was reduced if the mutant cells were compromised for the ability to make HSPGs by additional removal of sulfateless (sfl), an enzyme required for heparan sulfate biosynthesis, or brother of tout-velu (botv), a heparan sulfate copolymerase required for HSPG biosynthesis. This suggests that some of the build-up of extracellular Wg is due to trapping by excess HSPGs, rather than to a defect in endocytic trafficking (Rives, 2006).

In the process of evaluating endocytosis-defective cells for changes in Wg signaling levels, cells were frequently observed undergoing apoptosis. This is not surprising, since endocytosis is an important means for the cell to acquire macromolecules essential for viability as well as to gauge the growth needs of the tissue in which it resides. The results are troubling, though, given the widespread use of shits, DRab5DN and ShiDN in the Drosophila community. Thus, it is necessary to monitor cell viability and assay for expression of control genes when using these reagents in order to draw accurate conclusions about signaling levels (Rives, 2006).

One notable question that was not addressed experimentally is whether endocytosis of Arrow or DFz2 is induced by Wg stimulation or proceeds continuously, independent of ligand. Some evidence for Wg-induced endocytosis of DFz2 has recently been presented (Piddini, 2005). Signal-induced endocytosis is well established, especially for RTK signaling, and plays an important role in controlling signal duration. Constitutive endocytosis and recycling provide a more general means of regulating receptor concentration at the cell surface but may also be used to downregulate signaling by clearing activated receptors, as suggested for the Tkv receptor in the developing wing. Future investigation of this issue will provide insight into the regulation of Wg signaling by endocytosis (Rives, 2006).

Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila; Go transduces a signal from Frizzled

During asymmetric division, a cell polarizes and differentially distributes components to its opposite ends. The subsequent division differentially segregates the two component pools to the daughters, which thereby inherit different developmental directives. In Drosophila sensory organ precursor cells, the localization of Numb protein to the cell's anterior cortex is a key patterning event and is achieved by the combined action of many proteins, including Pins, which itself is localized anteriorly. This study describes a role for the trimeric G protein Go in the anterior localization of Numb and daughter cell fate specification. Go is shown to interact with Pins. In addition to a role in recruiting Numb to an asymmetric location in the cell's cortex, Go transduces a signal from the Frizzled receptor that directs the position in which the complex forms. Thus, Go likely integrates the signaling that directs the formation of the complex with the signaling that directs where the complex forms (Katanaev, 2006 see full text of article).

Because Fz appears to act as the exchange factor for Go in the Wnt and PCP pathways (Katanaev, 1995), The effects of GoWT and GoGTP on wing margin bristles were examined when Fz levels were modulated. The effects of overexpression of GoWT fell to zero in fz–/– wings, but the GoGTP overexpression phenotypes were not reduced; rather, they were enhanced. Why the aberrations increased is not clear, but this result shows that GoGTP is a potent disturber of asymmetric division in the absence of Fz, whereas WT Go requires it. This finding suggests that Go requires Fz to convert it into the 'active' GTP-bound state and predicts that overexpression of Fz should enhance the potency of Go. Indeed, co-overexpression of Fz and GoWT enhances the asymmetric division defects. Overexpression of Fz alone produced orientation defects but no asymmetric division aberrations (Katanaev, 2006).

In Drosophila, Wnt-1 (Wingless, Wg) is transduced by the Go-dependent receptors Fz and Dfz2. Therefore whether co-overexpression of Dfz2 could also enhance the effects of overexpression of Go was tested. Overexpression of Dfz2 alone characteristically induced ectopic margin bristles (activation of the Wg pathway) that showed no asymmetric division defects. But when Dfz2 and GoWT were co-overexpressed, they mutually enhanced their respective phenotypes, suggesting that Go enhanced the ability of Dfz2 to ectopically activate Wg signaling, and Dfz2 potentiated the ability of Go to disturb the asymmetric divisions. Dfz2 is usually down-regulated in the SOP region of the wing margin and likely does not normally influence Go activity there, but its forced expression shows an ability to potentiate the effects of Go (by inference catalyzing it into the GTP-activated form). These results provide the first example of the ability of Dfz2 to activate signaling in a pathway other than 'canonical' Wnt cascade (Katanaev, 2006).

Gβ13F and Gγ1 likely represent the β- and γ-subunits of the Go trimeric complex. Receptor-catalyzed exchange of GDP for GTP occurs on Gα-subunits complexed with βγ. Thus, βγ-subunits should be required for the effects of GoWT overexpression. Indeed, GoWT overexpression effects were attenuated when one gene copy of Gγ1 was removed, arguing that these effects were not due to sequestration of βγ moieties from another α-subunit such as Gi. Ablation of Gβ13F or Gγ1 genes was reported to affect neuroblast divisions. It was also found that loss or overexpression of Gγ1 and Gβ13F (but not Gβ5) resulted in adult bristle defects similar to those of loss or overexpression of Go. Taken together, these observations suggest that Gβ13F and Gγ1 represent the β- and γ-subunits of the Go trimeric complex (Katanaev, 2006).

Various roles for trimeric G proteins have been reported for asymmetric cell divisions; for example, Caenorhabditis elegans Gα-subunits GOA-1 and GPA-16 redundantly regulate posterior displacement of the mitotic spindle required for the asymmetric division of the zygote, and β- and γ-subunits are involved in orientating the mitotic spindle. In Drosophila, evidence for trimeric G protein function in both the formation of the asymmetric spindle and the correct localization of various cell fate determinants have come from manipulation of βγ-subunits in the neuroblasts. Additionally, Gi is known to be involved in asymmetric divisions and to interact with Pins; cell fate determinant localizations are aberrant during metaphase but are restored by telophase (Katanaev, 2006).

In this report, strong and pervasive roles have been documented for Go in Drosophila asymmetric divisions. Five major points are made: (1) In SOP asymmetric divisions, there are two patterning mechanisms: the establishment of the asymmetric complexes and the orientation of the asymmetry. Go appears to act in both functions and is therefore a likely molecular integrator of the two. (2) Go appears to function in both the neuroblast-type and SOP divisions and is therefore likely used in all asymmetric divisions in Drosophila. (3) Go binds to and genetically interacts with Pins. One function of Go, then, is likely mediated by a direct interaction with Pins. (4) Hitherto, Gi was considered the major Gα-subunit functioning in asymmetric cell divisions. Go shows significantly stronger phenotypes, suggesting a greater role, but genetic interaction between the two suggests a degree of functional redundancy. (5) Both Fz and Dfz2 appear able to act as exchange factors for Go in the SOP divisions. The role for Fz is supported by many different results, but whether Dfz2 normally functions here remains unclear (Katanaev, 2006).

Go appears to play parallel bifunctional roles in the establishment of asymmetries in both SOPs and PCP, as evidenced by the following: (1) polarized structures form in both; in PCP, it is the focal organizer of hair outgrowth, and in SOPs, it is the Numb crescent; (2) in both processes, Fz signaling organizes the polarized distribution of 'core group' PCP proteins. For example, Fz itself becomes localized to the distal and posterior ends of PCP cells and SOPs, respectively, whereas Van Gogh/Strabismus is found proximal and anterior in PCP cells and SOPs, respectively. (3) In both processes, these Fz-dependent localizations do not critically contribute to the final polarized structures, because loss of Fz (or other core group proteins) only leads to randomization in the positioning of the (usually) single-hair focus or Numb complex. Thus, there appear to be two semiindependent mechanisms: (1) the polarization of the core group PCP proteins, which instructs (2) the position of the self-assembling complexes (Katanaev, 2006).

Go appears to work in both these mechanisms. Mildly Go-compromised cells lose correct orientation of hairs or Numb complexes, consistent with an orientation function. Cells with strongly disturbed Go function lose the ability to polarize; in the SOP, Numb becomes diffuse or forms a number of small foci; and in PCP, many hair initiation sites are produced. Phenotypes of fz or other core group mutants occasionally result in two hairs per cell, but Go mutants frequently induce cells with five or six hairs (Katanaev, 2006).

The question now arises as to whether Go functions in the same way in both processes. In terms of the Fz-mediated orientation step, it is likely that Go performs the same role; in both, Fz is directed to one end of the cell (distal or posterior), and Go itself becomes preferentially distributed to the other end (proximal or anterior). This local enrichment of Go possibly serves as the point of integration with the internal asymmetry formation step. In the SOP case, anterior Go may recruit Pins and seed the formation of the anterior Numb crescent. In the PCP case, Go localizes opposite to the site of hair growth, suggesting that the highest depletion of Go specifies the site of hair growth. In the absence of the Fz orienting information, it may be a stochastic increase of Go localization (or activity) that establishes the initial asymmetric bias. Alternatively, the asymmetric distribution of Go may only be a manifestation of the Fz-mediated orientation, being essentially irrelevant to the subsequent step. In this case, the activity of Go (rather than its site of accumulation) would be required for the formation of the Numb crescent or the hair initiation point (Katanaev, 2006).

Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP

The Wingless pathway plays an essential role during synapse development. Recent studies at Drosophila glutamatergic synapses suggest that Wingless is secreted by motor neuron terminals and binds to postsynaptic Drosophila Frizzled-2 (DFz2) receptors. DFz2 is, in turn, endocytosed and transported to the muscle perinuclear area, where it is cleaved, and the C-terminal fragment is imported into the nucleus, presumably to regulate transcription during synapse growth. Alterations in this pathway interfere with the formation of new synaptic boutons and lead to aberrant synaptic structures. This study shows that the 7 PDZ protein dGRIP is necessary for the trafficking of DFz2 to the nucleus. dGRIP is localized to Golgi and trafficking vesicles, and dgrip mutants mimic the synaptic phenotypes observed in wg and dfz2 mutants. DFz2 and dGRIP colocalize in trafficking vesicles, and a severe decrease in dGRIP levels prevents the transport of endocytosed DFz2 receptors to the nucleus. Moreover, coimmunoprecipitation experiments in transfected cells and yeast two-hybrid assays suggest that the C terminus of DFz2 interacts directly with the PDZ domains 4 and 5. These results provide a mechanism by which DFz2 is transported from the postsynaptic membrane to the postsynaptic nucleus during synapse formation and implicate dGRIP as an essential molecule in the transport of this signal (Ataman, 2006: full text of article).

At the NMJ, the Wg pathway is initiated by the secretion of Wg from the presynaptic cells and its binding to DFz2 receptors present at the postsynaptic muscle cells. Upon Wg binding to DFz2, the receptor is internalized and transported to the perinuclear area, where it is cleaved, and the C-terminal DFz2 fragment (DFz2-C) is imported into the nucleus. Although the evidence suggested that endocytosis at the postsynaptic membrane and transport via microtubules are required for DFz2 trafficking from synapses to the muscle nucleus, the exact molecular mechanisms of trafficking were unknown. In this study, evidence is provided that the transport of DFz2 to the nucleus depends on interactions between a PDZ-binding motif at the C terminus of DFz2 and PDZ domains of dGRIP. In postsynaptic muscles, dGRIP is present in Golgi bodies and in a subset of vesicles that is highly concentrated at the postsynaptic area. These vesicles move along microtubules and colocalize with internalized DFz2. DFz2 and dGRIP can directly interact when expressed in heterologous systems. Manipulations that lead to severe reduction of dGRIP mimic all of the synaptic phenotypes resulting from mutations in wg or dfz2. Furthermore, in these dgrip mutants, internalized DFz2 accumulates at the postsynaptic region and is not transported to the nucleus. It is suggested that dGRIP is required at synapses to mediate the trafficking of DFz2 to the nucleus to properly regulate the expansion of the NMJ during muscle growth (Ataman, 2006).

Studies suggest that GRIP is involved in the clustering and trafficking of AMPA receptors at mammalian synapses. GRIP has also been found to interact with Ephrin ligands and Eph receptors, neuronal RAS guanine nucleotide exchange factor (GRASP1), members of the Liprin-α/syd2 family of proteins, the KIF5 microtubule motor kinesin, and extracellular matrix protein FRAS1. This large number of partners identified is perhaps not surprising, given that GRIP contains at least seven modular protein-interaction domains (Ataman, 2006).

Studies have shown that, similar to dGRIP, rat GRIP is also localized to both presynaptic axons and postsynaptic dendritic structures and is enriched in vesicular profiles that closely associate with microtubules. Furthermore, a recent study shows that knockdown of GRIP-1 using siRNA in primary hippocampal neurons interfered with the formation and growth of dendrites in developing neurons and the maintenance of dendrites in mature neurons. In this study, it was similarly found that interfering with GRIP function hampered synaptic bouton formation and growth in larval glutamatergic synapses. Additionally, elimination of postsynaptic dGRIP led to loss of the postsynaptic apparatus and presynaptic active zones and, presumably, to either the retraction or deficient stabilization of new synaptic boutons. These results imply that GRIP family proteins have a conserved role in both the formation and the stabilization of synapses in the nervous system (Ataman, 2006).

In Drosophila, dGRIP is involved in the guidance of embryonic muscle precursors to establish the proper body-wall muscle pattern, whereas this study shows that dGRIP is required for synapse differentiation. These results are not surprising, given the recurrent theme that many molecules necessary for early pattern formation in the embryo, such as members of the TGF-β pathway, the Wg pathway, and the tumor suppressor proteins DLG and Scribble (Scrib) are used again during synapse development (Ataman, 2006).

Evidence that dGRIP and DFz2 might interact arises from the observation that manipulations that lead to alterations in both proteins give rise to remarkably similar phenotypes, including decreased NMJ expansion and the presence of ghost boutons, which lack all postsynaptic proteins studied and the subsynaptic reticulum, and are devoid of active zones but filled with synaptic vesicles. These ghost boutons may represent boutons that initiated their differentiation presynaptically but never fully matured by forming corresponding pre- and post-synaptic specializations. Alternatively, the ghost boutons may represent boutons that are initially formed (including differentiation of both pre- and post-synaptic specializations) but subsequently retracted. However, studies have suggested that retraction of mature boutons at the Drosophila NMJ is accompanied by the presence of 'synaptic footprints', in which postsynaptic proteins are still present, despite the absence of a presynaptic bouton. In mutants in the current study, the opposite phenotype was observed, where synapses have some presynaptic proteins but are devoid of a postsynaptic apparatus (Ataman, 2006).

Interestingly, synaptic footprints are observed when the retrograde signaling mediated by TGFβ is disrupted. In contrast, ghost boutons are observed when the Wg pathway is abnormal. The Wg pathway at the NMJ has been shown to function in an anterograde manner, but the possibility that it also functions in a retrograde manner has not been studied. These findings suggest that, at the Drosophila NMJ, synapse retraction can be induced both pre- and post-synaptically, as in the vertebrate NMJ. It would be interesting to determine whether anterograde Wg and retrograde TGF-β signaling pathways crosstalk and coordinate synapse stability during development. Taken together, these studies suggest that one function of dGRIP at the NMJ is in trafficking DFz2 to the nucleus, which, in turn, regulates synaptic growth (Ataman, 2006).

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

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

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

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

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

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

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

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

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

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

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

The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development

Synapse-to-nucleus signaling is critical for synaptic development and plasticity. In Drosophila, the ligand Wingless causes the C terminus of its Frizzled2 receptor (Fz2-C) to be cleaved and translocated from the postsynaptic density to nuclei. The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain. This study found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large. These developmental defects in imp-beta11 mutants could be overcome by expression of Fz2-C fused to a nuclear localization sequence that can bypass Importin-beta11. Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway (Mosca, 2010).

Both pre- and post-synaptic events shape the properties of synapses during their development and subsequent plastic changes. The present study examined a synapse-to-nucleus pathway that governs the anatomical maturation of the Drosophila neuromuscular junction, a model glutamatergic synapse. By analyzing the phenotypes of importin-β11 and importin-α2 mutants, the mechanism has been determined by which an activity-dependent synaptic signal, the C-terminus of the Wg receptor Fz2, enters postsynaptic nuclei. By selectively blocking this process, it was determined that nuclear translocation causes growth of the SSR, a specialized post-synaptic membrane. By circumventing the importins with an NLS-tagged C-terminal peptide (NLS-Fz2-C) it was demonstrated that this phenotype depends on this signal and not other unidentified nuclear cargoes that might use these importins (Mosca, 2010).

Wg signaling at the Drosophila NMJ is an activity-dependent process that involves secretion of the Wg ligand from nerve terminals. Activation of Fz2 receptors on the postsynaptic membrane may give rise to multiple signals in the muscle, but the selective block of one such signal, the nuclear import of Fz2-C, gave rise to a thinner SSR surrounding the terminals and more boutons not surrounded by the scaffolding protein Discs Large. The SSR is an exceptional structure in which the sarcolemma is extensively invaginated. The function of the SSR is unclear, but it may create biochemically isolated postsynaptic compartments, similar to dendritic spines. By increasing the extracellular volume at the synapse, it may also provide a 'sink' into which released glutamate can diffuse to terminate synaptic responses. The SSR may filter the electrophysiological consequences of receptor activation or otherwise alter electrical signals. The SSR is also closely associated with ribosomes and may be a site of local glutamate receptor translation. The SSR is absent from newly formed embryonic synapses and grows during the larval stages. The number of boutons also increases during larval life and increased neuronal activity causes further increases in bouton number. Thus SSR growth entails the elaboration of postsynaptic membranes in an on-going process at both existing synapses and newly added boutons. The activity-dependent secretion of Wg and nuclear translocation of Fz2-C may therefore assist in matching SSR growth to synapse expansion. The SSR is reduced by only 42% in the absence of nuclear Fz2-C, however. Therefore, this signaling pathway serves to up-regulate a process likely initiated by other signals. At present, the nuclear targets of Fz2-C are unknown and, in addition to the potentially subtle modulation of SSR component expression, may include unrelated targets that require modulation by presynaptically released Wg (Mosca, 2010).

Diminished SSR did not represent a broader impairment of muscle growth or membrane trafficking. Many muscle parameters, such as size, input resistance and quantal response, as well as glutamate receptor clustering were normal. This latter observation is consistent with previous studies showing that receptors cluster at embryonic synapses prior to formation of the SSR and independent of transmitter release. Considerable receptor addition also occurs later as synapses are strengthened and new boutons form. This process was not blocked by imp-β11 mutations or the loss of nuclear Fz2-C. Thus, the reduction in SSR is not likely due to impaired muscle health, but rather, to the specific impairment of one aspect of synaptic maturation (Mosca, 2010).

Both Importin-β11 and Importin-α2 are required in the muscle for Fz2-C import and are detected in a complex with Fz2, suggesting a direct role for both importins in nuclear Fz2-C import. Although Importin-α is chiefly found to interact with Importin-β1, in the present case Importin-α2 partnered with Importin-β11 and Importin-β1 could not substitute for it. Thus particular combinations of α and β importins may provide specificity to nuclear translocation of signals. Interestingly, mutations in importin-13 also have diminished SSR. Increased ghost boutons and decreased nuclear Fz2-C were observed in importin-13 larvae, suggesting that the active nuclear import complex may also contain this importin. It has not yet been possible to demonstrate a biochemical association of Importin-13 with the Fz2 receptor, so its exact relationship remains uncertain (Mosca, 2010).

Active nuclear import is not mandated by the size of Fz2-C (8 kDa), which is below the diffusionary limit for the nuclear pore. Diffusion may account for the ~10% of normal levels of Fz2-C puncta that are observed in the absence of the importins but insufficient to preserve normal SSR growth. The inadequacy of free diffusion may indicate that cytosolic Fz2-C is restrained by a binding partner and an active import system may help regulate the pathway. Such a step is implied by the finding that Fz2 is constitutively cleaved, but thought to require an unidentified Wg-triggered activation step for nuclear entry. Recruitment of the importins in response to Wg could represent this activation step. Indeed, when expressed in S2 cells, Fz2-C is cleaved and can interact with dGRIP but, in the absence of Wg, neither enters the nucleus nor binds the importins (Mosca, 2010).

Mutations of the Wnt pathway cause several NMJ phenotypes, including changes in bouton number, shape, and presynaptic microtubule arrangements, in addition to the appearance of ghost boutons and the disruption of SSR formation reported here. Determining the mechanisms for each has therefore been complicated. Mutation of the Fz2 cleavage site, for example, may disrupt other downstream pathways because the cleavage site is also a Disheveled (Dsh) binding site. Mathew (2005) suggested that a broad array of defects arose from the failure of DFz2-C nuclear import. However, more recent work showed that bouton number, size, and microtubule organization was predominantly, though not necessarily exclusively, controlled by a presynaptic and local Wnt signalling pathway similar to the mammalian cerebellum. This left unresolved the significance of nuclear Fz2-C and other potential Fz2-derived muscle signals. The identification of a nuclear import mechanism for Fz2-C allowed a separation of postsynaptic pathways and a directed assessment of nuclear Fz2-C in synaptic development (Mosca, 2010).

Both imp-β11 and imp-α2 mutants caused errors in postsynaptic development that resembled defects associated in wg and dfz2: the increase in boutons lacking DLG and the under-development of the SSR. In contrast, neither imp-β11 nor imp-α2 had the altered bouton size or cytoskeleton seen in wg and dfz2. Only development of the SSR was consistently linked to failures in Fz2-C nuclear import. The restoration of normal postsynaptic development by NLS-Fz2-C in dfz2 and imp-β11 confirmed that nuclear DFz2-C is necessary and sufficient for normal postsynaptic development in these mutant backgrounds (Mosca, 2010).

Fz2-C import may not fully account for Wg signaling in the muscle; nearly 1/3 of wg boutons completely lacked SSR while nearly all dfz2 and imp-β11 boutons retained some SSR. A likely explanation is the presence of a parallel pathway mediated by another Wg receptor, Fz, and Dsh. Dsh is present in muscle and Fz immunoreactivity was observed at the NMJ. Indeed, partial redundancy of Fz and Fz2 exists in embryonic patterning. Because Armadillo/β-catenin, the nucleus-targeted element of the canonical Wnt pathway, is not detectable in the muscle, the Fz/Dsh pathway may be local and cytoplasmic. It is tempting to speculate that the two pathways work in parallel, with the Fz/Dsh-dependent pathway driving SSR formation locally and Fz2-C in the nucleus supporting it by transcriptional changes (Mosca, 2010).

The nuclear translocation of a synaptic Fz2-C signal illustrates the importance of synapse-to-nucleus signaling and active nuclear import for synaptic development. This mode of signalling also occurs for learning and memory models and axonal regeneration. Receptor cleavage and nuclear translocation are well-established for many developmental signals, but have chiefly been identified in single-pass transmembrane proteins. For the 7-transmembrane domain Fz receptors, however, the Drosophila NMJ is the only known case. Wnt signaling influences synapse formation in mammalian brain, and Fz5 and Fz8, the closest mammalian homologues of Drosophila Fz2, are strongly expressed in the hippocampus. Both Fz5 and Fz8 are highly conserved with Drosophila Fz2 in the vicinity of the cleavage site and also have extended C-terminal sequences. Potentially, the signaling pathway that promotes postsynaptic development in Drosophila via Wnt signaling, receptor cleavage, and active nuclear import of the C-terminus may also occur in mammals. As at the fly NMJ, mammalian nuclear import factors are likely regulators of synapse-to-nucleus signals rather than passive facilitators of transit through the nuclear pore. By associating with synaptic receptors in a manner likely to depend on receptor activation, importins offer a means to coordinate gene regulation with synaptic activity (Mosca, 2010). <

Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. It has been previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. This study shows that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes (INM and ONM) in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules (Speese, 2012).

The canonical view of nucleocytoplasmic transport posits that the sole gateway into and out of the nucleus is the nuclear pore complex (NPC). Thus all RNAs and ribonucleoprotein particles (RNPs) synthesized and assembled in the nucleus are thought to access the cytoplasm by transiting the NPC. This study now provides evidence for an alternative RNP export pathway: nuclear envelope budding. This pathway was uncovered while investigating Wnt-dependent NMJ synapse development in Drosophila larval body wall muscles. It was found that C-terminal fragments of the Wg receptor DFz2 accumulate in nuclear foci in association with large RNA granules localizing to the space between the INM and ONM. These granules are found at sites of INM invaginations, are bounded by LamC, and can be seen leaving the nucleus. Further, the granules contain transcripts encoding postsynaptic proteins, and mutations interfering with foci formation prevent proper differentiation of synaptic boutons. Thus it is suspected that, after exiting the nucleus by budding, the DFz2C RNP granules translocate to sites of synapse formation where local translation of the encoded proteins contributes to synapse assembly (Speese, 2012).

In addition to describing a noncannonical pathway for nuclear export of endogenous RNPs, this work sheds light on the previously mysterious mechanisms by which mutations in nuclear lamins and INM proteins lead to muscular dystrophies and related movement disorders. Further, by showing that nuclear import of a membrane receptor fragment serves to promote export of mRNA transcripts, this work adds a twist to the molecular mechanisms governing membrane to nucleus communication in Wnt signaling pathways (Speese, 2012).

The nuclear budding pathway described here bears remarkable resemblance to the nuclear egress mechanism employed by herpes viruses. Herpes capsids containing dsDNA are assembled in the nucleus, where they form multimegadalton complexes much too large to pass through NPCs. Instead, they exit via INM envelopment and ONM de-envelopment. Until now this highly unusual nuclear export pathway had been thought unique to this family of viruses and not representative of any endogenous nuclear export pathway. Rather it was supposed that herpes viruses had hijacked the lamina disassembly pathway operational during nuclear replication (Speese, 2012).

Both herpes virus egress and replication-dependent nuclear envelope disassembly involve multiple phosphorylation events. In capsid egress, viral proteins pUL34 and pUL31 are targeted to the INM where they recruit viral pUS3 kinase and host PKCs. Both pUS3 and the host PKCs disrupt the nuclear lamina by phosphorylating lamins, including LMNA, and other lamina-associated proteins. Both pUL34 and pUL31 have also been suggested to induce INM curvature around the capsid, facilitating budding into the perinuclear space (Speese, 2012).

Multiple lines of evidence are presented supporting the idea that local lamina remodeling at sites of DFz2C granule formation is driven by the same mechanisms at work in viral capsid egress and that these mechanisms have profound implications for synapse development. First, atypical PKC is required for both lamina remodeling and formation of INM invaginations containing DFz2C granules. Second, the apparent phosphorylation level of a species with identical electrophoretic mobility to LamC paralleled changes in aPKC activity, and this same band was immunoprecipitated with antibodies to LamC. Third, the aPKC recruitment factor Baz also localized to DFz2C/LamC foci, and Baz downregulation in muscles prevented DFz2C/LamC foci formation. Fourth, altered aPKC activity through RNAi or expression of a constitutively active enzyme, as well as decreasing Baz levels, resulted in an increase in ghost bouton number, similar to other disruptions of the Frizzled Nuclear Import (FNI) pathway. Fifth, downregulation of the FNI pathway through LamC-RNAi, or DFz2 overexpression in muscles, led to an increase in GluRIIA clustering similar to that observed in mutations in dapkc and baz. This increase in GluR clustering was reflected by an elevation of mEJP amplitude, as also observed in dapkc and baz mutants. Finally, immunofluorescence from an antibody recognizing phosphorylated PKC substrates was greatly enhanced at DFz2C/LamC foci. Thus it is proposed that herpes viruses have in fact hijacked a nuclear export pathway employed by endogenous RNPs (Speese, 2012).

That nuclear envelope budding has not been previously investigated as a means for endogenous RNP egress raises the question of whether this is a highly specialized mechanism utilized only by Drosophila larval muscle cells or is a more widespread phenomenon. The results and reports in the literature strongly support the latter view. Notably, DFz2C/LamC foci were observed in both Drosophila salivary gland and in S2 cell nuclei, both of which appear to utilize the FNI signaling pathway. Others have also reported INM infoldings containing electron dense granules (suggested to be aggregates of RNPs) in Drosophila salivary glands and midgut cells at specific developmental stages. Similar perinuclear granules have likewise been observed in diverse contexts, from plants to mammals, where they are particularly prevalent in early embryonic stages. Indeed, that such granules might reflect an alternate mode of nucleocytoplasmic export has been previously proposed, but it had not been experimentally validated until now. The combination of the current data with the numerous cytological reports of perinuclear granules strongly supports the notion that nuclear envelope budding is a mechanism for nuclear export of large RNP granules in many cell types (Speese, 2012).

Mutations across the human LMNA gene lead to a diverse set of disorders termed laminopathies, with extreme variability in their tissue specificity and pathogenesis. Some manifest as muscular dystrophies affecting specific skeletal muscles. Although laminopathies affecting muscles have been historically classified as myopathies, recent evidence in mice indicates that gross NMJ defects are detectable well before any signs of muscle degeneration. Similarly, in this study no alterations were observed in Drosophila larval muscle morphology or organization in the same lamC mutants, wherein defects at the NMJ were clearly evident. Thus, the underlying basis of many muscle-specific laminopathies could be disruption of nuclear budding leading to improper NMJ development. Consistent with this are observations that insertion of GFP sequences in the highly conserved rod domain of fly LamC (this report and Schulze, 2009) results in formation of LMNA-positive 'O-ring'structures in the nucleus, as well as disruption of both DFz2C granule organization and NMJ development. Similar O-rings have been observed in humans with autosomal-dominant AD-EDMD, which is caused by mutations in this same rod domain (Speese, 2012).

An additional link between congenital neuromuscular disease and nuclear budding is provided by studies of dystonia, a sustained muscle contraction disorder. Dystonia symptoms are ameliorated by botulinum toxin treatment, suggesting defects in neurotransmission. The AAA+ ATPase TorsinA is responsible for most cases of early-onset autosomal-dominant primary dystonia. Besides its role in synaptic vesicle recycling, a recent study indicates that TorsinA is required for HSV nuclear egress. Moreover, ultrastructural analysis of developing motorneurons in torsinA mutant mice revealed accumulations of vesicular structures in the perinuclear space. Taken together, these results raise the intriguing possibility that TorsinA functions to promote nuclear envelope scission during nuclear budding, and that alterations in RNP granule export might contribute to the phenotypic characteristics of Dystonia (Speese, 2012).

This study has identified several transcripts colocalizing with DFz2C/LamC foci encoding postsynaptically localized proteins required for synapse development and plasticity. Packaging of these RNAs into DFz2C granules appeared quite specific because numerous transcripts encoding other synaptic proteins were absent from the foci. The observation that RNAs in DFz2C foci exit the nucleus and that at least one of the transcripts, Par6, is localized to synaptic boutons, suggests that mRNAs exported by nuclear envelope budding translocate to postsynaptic sites for local translation, as has been well documented for large RNA granules in neurons. However, where such RNA granules are initially formed has not been established. The current results suggest that they could form inside the nucleus (Speese, 2012).

An important future direction will be to determine whether each granule contains a single mRNA, or is a combination of transcripts. Further, the exact role of DFz2C in assembly and/or transport of granules is at present unclear. Neither is it known whether DFz2C remains associated with the granules after nuclear egress. Intriguingly, DFz2C contains a C-terminal PDZ binding motif, raising the possibility that it may provide a zip code for targeting the granules to postsynaptic sites (Speese, 2012).

Combined, these results provide insight into how synapses communicate with the nucleus to regulate both gene expression and nuclear envelope architecture. In the future it will be of great interest to determine the extent to which the nuclear budding pathway extends to other Wnt receptors and whether it contributes to localized protein expression in response to other signal transduction pathways (Speese, 2012).

A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling

A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product,functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation, were characterized. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects (Dani, 2012).

It is well known that synaptic interfaces harbor heavily-glycosylated membrane proteins, glycolipids and ECM molecules, but understanding of glycan-mediated mechanisms within this synaptomatrix is limited. A genomic screen aimed to systematically interrogate glycan roles in both structural and functional development in the genetically-tractable Drosophila NMJ synapse. 130 candidate genes were screened, classified into 8 functional families: N-glycan biosynthesis, O-glycan biosynthesis, GAG biosynthesis, glycoprotein/proteoglycan core proteins, glycan modifying/degrading enzymes, glycosyltransferases, sugar transporters and glycan-binding lectins. From this screen, 103 RNAi knockdown conditions were larval viable, whereas 27 others produced early developmental lethality. 35 genes had statistically significant effects on different measures of morphological development: 27 RNAi-mediated knockdowns increased synaptic bouton number, 9 affected synapse area (2 increased, 7 decreased) and 2 genes increased synaptic branch number. These data suggest that overall glycan mechanisms predominantly serve to limit synaptic morphogenesis. 13 genes had significant effects on the functional differentiation of the synapse, with 12 increasing transmission strength and only 1 decreasing function upon RNAi knockdown. Thus, glycan-mediated mechanisms also predominantly limit synaptic functional development. A very small fraction of tested genes (CG1597; pgant35A, CG7480; veg, CG6657; hs6st, CG4451; sulf1, CG6725 and CG11874) had effects on both morphology and function. A large percentage of genes (~30%) showed morphological defects with no corresponding effect on function, while only 7% of genes showed functional alterations without morphological defects, and <5% of all genes affect both. These results suggest that glycans have clearly separable roles in modulating morphological and functional development of the NMJ synapse (Dani, 2012).

A growing list of neurological disorders linked to the synapse are attributed to dysfunctional glycan mechanisms, including muscular dystrophies, cognitive impairment and autism spectrum disorders. Drosophila homologs of glycosylation genes implicated in neural disease states include ALG3 (CG4084), ALG6 (CG5091), DPM1 (CG10166), FUCT1 (CG9620), GCS1 (CG1597), MGAT2 (CG7921), MPDU1 (CG3792), PMI (CG33718) and PPM2 (CG12151). Two of these genes, Gfr (CG9620) and CG1597, showed synaptic morphology phenotypes in the RNAi screen. Given that connectivity defects are clearly implicated in cognitive impairment and autism spectrum disorders, it would be of interest to explore the glycan mechanism affecting synapse morphology in Drosophila models of these disease states. Glycans are well known to modulate extracellular signaling, including ligands of integrin receptors, to regulate intercellular communication. In the genetic screen, several O-glycosyltransferases mediating this mechanism were identified to show morphological (GalNAc-T2, CG6394; pgant35A, CG7480, O-fut2, CG14789; rumi, CG31152) and functional (pgant5, CG31651; pgant35A, CG7480) synaptic defects upon RNAi knockdown. These findings suggest that known integrin-mediated signaling pathways controlling NMJ synaptic structural and functional development are modulated by glycan mechanisms. The screen showed CG6657 RNAi knockdown affects functional differentiation, consistent with reports that this gene regulates peripheral nervous system development. The corroboration of the screen results with published reports underscores the utility of RNAi-mediated screening to identify glycan mechanisms, and supports use of the screen results for bioinformatic/meta-analysis to link observed phenotypes to neurophysiological/pathological disease states and to direct future glycan mechanism studies at the synapse (Dani, 2012).

From this screen, the two functionally-paired genes sulf1 and hs6st were selected for further characterization. As in the RNAi screen, null alleles of these two genes had opposite effects on synaptic functional differentiation but similar effects on synapse morphogenesis, validating the corresponding screen results. The two gene products have functionally-paired roles; Hs6st is a heparan sulfate (HS) 6-O-sulfotransferase, and Sulf1 is a HS 6-O-endosulfatase. These activities control sulfation of the same C6 on the repeated glucosamine moiety in HS GAG chains found on heparan sulfate proteoglycans (HSPGs). At the Drosophila NMJ, two HSPGs are known to regulate synapse assembly; the GPI-anchored glypican Dally-like protein (Dlp), and the transmembrane Syndecan (Sdc). In contrast, the secreted HSPG Perlecan (Trol) is not detectably enriched at the NMJ, and indeed appears to be selectively excluded from the perisynaptic domain. In other developmental contexts, the membrane HSPGs Dlp and Sdc are known to act as co-receptors for WNT and BMP ligands, regulating ligand abundance, presentation to cognate receptors and therefore signaling. Importantly, the regulation of HSPG co-receptor abundance has been shown to be dependent on sulfation state mediated by extracellular sulfatases. Consistently, upregulation of Dlp and Sdc was observed in sulf1 null synapses, whereas Dlp was reduced in hs6st null synapses. In the developing Drosophila wing disc, HSPG co-receptors increase levels of the Wg ligand due to extracellular stabilization, and the primary function of Dlp in this developmental context is to retain Wg at the cell surface. Likewise, in developing Drosophila embryos, a significant fraction of Wg ligand is retained on the cell surfaces in a HSPG-dependent manner, with the HSPG acting as an extracellular co-receptor. Syndecan also modulates ligand-dependent activation of cell-surface receptors by acting as a co-receptor. At the NMJ, regulation of both these HSPG co-receptors occurs in the closely juxtaposed region between presynaptic bouton and muscle subsynaptic reticulum, in the exact same extracellular space traversed by the secreted trans-synaptic Wg and Gbb signals. It is therefore proposed that altered Dlp and Sdc HSPG co-receptors in sulf1 and hs6st mutants differentially trap/stabilize Wg and Gbb trans-synaptic signals at the interface between motor neuron and muscle, to modulate the extent and efficacy of intercellular signaling driving synaptic development (Dani, 2012).

HS sulfation modification is linked to modulating the intercellular signaling driving neuronal differentiation . In particular, WNT and BMP ligands are both regulated via HS sulfation of their extracellular co-receptors, and both signals have multiple functions directing neuronal differentiation, including synaptogenesis. In the Drosophila wing disc, extracellular WNT (Wg) ligand abundance and distribution was recently shown to be strongly elevated in sulf1 null mutants. Moreover, sulf1 has also recently been shown to modulate BMP signaling in other cellular contexts. Consistently, this study has shown increased WNT Wg and the BMP Gbb abundance and distribution in sulf1 null NMJ synapses. The hs6st null also exhibits elevated Wg and Gbb at the synaptic interface, albeit the increase is lower and results in differential signaling consequences. In support of this contrasting effect, extracellular signaling ligands are known to bind HSPG HS chains differentially dependent on specific sulfation patterns. It is important to note that the sulf1 and hs6st modulation of trans-synaptic signals is not universal, as Jelly Belly (Jeb) ligand abundance and distribution was not altered in the sulf1 and hs6st null conditions. This indicates that discrete classes of secreted trans-synaptic molecules are modulated by distinct glycan mechanisms to control NMJ structure and function (Dani, 2012).

At the Drosophila NMJ, Wg is very well characterized as an anterograde trans-synaptic signal and Gbb is very well characterized as a retrograde trans-synaptic signal. In Wg signaling, the dFz2 receptor is internalized upon Wg binding and then cleaved so that the dFz2-C fragment is imported into muscle nuclei. In hs6st nulls, increased Wg ligand abundance at the synaptic terminal corresponds to an increase in dFz2C punctae in muscle nuclei as expected. In contrast, the increase in Wg at the sulf1 null synapse did not correspond to an increase in the dFz2C-terminus nuclear internalization, but rather a significant decrease. One explanation for this apparent discrepancy is the 'exchange factor' model based on the biphasic ability of the HSPG co-receptor Dlp to modulate Wg signaling. In the Drosophila wing disc, this model suggests that the transition of Dlp co-receptor from an activator to repressor of signaling depends on Wg cognate receptor dFz2 levels, such that a low ratio of Dlp:dFz2 potentiates Wg-dFz2 interaction, whereas a high ratio of Dlp:dFz2 prevents dFz2 from capturing Wg. In sulf1 null synapses, a very great increase was observed in Dlp abundance (~40% elevated) with no significant change in the dFz2 receptor. In contrast, at hs6st null synapses there is a decrease in Dlp abundance (15% decreased) together with a significant increase in dFz2 receptor abundance (~25% elevated). Thus, the higher Dlp:dFz2 ratio in sulf1 nulls could explain the decrease in Wg signal activation, evidenced by decreased dFz2-C terminus import into the muscle nucleus. In contrast, the Dlp:Fz2 ratio in hs6st is much lower, supporting activation of the dFz2-C terminus nuclear internalization pathway. This previously proposed competitive binding mechanism dependent on Dlp co-receptor and dFz2 receptor ratios predicts the observed synaptic Wg signaling pathway modulation in sulf1 and hs6st dependent manner (Dani, 2012).

At the Drosophila NMJ, Gbb is very well characterized as a retrograde trans-synaptic signal, with muscle-derived Gbb causing the receptor complex Wishful thinking (Wit), Thickveins (Tkv) and Saxaphone (Sax) to induce phosphorylation of the transcription factor mothers against Mothers against decapentaplegic (P-Mad). Mutation of Gbb ligand, receptors or regulators of this pathway have shown that Gbb-mediated retrograde signaling is required for proper synaptic differentiation and functional development. Further, loss of Gbb signaling results in significantly decreased levels of P-Mad in the motor neurons. This study shows that accumulation of Gbb in sulf1 and hs6st null synapses causes elevated P-Mad signaling at the synapse and P-Mad accumulation in motor neuron nuclei. Importantly, sulf1 null synapses show a significantly higher level of P-Mad signaling compared to hs6st null synapses, and this same change is proportionally found in P-Mad accumulation within the motor neuron nuclei. These findings indicate differential activation of Gbb trans-synaptic signaling dependent on the HS sulfation state is controlled by the sulf1 and hs6st mechanism, similar to the differential effect observed on Wg trans-synaptic signaling. Genetic interaction studies show that these differential effects on trans-synaptic signaling have functional consequences, and exert a causative action on the observed bi-directional functional differentiation phenotypes in sulf1 and hs6st nulls. Genetic correction of Wg and Gbb defects in the sulf1 null background restores elevated transmission back to control levels. Similarly, genetic correction of Wg and Gbb in hs6st nulls restores the decreased transmission strength back to control levels. These results demonstrate that the Wg and Gbb trans-synaptic signaling pathways are differentially regulated and, in combination, induce opposite effects on synaptic differentiation (Dani, 2012).

Both wg and gbb pathway mutants display disorganized and mislocalized presynaptic components at the active zone (e.g. Bruchpilot; Brp) and postsynaptic components including glutamate receptors (e.g. Bad reception; Brec/GluRIID). Consistently, the bi-directional effects on neurotransmission strength in sulf1 and hs6st mutants are paralleled by dysregulation of these same synaptic components. Changes in presynaptic Brp and postsynaptic GluR abundance/distribution causally explain the bi-directional effects on synaptic functional strength between sulf1 and hs6st null mutant states. Alterations in active zone Brp and postsynaptic GluRs also agree with assessment of spontaneous synaptic activity. Null sulf1 and hs6st synapses showed opposite effects on miniature evoked junctional current (mEJC) frequency (presynaptic component) and amplitude (postsynaptic component). Further, quantal content measurements also support the observation of bidirectional synaptic function in the two functionally paired nulls. Genetic correction of Wg and Gbb defects in both sulf1 and hs6st nulls restores the molecular composition of the pre- and postsynaptic compartments back to wildtype levels. When both trans-synaptic signaling pathways are considered together, these data suggest that HSPG sulfate modification under the control of functionally-paired sulf1 and hs6st jointly regulates both WNT and BMP trans-synaptic signaling pathways in a differential manner to modulate synaptic functional development on both sides of the cleft (Dani, 2012).

This paper has presented the first systematic investigation of glycan roles in the modulation of synaptic structural and functional development. A host of glycan-related genes were identified that are important for modulating neuromuscular synaptogenesis, and these genes are now available for future investigations, to determine mechanistic requirements at the synapse, and to explore links to neurological disorders. As proof for the utilization of these screen results, this study has identified extracellular heparan sulfate modification as a critical platform of the intersection for two secreted trans-synaptic signals, and differential control of their downstream signaling pathways that drive synaptic development. Other trans-synaptic signaling pathways are independent and unaffected by this mechanism, although it is of course possible that a larger assortment of signals could be modulated by this or similar mechanisms. This study supports the core hypothesis that the extracellular space of the synaptic interface, the heavily-glycosylated synaptomatrix, forms a domain where glycans coordinately mediate regulation of trans-synaptic pathways to modulate synaptogenesis and subsequent functional maturation (Dani, 2012).

Shank modulates postsynaptic wnt signaling to regulate synaptic development

Prosap/Shank scaffolding proteins regulate the formation, organization, and plasticity of excitatory synapses. Mutations in SHANK family genes are implicated in autism spectrum disorder and other neuropsychiatric conditions. However, the molecular mechanisms underlying Shank function are not fully understood, and no study to date has examined the consequences of complete loss of all Shank proteins in vivo. This study characterized the single Drosophila Prosap/Shank family homolog. Shank is enriched at the postsynaptic membrane of glutamatergic neuromuscular junctions and controls multiple parameters of synapse biology in a dose-dependent manner. Both loss and overexpression of Shank result in defects in synaptic bouton number and maturation. It was found that Shank regulates a noncanonical Wnt signaling pathway in the postsynaptic cell by modulating the internalization of the Wnt receptor Fz2. This study identifies Shank as a key component of synaptic Wnt signaling, defining a novel mechanism for how Shank contributes to synapse maturation during neuronal development (Harris, 2016).

The postsynaptic density (PSD) of excitatory synapses contains a complex and dynamic arrangement of proteins, allowing the cell to respond to neurotransmitter and participate in bidirectional signaling to regulate synaptic function. Prosap/Shank family proteins are multidomain proteins that form an organizational scaffold at the PSD. Human genetic studies have implicated SHANK family genes as causative for autism spectrum disorder (ASD) (Uchino and Waga, 2013; Guilmatre, 2014), with haploinsufficiency of SHANK3 considered one of the most prevalent causes (Betancur and Buxbaum, 2013). Investigations of Shank in animal models have identified several functions for the protein at synapses, including regulation of glutamate receptor trafficking, the actin cytoskeleton, and synapse formation, transmission, and plasticity (Grabrucker, 2011; Jiang and Ehlers, 2013). However, phenotypes associated with loss of Shank are variable, and it has been challenging to fully remove Shank protein function in vivo as a result of redundancy between three Shank family genes and the existence of multiple isoforms of each Shank. There is a single homolog of Shank in Drosophila (Liebl and Featherstone, 2008), presenting the opportunity to characterize the function of Shank at synapses in vivo in null mutant animals (Harris, 2016).

Wnt pathways play important roles in synaptic development, function, and plasticity. Like Shank and several other synaptic genes, deletions and duplications of canonical Wnt signaling components have been identified in individuals with ASD. A postsynaptic noncanonical Wnt pathway has been characterized at the Drosophila glutamatergic neuromuscular junction (NMJ), linking release of Wnt by the presynaptic neuron to plastic responses in the postsynaptic cell. In this Frizzled-2 (Fz2) nuclear import (FNI) pathway, Wnt1/Wg is secreted by the neuron and binds its receptor Fz2 in the postsynaptic membrane. Surface Fz2 is then internalized and cleaved, and a C-terminal fragment of Fz2 (Fz2-C) is imported into the nucleus in which it interacts with ribonucleoprotein particles containing synaptic transcripts. Mutations in this pathway result in defects of synaptic development at the NMJ (Harris, 2016 and references therein).

In this study, a null allele of Drosophila Shank was created, allowing investigation of the consequences of removing all Shank protein in vivo. Loss of Shank is shown to impair synaptic bouton number and maturity and results in defects in the organization of the subsynaptic reticulum (SSR), a complex system of infoldings of the postsynaptic membrane at the NMJ. It was also demonstrated that overexpression of Shank has morphological consequences similar to loss of Shank and that Shank dosage is critical to synaptic development. Finally, the results indicate that Shank regulates the internalization of Fz2 to affect the FNI signaling pathway, revealing a novel connection between the scaffolding protein Shank and synaptic Wnt signaling (Harris, 2016).

By generating Drosophila mutants completely lacking any Shank protein, this study identified a novel function of this synaptic scaffolding protein in synapse development. Aberrant expression of Shank results in defects affecting synapse number, maturity, and ultrastructure, and a subset of these defects is attributable to a downregulation of a noncanonical Wnt signaling pathway in the postsynaptic cell (Harris, 2016).

The defects observed in Shank mutants are mostly consistent with defects described from in vivo and in vitro rodent models of Shank. Synaptic phenotypes reported from Shank mutants vary, likely reflecting incomplete knockdown of Shank splice variants, and heterogeneity in the requirement for Shank between the different brain regions and developmental stages analyzed (for review, see Jiang and Ehlers, 2013). Nevertheless, taken collectively, analyses of Shank1-Shank3 mutant mice indicate that Shank genes regulate multiple parameters of the structure and function of glutamatergic synapses, including the morphology of dendritic spines and the organization of proteins in the PSD (Harris, 2016 and references therein).

By removing all Shank protein in Drosophila, this study identified essential functions for Shank at a model glutamatergic synapse. Shank mutants exhibit prominent abnormalities in synaptic structure, including a decrease in the total number of synaptic boutons, which results in an overall decrease in the number of AZs. In addition, a subset of synaptic boutons fails to assemble a postsynaptic apparatus. Finally, even in mature boutons, the SSR has fewer membranous folds and makes less frequent contact with the presynaptic membrane, indicating a defect in postsynaptic development. The SSR houses and concentrates important synaptic components near the synaptic cleft, including scaffolding proteins, adhesion molecules, and glutamate receptors. Thus, defects in SSR development can affect the assembly and regulation of synaptic signaling platforms. These findings indicate that Shank is a key regulator of synaptic growth and maturation (Harris, 2016).

The findings also indicate that gene dosage of Shank is critical for normal synapse development at Drosophila glutamatergic NMJs. The morphological phenotypes that were observed scale with the level of Shank expression, with mild phenotypes seen with both 50% loss and moderate overexpression of Shank, and severe phenotypes seen with both full loss and strong overexpression of Shank. The observation of synapse loss in heterozygotes of the Shank null allele is significant, because haploinsufficiency of SHANK3 is well established as a monogenic cause of ASD (Harris, 2016).

Consistent with the observation that excess Shank is detrimental, duplications of the SHANK3 genomic region (22q13) are known to cause a spectrum of neuropsychiatric disorders. Large duplications spanning SHANK3 and multiple neighboring genes have been reported in individuals with attention deficit-hyperactivity disorder (ADHD), schizophrenia, and ASD. Smaller duplications, spanning SHANK3 and only one or two adjacent genes, have been reported in individuals with ADHD, epilepsy, and bipolar disorder. Furthermore, duplication of the Shank3 locus in mice results in manic-like behavior, seizures, and defects in neuronal excitatory/inhibitory balance. Thus, the requirement for proper Shank dosage for normal synaptic function may be a conserved feature (Harris, 2016).

One unexpected finding from this study was the identification of a previously unappreciated aspect of Shank as a regulator of Wnt signaling. Shank regulates the internalization of the transmembrane Fz2 receptor, thus affecting transduction of Wnt signaling from the plasma membrane to the nucleus. Downregulation of this pathway is implicated in impaired postsynaptic organization, including supernumerary GBs and SSR defects. The physical proximity of Shank and Fz2 at the postsynaptic membrane suggests that Shank directly or indirectly modulates the internalization of Fz2. Shank is a scaffolding protein with many binding partners that could contribute to such an interaction. One intriguing possibility is the PDZ-containing protein Grip. Shank2 and Shank3 have been reported to bind Grip1. Furthermore, Drosophila Grip transports Fz2 to the nucleus on microtubules to facilitate the FNI pathway (Ataman, 2006). Thus, an interaction between Shank, Fz2, and Grip to regulate synaptic signaling is an attractive model (Harris, 2016).

Although loss of Shank is associated with impaired internalization of the Fz2 receptor, how excess Shank leads to FNI impairment remains an open question. One possibility is that an increase in the concentration of the Shank scaffold at the synapse physically impedes the transport of Fz2 or other components of the pathway or saturates binding partners that are essential for Fz2 trafficking. Both overexpression and loss of function of Shank ultimately lead to a failure to accumulate the cleaved Fz2 C terminus within the nucleus, in which it is required to interact with RNA binding proteins that facilitate transport of synaptic transcripts to postsynaptic compartments. Although Shank and Wnt both play important synaptic roles, this study is the first demonstration of a functional interaction between Shank and Wnt signaling at the synapse (Harris, 2016).

Intriguingly, no obvious defects were found in glutamate receptor levels or distribution in the absence of Shank. This was surprising given the role for Shank in regulating the FNI pathway, and downregulation of FNI was shown previously to lead to increased GluR field size. Several studies have reported changes in the levels of AMPA or NMDA receptor subunits in Shank mutant mice, although others have also observed no changes. Levels of metabotropic glutamate receptors are also affected in some Shank mutant models. Moreover, transfected Shank3 can recruit functional glutamate receptors in cultured cerebellar neurons. It is possible that Drosophila Shank mutants have defects in GluRs that are too subtle to detect with current methodology. Another possibility is that Shank is involved in signaling mechanisms that are secondary to FNI and that lead to compensatory changes in GluRs at individual synapses. Indeed, the results are consistent with Shank having additional functions at the synapse in addition to its role in FNI, particularly affecting synaptic bouton number. In conclusion, this study has fpind that the sole Drosophila Shank homolog functions to regulate synaptic development in a dose-dependent manner, providing a new model system to further investigate how loss of this scaffolding protein may underlie neurodevelopmental disease (Harris, 2016).

frizzled2: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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