Interaction of Wingless with its receptors, Frizzled and Frizzled2 and Dfrizzled-3

Frizzled2, a Drosophila homolog of the frizzled family, is considered to be the receptor for Wingless. Schneider (S2) cultured cells fail respond to Wingless protein, that is they fail to stabilize Armadillo in response to added WG. S2 cells fail to respond to added WG because they do not express fz2. A plasmid was constructed carrying the fz2 gene was transfected into S2 cells. When fz2-transfected cells are incubated in the presence of WG, the level of faster migrating (hypophosphorylated) ARM increases. In addition, transfected S2 cells show strong surface staining when incubated with WG and anti-WG antibodies, indicating a direct effect of fz2-transfection on WG binding (Bhanot, 1996).

FZ2 proteins possess a conserved region of 120 amino acids with an invariant pattern of ten cysteine residues (the cysteine rich domain, CRD). The binding of Wingless to the FZ2 CRD extracellular domain could result in a rearrangement of transmembrane domains and activation of downstream cytoplasmic proteins. When a truncated form of FZ2 carrying an isolated CRD segment and part of the adjacent linker region is displayed on the surface of cultured cells and incubated with WG and anti-WG antibodies, strong surface staining is detected. This provides evidence that the CRD constitutes a significant part of the WG binding domain (Bhanot, 1996).

Mutations in the wg gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with DFz2 (Frizzled 2), a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if DFz2 functions in the Wg signaling pathway during fly development. Overexpression of DFz2 is shown to increase Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. Alongside the anterior margin, supernumerary stout mechanosensory bristles are often observed one or more cell diameters away from the normal stout bristle row. Within the wing, supernumerary chemosensory and slender mechanosensory bristles are detected. The bristle types (stout, slender and chemosensory) are located on the appropriate dorsal or ventral surface of the wing, and are restricted to the anterior region of the wing. Alongside the posterior wing margin, supernumerary non-sensory bristles are observed several cell diameters distant from the margin where these bristles are normally located. The formation of supernumerary bristles by DFz2 does not appear to result from a disturbance in lateral inhibition between neighboring bristle precursor cells. Contrary to what is typically seen when lateral inhibition is perturbed, there is no overall change in margin bristle density. The spacing between bristles within the three regular rows of anterior bristles and within the single row of posterior bristles of wings overexpressing DFz2 is indistinguishable from that of wild type. No ectopic bristles are observed when Fz2 is overexpressed. However, the polarity of margin bristles is abnormal, and wing hair polarity is deranged throughout the plane of the wing blade. It is suggested that the results of overexpression reflect an independence between Fz and DFz2 pathways during wild-type wing development (Zhang, 1998).

Overexpression of a truncated form of DFz2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length DFz2 but not full-length Fz. These results suggest that DFz2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development; the truncated form of DFz2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or DFz2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity (Zhang, 1998).

How does the overexpression of DFz2 lead to ectopic bristle induction? One possibility is that cells at some distance from the narrow stripe of Wg-secreting margin cells are normally exposed to a low level of Wg, insufficient to induce bristle determination. Overexpression of DFz2, combined with endogenous receptors, may increase Wg signal transduction in some of these cells to a level comparable to those nearer the wing margin, thus inducing ectopic margin bristles. It is worth noting that at the anterior margin, most ectopic bristles induced close to the margin are of the stout mechanosensory type, which are normally present in a row of cells closest to the stripe of Wg secretion. The ectopic bristles further away from the wing margin are usually of the slender or chemosensory types, which are normally present in two rows of cells more distant from the Wg stripe than the stout row. This suggests that Wg is normally present in the wing as a concentration gradient surrounding the wing margin. Overexpression of DFz2 increases the level of Wg signal transduction in cells that are exposed to a particular concentration of Wg. Consequently, cells adopt fates that are normally induced only at a higher concentration of Wg. Cells that would have normally adopted slender or chemosensory bristle fates instead become stout bristles; cells that would have normally failed to adopt any bristle fate instead become slender or chemosensory bristles. This interpretation is consistent with observations that Wg acts as a gradient morphogen to induce expression of different target genes near the wing margin (Zhang, 1998).

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).

A study of wingless mutants has begun to unravel the Wingless protein structural requirement for signaling. Signaling by Wingless involves binding to the cell surface. A study of Wingless structural protein mutations reveals that mutant wg can bind to the cell surface and be transported in vesicles but cannot signal. Endocytosis is dispensible for wild type levels of signaling in cells that are immediately adjacent to wg-expressing cells (Bejsovec, 1995).

Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the Wingless pathway

Recently, double-stranded RNA (dsRNA) has been found to be a potent and specific inhibitor of gene activity in the nematode Caenorhabditis elegans (Fire, 1998). The potential of dsRNA to interfere with the function of genes in Drosophila, termed RNA inhibition or RNAi) has been investigated. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. dsRNA corresponding to four genes with previously defined functions was introduced. dsRNA is shown to potently and specifically inhibits the activities of wg, fushi tarazu (ftz), even-skipped (eve), and tramtrack (ttk). The reception mechanism of the morphogen Wingless was determined using dsRNA. Interference of the frizzled and Drosophila frizzled 2 genes together produces defects in embryonic patterning that mimic the loss of wingless function. Interference with the function of either gene alone has no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. These results demonstrate that dsRNA interference can be used to analyze many aspects of gene function (Kennerdell, 1998).

The potency and specificity of dsRNA interference on gene activity suggests that it might be a useful means to eliminate Frizzled2 activity. Although the null phenotype of Fz2 is unknown, it was reasoned that if Fz2 encodes the Wg receptor, then its mutant phenotype should resemble wg loss-of-function mutants. Larvae that lack wg activity are completely covered with denticles on the ventral cuticle, unlike wild-type larvae in which ventral cuticle is an alternating pattern of naked cuticle and denticles. When dsRNA corresponding to the wg gene is injected, the region around the site of injection exhibits a wg-like mutant phenotype, and the remainder of the embryo was wild type. Surprisingly, no animals exhibit a null phenotype despite the injection of twice as much dsRNA as for other genes. The RNAi (RNA inhibition) effect is localized, and the range of phenotypes is limited by the size of the region with ectopic denticles. When dsRNA corresponding to the 5' UTR of Fz2 is injected, no effect on denticle patterning is observed. Ectopic denticles are not observed in embryos injected with dsRNA corresponding to the 5' UTR of the fz gene. In contrast, an equimolar mixture of ds-fz and ds-Fz2 RNAs causes localized transformation of naked cuticle into denticles. The RNAi effect is limited to the site of injection even at high doses of dsRNA, and its potency is highly similar to the potency of ds-wg RNA. Denticles in the affected regions resemble those typical of the fifth row in a wild-type abdominal segment, and the denticles are oriented either toward the midline or along the anteroposterior axis with reversed polarity. These features are precisely those observed in wg mutant embryos and embryos treated with ds-wg RNA (Kennerdell, 1998).

Engrailed expression initiates normally in wg mutants but fails to be maintained. To examine whether Fz and Fz2 have a similar function, embryos were injected with ds-fz and ds-Fz2 RNAs. After further development, the embryos were stained with an anti-Engrailed antibody. Expression of engrailed os absent in lateral ectoderm within the affected region. This discontinuous loss of engrailed expression resembles loss of functional wg (Kennerdell, 1998).

The interfering activity of ds-fz and ds-Fz2 RNA mixtures could mean that the fz and Fz2 genes act redundantly, and the activities of both genes must be blocked before a phenotype is observed. Alternatively, it could reflect some other synergy between the injected RNAs. ds-Fz2 RNA was injected alone into embryos mutant for fz and was found to possess interfering activity that is comparable to the interfering activity of mixed ds-fz and ds-Fz2 RNAs. These data are most consistent with the fz and Fz2 genes acting redundantly to pattern the ventral epidermis (Kennerdell, 1998).

Experiments in cell culture have suggested that Fz2 acts as a receptor in the Wg signal transduction pathway. Do Fz and Fz2 act between Wg and the intracellular components of its signal transduction pathway? Genetic epistasis can determine the order of action of genes in a common pathway. If fz and Fz2 function downstream of wg, then interference of fz and Fz2 activities should suppress activating mutations of wg. A transgenic strain that expresses high levels of Wg in all epidermal cells causes those cells to secrete naked cuticle. This strain was used to determine whether fz and Fz2 are required for wg action. When these animals are injected with ds-fz and ds-Fz2 RNAs, the formation of ectopic naked cuticle is suppressed. The injected transgenic embryos are distinct from wild-type embryos injected with RNA and from the uninjected transgenic strain. They resembled wild-type embryos injected with dsRNA in that they have denticles alternating with naked cuticle plus some localized patches of continuous denticle lawn. However, they do not usually have the complete complement of denticles. This incomplete suppression is attributed to the fact that interference of fz and Fz2 is primarily localized to regions close to the site of injection. Nevertheless, this result provides genetic evidence for a function of fz and Fz2 downstream of wg (Kennerdell, 1998).

Transduction of a Wg signal antagonizes the Shaggy/Zw3 kinase, which functions to modulate levels of Arm. Do fz and Fz2 act between wg and shaggy, as would be predicted for the Wg receptor? Loss of shaggy activity results in all epidermal cells adopting posterior segmental fates, and mutant embryos lack ventral denticles. When shaggy mutant embryos are injected with ds-fz and ds-Fz2 RNAs, there is no change in their phenotype; they resemble shaggy embryos. The similarity of the phenotypes of shaggy with or without fz and Fz2 interference suggests that fz and Fz2 function upstream of zw3 in wg signaling (Kennerdell, 1998).

Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants

The absence of Drosophia Frizzled-3 produces no apparent phenotype. Binding studies reveal that Wg can interact with Dfz3 in cultured cells. In order to reveal a role for Dfz3 in development, the possiblity of a genetic interaction of Dfz3 with wingless has been investigated. Dfz3 may be involved in Wg signaling required for adult appendage formation. For example, Dfz3 may serve as an attenuator of Wg signaling, at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation. For analysis of this possiblility, a study was made to find possible interaction between Dfz3 and Wg signaling in various wg mutant backgrounds. Wing blades are frequently absent from flies mutant for wg 1. Thus, the first question to be examined was is the wg 1 phenotype affected by the absence of Dfz3? The absence of wing blades is partially rescued through the elimination of Dfz3 activity. On a wg 1/wg CX4 background, fractions of flies with two wings increased from 46% to 87%, while those flies with one wing and wing-less flies, respectively, reduced from 44% and 10% to 13% and 0.5%. The wing-less phenotype of wg 1 is enhanced in a heterozygous apterous (ap) mutant background: no wing blade is generated at approx. 90% of the presumptive wing-blade-forming sites in wg 1 homozygous flies heterozygous for ap. Wing blade formation increases 3-fold in the absence of Dfz3 activity. Since wg CX4 and wg 1 are null and regulatory mutant alleles, respectively, these effects are not due to possible change in Wg protein conformation. Thus, wild-type Dfz3 may serve as an attenuator of Wg signaling at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation (A. Sato, 1999).

To confirm that Dfz3 attenuates Wg signaling, an examination was made of the effects of Dfz3 absence in a different developmental context. Nearly all wg11en/wgCX4 flies lack antennal structures. This antenna-less phenotype is significantly rescued by removing Dfz3 activity; complete antennal structures, as well as incomplete ones, areregenerated at more than 70% of putative antennal sites. Distal antennal segment formation requires the circular expression of Bar homeobox genes. Dachshund (Dac) is required for the formation of proximal leg structures and expressed circularly in leg and antenna discs. Thus, wg 11en/wgCX4 fly discs with or without Dfz3 activity were stained for Wg, BarH1 and Dac. When there is Dfz3 activity, antennal discs are small and no or little expression of BarH1 and Dac is detected. In the absence of Dfz3 activity, about 10% of the discs, probably corresponding to the completely rescued type, exhibit circular BarH1 and Dac expression similar to that of wild-type discs. In about 50% of discs, presumably corresponding to the partially rescued type, Dac expression is partially restored without recovery of BarH1 expression. In contrast to BarH1 and Dac, no Wg expression is detected in the rescued mutant discs, indicating that wg expression is not enhanced by the absence of Dfz3. That wgCX4 and wg 11en are regulatory mutant alleles of wg suggests again that the genetic interactions found here would not be due to possible change in Wg protein structure, but simply to reduction in transcription products of wg. Thus it follows that in wg hypomorphic mutants, Dfz3 reduces Wg signaling activity required for antennal formation without changing wg expression; accordingly, Dfz3 would appear to function as a negative factor or attenuator of Wg signaling at least on a wg hypomorphic mutant background (A. Sato, 1999).

To determine whether Dfz3 is capable of binding to Wg and transducing Wg signals, fz2 and Dfz3 were expressed under the control of the metallothionein promoter in Schneider line 2 (S2) cells. To examine Wg binding, either fz2 or Dfz3 was transiently expressed in S2 cells. Transfectants were then incubated with Wg-conditioned medium and stained with anti-Wg antibody. Not only fz2- but also Dfz3-transfectants showed strong surface staining while no surface signals could be detected in pMK33-transfected cells, indicating tight binding of Wg to Dfz3. To determine whether Dfz3 is capable of transducing Wg signals, Arm stabilization in response to added Wg was assessed. Arm is phosphorylated by Zeste-White 3 kinase and the phosphorylation is suppressed by Dsh (activated by Wg signals), thus permitting assay of Wg signaling activity by Western blotting using anti-Arm antibody. S2 cells were transfected by fz2 or Dfz3 cDNA, whose expressions were controlled by the metallothionein promoter. Arm always accumulates in fz2-expressing cells in a Wg-dependent manner. In Dfz3-expressing cells, there is little or no Wg-dependent Arm accumulation; Wg-dependent Arm accumulation in S2/Dfz3-C1 and C2 is marginal while that in S2/Dfz3-C3 and C4, is low but significant. Thus it may be, at least in cultured S2 cells, that Dfz3 is capable of serving as a transducer of Wg signals but its activity is much less than that of Fz2 (A. Sato, 1999).

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic Wingless signal transduction

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).

Wingless capture by Frizzled and Frizzled2 in Drosophila embryos

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 discs. 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 discs 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).

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).

Wnt signals across the plasma membrane to activate the ß-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP

Wnt-induced signaling via ß-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes ß-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. Mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for ß-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells were used to study transmission of a Wnt signal across the plasma membrane. The findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for ß-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled, and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the ß-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors (Cong, 2004).

What is the mechanism by which Frizzled transduces a Wnt signal? Mutations that disrupt the signaling activity of Frizzled also affect the ability of Frizzled to induce membrane translocation of Dvl and reduce physical interaction between Frizzled and Dvl, suggesting that a physical interaction between Frizzled and Dvl is required for the signaling activity of Frizzled. It is proposed that Frizzled might function as a docking site for Dvl in ß-catenin signaling. The results are consistent with the finding that the Lys-Thr-x-x-x-Trp motif at the C-terminal tail of Frizzled is not only required for activating ß-catenin signaling, but also for inducing Dvl membrane translocation. The PDZ domain of Dvl has been shown to directly bind to a peptide of C-terminal region of Frizzled containing the Lys-Thr-x-x-x-Trp motif, and this peptide can inhibit Wnt/ß-catenin signaling in Xenopus. However, the binding is relatively weak (Kd~10 microM). The current results suggest that multiple regions of Frizzled might be involved in the binding with Dvl and could increase the binding affinity (Cong, 2004).

The same structural elements may be required for Frizzled to function in both the planar polarity and the ß-catenin pathways, since membrane translocation of Dvl has been implicated in planar polarity signaling, and residues essential for the activity of Frizzled in ß-catenin signaling are also important for Frizzled-induced translocation of Dvl to the plasma membrane. It is possible that other proteins in the Frizzled-Dvl complex, such as LRP in ß-catenin signaling and Flamingo in planar polarity signaling, determine the signaling consequences of interaction between Frizzled and Dvl (Cong, 2004).

What is the role of LRP in transmitting the Wnt signal and what is the function of its extracellular domain of LRP for receiving the Wnt signal? An in vitro binding assay has suggested that Wnt1 is able to bind to the extracellular domain of LRP, but analogous binding was not observed in studies with Wg protein. Results from in vitro binding assays need to be treated cautiously, as the concentrations of ligands and receptors in these assays could be significantly higher than in physiological situations, and certain components normally involved in formation of the receptor complex could be missing in these assays. Therefore, functional data are necessary to address the significance of potential binding between Wnt and LRP. The extracellular domain of LRP can be functionally replaced by the extracellular domain of Frizzled, suggesting a physiological role for a direct, or indirect, interaction of Wnt with the extracellular domain of LRP (Cong, 2004).

LRP can also transmit a signal via ß-catenin without a requirement for Wnt. Advantage was taken of two commonly used inducible oligomerization strategies to demonstrate that oligomerization of LRP6 increases its signaling activity and its interaction with Axin. Interestingly, it has been shown that the second cysteine-rich domain of DKK2 stimulates ß-catenin signaling via LRP independently of Dvl. Further experiments are needed to determine whether this DKK2 fragment activates LRP by altering the oligomerization status of LRP (Cong, 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 junctions (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).

Notum coordinates synapse development via extracellular regulation of Wnt Wingless trans-synaptic signaling

Synaptogenesis requires orchestrated communication between pre- and postsynaptic cells via coordinated trans-synaptic signaling across the extracellular synaptomatrix. The first discovered Wnt signaling ligand Drosophila Wingless (Wg; Wnt-1 in mammals) plays critical roles in synaptic development, regulating synapse architecture as well as functional differentiation. This study investigated synaptogenic functions of the secreted extracellular deacylase Notum, which restricts Wg signaling by cleaving an essential palmitoleate moiety. At the glutamatergic neuromuscular junction (NMJ) synapse, Notum secreted from the postsynaptic muscle was found to act to strongly modulate synapse growth, structural architecture, ultrastructural development and functional differentiation. In notum nulls, upregulated extracellular Wg ligand and nuclear trans-synaptic signal transduction was found, as well as downstream misregulation of both pre- and postsynaptic molecular assembly. Structural, functional and molecular synaptogenic defects are all phenocopied by Wg over-expression, suggesting Notum acts solely through inhibiting Wg trans-synaptic signaling. Moreover, these synaptic development phenotypes are suppressed by genetically correcting Wg levels in notum null mutants, indicating that Notum normally functions to coordinate synaptic structural and functional differentiation via negative regulation of Wg trans-synaptic signaling in the extracellular synaptomatrix (Kopke, 2017).

In the developing nervous system, Wnt signaling ligands act as potent regulators of multiple stages of neuronal connectivity maturation, stabilization and synaptogenesis, including sculpting structural architecture and determining neurotransmission strength. Drosophila Wingless is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ), to bind Frizzled-2 (Fz2) receptors in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the Frizzled Nuclear Import (FNI) signaling pathway, which involves Fz2 endocytosis followed by Fz2 cleavage and Fz2 C-terminus nuclear import (Mathew, 2005). Fz2-C trafficked in nuclear ribonucleoprotein (RNP) granules regulates translation of synaptic mRNAs, thereby driving expression changes that modulate synapse structural and functional differentiation (Speese, 2012). In the presynaptic neuron, Wg binding to Fz2 activates a divergent canonical pathway inhibiting the Glycogen Synthase Kinase 3β (GSK3β) homolog Shaggy (Sgg) to regulate microtubule cytoskeleton dynamics via Microtubule-Associated Protein 1B (MAP1B) homolog Futsch. Futsch binding to microtubules regulates architectural changes in synaptic branching and bouton formation. Such multifaceted Wg functions require tight management throughout synaptic development (Kopke, 2017).

A highly conserved extracellular Wg regulator is the secreted deacylase Notum. The notum gene was discovered in a Drosophila gain-of-function (GOF) mutant screen targeting wing development. Under scalloped-Gal4 control, notum GOF causes loss of the wing and duplication of the dorsal thorax. In the developing wing disc, Notum acts as a secreted, extracellular feedback inhibitor of Wg signaling. Notum function was recently re-defined as a carboxylesterase that cleaves an essential Wg lipid moiety (palmitoleic acid attached to conserved serine), leaving it unable to bind to Fz2 and activate downstream signaling (Kakugawa, 2015). This Wnt palmitoleate moiety is similarly cleaved by human Notum acting as a highly conserved secreted feedback antagonist in the extracellular space to inactivate Wnt signaling (Langton, 2016; Kakugawa, 2015). At the Drosophila NMJ, extracellular regulation of Wg trans-synaptic signaling has been found to play key roles in synaptogenesis (Dani, 2012; Parkinson et al., 2013). For example, extracellular matrix metalloproteinase (MMP) enzymes cleave heparan sulfate proteoglycan (HSPG) co-receptors to regulate Wg trans-synaptic signaling that controls structural and functional synaptic development. Impairment of this mechanism is causative for Fragile X syndrome (FXS) synaptogenic defects. Similarly, misregulated extracellular mechanisms impair Wg trans-synaptic signaling in both Congenital Disorder of Glycosylation (CDG) and Galactosemia disease states, causing NMJ synaptogenic defects underlying coordinated movement disorders. Given these insights, this study investigated the putative roles for Notum as a new secreted Wg antagonist regulating synaptogenesis (Kopke, 2017).

This study utilized the well-characterized Drosophila NMJ glutamate synapse model to study Notum requirements in synaptic development. Notum, secreted from muscle and glia, is resident in the extracellular space surrounding developing synaptic boutons, where it negatively regulates Wg trans-synaptic signaling. In notum mutants, extracellular Wg ligand levels and downstream Wg signaling are elevated. Null mutants display both increased synapse number and strength, altered synaptic vesicle cycling, and synaptic ultrastructural defects including a decrease in SSR/bouton ratio, decreased synaptic vesicle density and an increase in the size of vesicular organelles. Cell-targeted RNAi studies reveal both postsynaptic and perisynaptic requirements, with muscle and glial notum knockdown resulting in overelaborated NMJ architecture, but neuronal-driven notum knockdown causing no detectable effects on synaptogenesis. Null notum defects are all phenocopied by neuronal Wg overexpression, suggesting that synaptogenic phenotypes arise from lack of Wg inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes, demonstrating that Notum functions solely as a negative regulator of Wg signaling. Taken together, these results identify Notum as a secreted Wnt inhibitor resident in the extracellular synaptomatrix with critical functions regulating trans-synaptic Wnt signaling to coordinate structural and functional synaptogenesis (Kopke, 2017).

Tightly coordinated trans-synaptic signals are required for proper development of the pre- and postsynaptic apparatus to ensure efficient communication at the synapse. This signaling is both coordinated and controlled in the extracellular space through the actions of secreted and transmembrane glycans, heparan sulfate proteoglycan (HSPG) co-receptors and secreted enzymes, such as matrix metalloproteinase (Mmp) classes. Wg (Wnt-1) mediates a critical trans-synaptic signaling pathway regulated by these extracellular synaptic mechanisms, with key roles in both structural and functional synaptogenesis. This study proposes that Notum is a novel extracellular regulator limiting Wg trans-synaptic signaling to control NMJ synaptogenesis. Wg is post-translationally modified by addition of palmitoleate on a conserved serine (S239) by membrane-bound O-acyltransferase (MBOAT) Porcupine. This lipidation event is required for Fz2 receptor binding and essential for signaling. At the synaptic interface, the GPI-anchored glypican Dally-like Protein (Dlp) regulates Wg trans-synaptic signaling, and Notum was initially described as cleaving such GPI-anchored glypicans from the cell surface, affecting their ability to interact with the Wg ligand. However, Notum was recently redefined as a secreted carboxylesterase, not a phospholipase (Kakugawa, 2015), with structural studies showing a hydrophobic pocket that binds and then cleaves palmitoleate (Kopke, 2017).

Notum is consistently reported to act primarily as an extracellular Wg feedback inhibitor. The current studies support this function within the synaptomatrix during synaptogenesis. At the Drosophila NMJ, Wg is secreted from both presynaptic neurons and associated peripheral glia (Kerr, 2014), with the glial function specifically regulating synaptic transmission strength and postsynaptic glutamate receptor clustering. This analyses suggest that Notum is secreted from both postsynaptic muscle and peripheral glia, establishing a dynamic, Wg-like expression pattern surrounding synaptic boutons. In notum null mutants, Wg signaling is increased at the developing NMJ, revealed by both decreased Fz2 receptor in the synaptic membrane (Wg-driven endocytosis) and an increase in nuclear Fz2-C punctae (FNI pathway). These findings are consistent with Notum function limiting Wg signaling, as established in other developmental contexts. Notum appears to provide a fascinating directional regulation of Wg trans- synaptic signaling, affecting the anterograde FNI signaling pathway in muscles, but not the autocrine divergent canonical pathway in neurons. Despite the strong elevation in synaptic Wg ligand levels in notum null mutants, no evidence is seen of altered presynaptic MAP1B homolog Futsch or changes in the microtubule cytoskeleton. However, Notum strongly limits Fz2 C-terminus nuclear import into the postsynaptic nuclei, which is known to drive ribonucleoprotein (RNP) translational regulation of synaptic mRNAs to control synapse structural and functional differentiation (Kopke, 2017).

Synaptic morphogenesis and architectural development is strongly perturbed in notum null mutants, including increased NMJ area, branching and bouton formation, consistent with Notum function inhibiting Wg trans-synaptic signaling. Elevating presynaptic Wg closely phenocopies notum synaptic defects, including expanded innervation area, more branching and supernumerary synaptic boutons. The results show that Notum secreted from muscle and peripheral glia controls Wg in the extracellular space, with targeted notum RNAi resulting in a similar NMJ expansion to notum nulls, whereas neuronal notum knockdown produces no effects. Interestingly, the glial-targeted RNAi increases boutons with no change in branching, whereas muscle knockdown has a stronger impact also affecting branching. Presynaptic Futsch/Map1B microtubule loops have been proposed to mediate Wg-dependent branching and bouton formation. However, neuronal Wg overexpression has no discernable effect on Futsch-positive microtubule loops. Consistently, Notum LOF also does not impact this pathway, with notum mutants displaying no change in Futsch-labeled looped, bundled, punctate or splayed microtubules. Wg binding to the presynaptic Fz2 receptor may activate another divergent Wnt pathway that does not involve Futsch. Alternatively, Wg signaling via muscle Fz2 may produce a retrograde signal back to the neuron to alter presynaptic development. To test these two possibilities, future studies will employ cell-targeted Fz2 knockdown in notum nulls to assay for suppression of the synaptic overgrowth phenotypes (Kopke, 2017).

Measures of functional synaptic differentiation reveal elevated neurotransmission and faster motor output function with both notum knockout and Wg over-expression. These results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles of Wg in NMJ functional development. Notum LOF increases presynaptic function selectively with an elevated mEJC frequency, greater EJC quantal content and heightened synaptic vesicle release during maintained high- frequency stimulation. Some of these effects may map to the increased synaptic bouton numbers. Both Notum LOF and Wg GOF also cause NMJ boutons to spatially clump together, with ultrastructural studies showing multiple boutons sharing one SSR profile. These are not satellite boutons, but rather aberrantly developing boutons that may result in functional defects. Notum knockdown in glia does not cause detectable mEJC/EJC changes, although Wg from glia regulates NMJ functional properties. Interestingly, loss of Notum appears to improve motor performance, and repo-targeted notum RNAi shows that glial Notum contributes to this function. This is an unusual outcome in a mutant condition, and it is assumed that there must be a counter-balancing cost for increasing neuromuscular function. Live FM dye imaging reveals that notum mutants load less dye into synaptic boutons upon nerve stimulation, indicating a role in synaptic vesicle endocytosis and/or the developmental regulation of synaptic vesicle pool size. These results show Notum function limits Wg trans-synaptic signaling to control presynaptic differentiation critical for synapse function and motor output. As with Wg, the source of Notum (muscle vs. glia) appears to be important for distinct synaptogenic functions. Notum from peripheral glia regulates only bouton formation, whereas Notum from muscle regulates both NMJ growth and function (Kopke, 2017).

Electron microscopy reveals a very strong decrease in synaptic vesicle density in notum null boutons, providing an explanation for the live FM1-43 dye imaging defects. One of the most striking ultrastructural phenotypes is numerous, enlarged synaptic vesicular bodies. These organelles are highly reminiscent of bulk endosomes, in which a large area of presynaptic membrane is internalized, and will subsequently bud off synaptic vesicles. This pathway is usually driven by intense stimulation during activity-dependent bulk endocytosis (ADBE), as first observed at the frog neuromuscular junction. This pathway is induced by high frequency trains of stimulation, and several proteins have been identified that affect the formation of bulk endosomes, including Syndapin and Rolling Blackout (RBO). At the Drosophila NMJ, conditional rbots mutants block ADBE, reducing the number and size of bulk endosomes (Vijayakrishnan, 2009). It will be interesting to test Wg GOF for enlarged endosomal structures, and study their involvement in Wg-dependent synaptic maturation. On the postsynaptic side, Notum also drives proper differentiation. Notum LOF reduces the postsynaptic DLG scaffold and postsynaptic SSR layering. The reduced SSR area in notum mutants is surprising, given that a reduction in postsynaptic Wg signaling also results in fewer SSR layers. However, SSR architecture has not been studied following Wg over-expression. Postsynaptic SSR formation may be sensitive to bidirectional Wg changes, and may be reduced if Wg is tipped in either direction (Kopke, 2017).

Mechanistically, Notum controls both pre- and postsynaptic molecular assembly, with LOF defects phenocopied by Wg over-expression. The results are consistent with Notum function inhibiting Wg trans-synaptic signaling, and consistent with previously characterized roles for Wg in synaptic molecular development. This study analyzed both the presynaptic active zone protein Bruchpilot and the two postsynaptic GluR classes. Both presynaptic Brp and postsynaptic GluRs are misregulated in notum nulls, with an increase in synapse number but not density. Importantly, both Notum LOF and Wg GOF elevates synapse number. Consistently, Wnt7a over-expression in mouse cerebellar cells also increases the number of synaptic sites and causes accumulation of presynaptic proteins required for synaptic vesicle function. The increased synapse density per NMJ may compensate for reduced neurotransmission per bouton, leading to a net stronger overall NMJ function. In notum mutants, this could reconcile the elevated synaptic strength measured by electrophysiology compared to compromised single bouton function measured by FM dye imaging and impaired TEM ultrastructure. In any case, synaptic assembly during development is regulated by Notum function limiting Wg trans-synaptic signaling (Kopke, 2017).

Genetically reducing Wg by combining a heterozygous wg null mutation into the homozygous notum null background reduces extracellular synaptic Wg back to control levels. Wg reduction suppresses synaptogenic defects, restoring increased NMJ area, branching and bouton numbers completely back to normal. Both notumKO and Wg GOF causes hyperactive movement, with roll-over speeds supporting synaptogenic defects of larger, stronger NMJs in both mutant conditions. However, notumKO motor function is only partially restored by correcting Wg levels. One explanation for incomplete rescue is that multiple Wnts may contribute to motor behavior. Serine lipidation is conserved for all Wnts, and at least two other Wnts have been suggested to act at the Drosophila NMJ (Wnt2, Wnt5). Wnts are the only secreted ligands suggested to be O-palmitoleated on a serine to function as Notum substrates (Kopke, 2017).

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the Wnt ligands. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. fz CRD mutant transgenes were generated and tested for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. Replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. It is therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. This model was tested by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. It is proposed that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling (Povelones, 2005).

The principle finding of this study is that the Fz CRD is required for efficient Arm signaling. Fz transgenes carrying CRD mutations have compromised Arm signaling function in cell culture and cannot fully restore Arm signaling to fz,fz2 mutants in vivo. In addition, adding a heterologous Wnt-binding domain (WIF) to a CRD-deleted fz restores its ability to activate Arm signaling via Wg in cell culture. Based on the manipulations and results, it is hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor, a function that can be taken over by other Wnt-binding domains. This idea was tested by creating a transgene fusing Wg to Fz, eliminating the CRD in the process; this results in a constitutively active receptor (Povelones, 2005).

While both in vivo and in vitro tests reveal that mutants with a defective Wnt interaction domain are compromised for Arm signaling, the requirement for the CRD is most evident in cell culture where all of the mutants show a reduced activity, particularly the one where the entire CRD is lacking. In the cell culture experiments, where the Wg signaling can be measured in a quantitative manner, a range of responses were found to the CRD mutants, corresponding to the differences in Wnt-binding strength. A range of phenotypes was noticed after examining cuticles in vivo and the abilities of the CRD mutants to restore signaling. While these rescue data are more difficult to measure, the phenotypes correspond in strength to the in vitro signaling levels. It is inferred from this relationship that signaling operates through the same mechanism in vivo as in cell culture. As an extension of this argument, it is suggested that the CRD plays a similar role in cell culture as in the embryo. However, signaling in vivo is less stringently dependent on the presence of the CRD, suggesting that its absence is being compensated for by other factors. If the function of the CRD (or other Wnt-binding domains such as the WIF) is, as proposed, to bring Wg in close proximity to the membrane domain of Fz, it is possible this function is taken over by other molecules acting in trans and that these factors are not present in vitro. Candidates for such molecules are members of the CRD containing ROR family and the RYK receptor tyrosine kinase, which has a WIF domain. It is also possible that extracellular matrix molecules provide such an accessory function, by presenting or concentrating Wg close to the Fz signaling domain (Povelones, 2005).

Is the only function of the CRD (or another Wg-binding domain, such as WIF) to capture Wg and to present it to the coreceptor Arrow? In that view, there would be no need for the seven-transmembrane domain of the Fz receptors; Fz would solely act to promote Wg interacting with Arrow. This was found to be unlikely; there are several studies that point to a requirement of specific residues in the Fz membrane domain in signaling. Mutations in those residues, either engineered or present in natural alleles, disrupt signaling. In addition, it has been recently proposed that in Drosophila, fz activates PCP and Arm signaling through heterotrimeric G proteins. Finally, expressing the CRD on the cell's surface as a GPI-linked membrane molecule does not promote signaling, but instead acts as a dominant negative. Taken together, these data suggest that the transmembrane portion of fz is a dynamic signal activating molecule and not merely a Wg presentation module (Povelones, 2005).

Overexpression of fzWIF in the Drosophila wing leads to both gain-of-function PCP and Arm signaling phenotypes. This is the composite of the consequences of fz and fz2 overexpression, which individually activate PCP and Arm signaling, respectively. There is much interest in determining how each receptor couples to a particular pathway. Although there is some disagreement in these studies, it is generally concluded that the transmembrane portion of fz, including the cytoplasmic tail, couples it to PCP signaling. Since fzWIF contains this portion of fz, it is not surprising that it too affects PCP signaling. What structural feature of fz2 is responsible for coupling it exclusively to Arm signaling? It was found that specifically replacing the fz CRD with the WIF domain results in a receptor that, like fz2, can activate Arm signaling. This finding is consistent with a study of fz/fz2 chimeras where the ability to activate Arm signaling was shown to be a property of the fz2 CRD. It was proposed that the feature conferring Arm coupling was the 10-fold higher affinity of the fz2 CRD for the Wg protein. By analogy, the WIF domain, like the fz2 CRD, may have a higher affinity for Wg than the fz CRD (Povelones, 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).

Lipoprotein particles are required for Hedgehog and Wingless signalling

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. The Drosophila lipoprotein particle, Lipophorin (Retinoid- and fatty acid-binding glycoprotein), bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles marked by lipophorin, and co-localize with these particles in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. A novel function is proposed for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins (Panakova, 2005).

In the developing wing of Drosophila, Hedgehog activates short-range target gene expression up to five cells away from its source of production, and longer-range targets over more than twelve cell diameters. Wingless can signal through a range of over 30 cell diameters. These morphogens are anchored to the membrane via covalent lipid modification. The mechanisms that allow long-range movement of molecules with such strong membrane affinity are unclear (Panakova, 2005).

Like Wingless and Hedgehog, glycophosphatidylinositol (gpi)-linked proteins transfer between cells with their lipid anchor intact. Gpi-linked green fluorescent protein (GFP) expressed in Wingless-producing cells spreads into receiving tissue at the same rate as Wingless, where it co-localizes with Wingless in endosomes. Thus, it is proposed that these proteins travel together on a membranous particle, which has been called an argosome. How might argosomes form? One possibility is that argosomes are membranous exovesicles. Such particles could be generated by plasma membrane vesiculation, or by an exosome-related mechanism. Alternatively, argosomes might resemble lipoprotein particles like low-density lipoprotein (LDL). Vertebrate lipoprotein particles are scaffolded by apolipoproteins and comprise a phospholipid monolayer surrounding a core of esterified cholesterol and triglyceride. Insects construct similar particles called lipophorins. Lipid-modified proteins of the exoplasmic face of the membrane (such as GFPgpi, Wingless or Hedgehog) might insert into the outer phospholipid monolayer of such a particle via their attached lipid moieties. This study use biochemical fractionation to determine the sort of particle with which lipid-linked proteins associate, and genetic means to address its function (Panakova, 2005).

Lipid-linked proteins copurify with lipophorin: Sedimentation of Wingless, Hedgehog and gpi-linked proteins were compared to that of transmembrane proteins, exosomes and lipophorin particles. To mark exosomes, flies were used expressing a vertebrate CD63:GFP fusion construct. CD63 is a tetraspanin that localizes to internal vesicles of multivesicular endosomes, and is released on exosomes. In Drosophila imaginal discs, CD63:GFP localizes to late endosomes in producing cells, consistent with vertebrate studies. It is released and endocytosed by neighbouring cells between one and three cell diameters away, indicating that it is present on exosomes (Panakova, 2005).

To mark lipoprotein particles, antibodies were made to Drosophila apolipophorins I and II (ApoLI and ApoLII); these proteins are generated by cleavage of the precursor pro-Apolipophorin (Sundermeyer, 1996; Kutty, 1996). Lipophorin is produced in the fat body (Kutty, 1996); consistent with this, apolipophorin transcripts cannot be detected in imaginal discs. Nevertheless, the ApoLI and ApoLII proteins are as abundant in discs as in the fat body (Panakova, 2005).

Plasma membrane and exosomal markers are completely pelleted after centrifugation for 3 h at 120,000g, whereas most ApoLII remains in the supernatant. Most Wingless:GFP and Hedgehog is present in the pellet, as are the gpi-linked proteins Fasciclin, Connectin, Klingon and Acetylcholineasterase; this is not unexpected, because these proteins localize to the plasma membrane and internal membrane compartments. Surprisingly, however, some Wingless:GFP (6%), Hedgehog (2%) and gpi-linked proteins (14%-22%) remain in the supernatant (Panakova, 2005).

The 120,000g supernatant (S120) contains both free soluble proteins and lipoprotein particles. To separate them, isopycnic density centrifugation was performed. In these gradients, lipophorin moves to the top low-density fraction whereas soluble proteins are present in higher-density fractions. Gpi-linked proteins are found almost entirely in the top fraction with lipophorin. Treating the S120 with Phosphatidylinositol-specific phospholipase C (PI-PLC) before density centrifugation shifts their migration to higher-density fractions. This suggests that gpi-linked proteins associate with low-density particles via their gpi anchor (Panakova, 2005).

Similarly, when S120s from larvae that express Wingless:GFP or Hedgehog:HA in imaginal discs are subjected to isopycnic density centrifugation, these proteins are found in the lowest-density fraction with ApoLII, as is endogenous Hedgehog. Antibodies to endogenous Wingless detect a doublet in the top fraction and a band of somewhat higher mobility in high-density fractions. These data indicate that non-membrane-bound Wingless and Hedgehog associate with low-density particles in imaginal discs in vivo; other larval tissues may secrete Wingless in a non-lipophorin-associated form (Panakova, 2005).

To ask whether lipid-linked proteins associate with lipophorin, or with some other low-density particle, ApoLII was immunoprecipitated from larval S120s and precipitates were probed for Wingless, Hedgehog or GFPgpi. These proteins are immunoprecipitated by anti-ApoLII. Hedgehog and Fas-1 also immunoprecipitate with ApoLII from the more purified top fraction of KBr gradients. Thus, lipid-linked morphogens and gpi-linked proteins associate directly with lipophorin particles (Panakova, 2005).

Lipophorin-RNAi perturbs lipid transport: To assess the role of lipophorin in larval growth and development, the levels of ApoLI and II were reduced by RNA interference directed against two different regions of the apolipophorin messenger RNA. Similar phenotypes were produced by each construct. To express double-stranded (ds)RNA, a modified GAL4:UAS system was used in which expression of inverted repeats can be temporally controlled by heat-shock-dependent excision of an intervening HcRed cassette by the flippase (FLP) recombinase. Extracts were tested from wild-type larvae or larvae harbouring hs-flp, GAL4 driver and UAS dsRNA constructs at various times after heat shock to see how fast lipophorin levels were reduced. Larvae of the latter genotype made only 50% of the wild-type level of ApoLII, even in the absence of heat shock; basal activity of the heat-shock promoter in the fat body causes HcRed excision in approximately 50% of fat-body cells, although excision strictly depends on heat shock in other larval tissues. Although they survive less frequently, these flies have no obvious phenotype (Panakova, 2005).

After heat shock, all fat-body cells excise the HcRed cassette and ApoLII levels decrease further. After four days, ApoLII is reduced to 5% of wild-type levels. ApoLI levels are reduced with similar kinetics. These animals prolong the third larval instar and rarely pupariate. All the experiments described below were performed on third-instar larvae 4-6 days after heat shock (Panakova, 2005).

To investigate the requirement for lipophorin in lipid transport, the accumulation of neutral lipids in larval tissues was assessed by staining them with Nile Red. Cells of the posterior midgut normally contain many small lipid droplets. Lipophorin reduction causes a dramatic expansion of these droplets, suggesting that lipophorin is required for the efficient extraction of lipid from the midgut (Panakova, 2005).

The wild-type fat body contains both small and large lipid droplets. Fat bodies of lipophorin-RNAi larvae are reduced in size and have fewer small lipid droplets, although larger droplets appeared normal. These data suggest that lipophorin delivers lipid to the fat body (Panakova, 2005).

Lipid droplets in discs from lipophorin-RNAi larvae are fewer and smaller than in the wild type. Their discs are also reduced in size, particularly in the wing pouch. Thus, discs require lipophorin for accumulation of lipid droplets and for growth. Neither Caspase3 activation nor membrane phosphatidylinositol 3,4,5-phosphate (PIP3) accumulation is altered in lipophorin- RNAi discs , suggesting that their small size is not due to cell death or reduced insulin signalling (Panakova, 2005).

Hedgehog function requires lipophorin: To test whether lipophorin association is required for Hedgehog function, Hedgeghog distribution and signalling was examined in lipophorin-RNAi larval discs. In wild-type discs, Hedgehog expressed in the posterior compartment moves across the anterior-posterior (AP) compartment boundary and activates transcription of short and long-range target genes. Cells closest to the source respond by activating the transcription of collier and patched. Further away, Hedgehog activates transcription of decapentaplegic. Levels of Collier and a decapentaplegic reporter construct (dpplacZ) were monitored in wild-type and lipophorin-RNAi discs stained in parallel and imaged under identical conditions. Discs from lipophorin-RNAi larvae activate collier at least as efficiently as those of the wild type. In contrast, the range of activation of dppLacZ is significantly narrowed in lipophorin RNAi discs. dppLacZ is expressed up to 11 cells away from the AP boundary in wild-type discs, but only up to six cells away in lipophorin-RNAi larvae. These data suggest that lipophorin knockdown decreases the range of Hedgehog signalling (Panakova, 2005).

To discover whether Hedgehog trafficking was altered, discs were stained for Hedgehog and Patched. In wild-type discs, Hedgehog moves into the anterior compartment, where it is found in endosomes, often with Patched. Patched-mediated endocytosis is thought to sequester Hedgehog and limit its spread. Hedgehog is most abundant up to five cell rows away from the AP boundary; although Hedgehog signals over a wider range, specific staining there cannot be distinguished from background. In lipophorin-RNAi discs, Hedgehog accumulates to abnormally high levels in the first five rows of anterior cells. 380 Hedgehog spots were found in the most apical 10 µm of the wild-type disc. The lipophorin-RNAi disc contained 1,208 Hedgehog spots in the same region. Most accumulated Hedgehog colocalizes with Patched in endosomes. Furthermore, Patched co-accumulates more extensively with Hedgehog in endosomes than it does in wild-type. These data indicate that lipophorin RNAi either increases the susceptibility of Hedgehog to Patched-mediated endocytosis, or prevents subsequent degradation of the protein (Panakova, 2005).

Drosophila cannot synthesize sterols and relies on dietary sources. To assess whether reduced uptake of sterols or other lipids might cause the changes seen, the effects of lipid deprivation on larval development were explored. Larvae were allowed to hatch and feed on sucrose/agarose plates supplemented with yeast for 2-3 days, then transferred to plates containing chloroform-extracted yeast autolysate, rather than yeast. These larvae are developmentally delayed; after 7 days of lipid deprivation, their discs are much smaller than those of younger late-third-instar larvae. In contrast, yeast-fed siblings pupariate and begin to eclose by this time. Those flies that infrequently eclose after larval lipid depletion are small (35%-60% of normal body weight) but normally patterned. Thus, lipid depletion stalls imaginal growth (Panakova, 2005).

To discover whether lipid starvation affected Hedgehog trafficking or signalling, larvae were deprived of lipid 2 days after hatching and their discs were stained 6 days later. No changes in Hedgehog or Patched distribution are apparent in these discs compared with younger yeast-fed discs of similar size. Furthermore, the range of dpp and collier expression does not differ in lipid-starved and yeast-fed discs. Thus, lipid starvation does not mimic the effects of lipophorin knockdown. It is speculated that lipid-starvation-induced growth arrest prevents membrane sterol from dropping to levels that would interfere with the Hedgehog pathway. Thus, lipophorin does not indirectly affect the Hedgehog pathway via lipid deprivation (Panakova, 2005).

Wingless function requires lipophorin: To discover whether lipophorin RNAi perturbed Wingless trafficking, Wingless distribution was examined. In lipophorin- RNAi discs, extracellular Wingless is less abundant on both the apical and basolateral epithelial surfaces and spreads over shorter distances. However, no consistent alterations were detected in intracellular Wingless. Thus, lipophorin promotes accumulation of extracellular Wingless (Panakova, 2005).

To investigate whether Wingless signalling requires lipophorin, the activation of two target genes was examined. Senseless is produced only in cells near the Wingless source and its expression is unaffected by lipophorin RNAi. Distalless is normally produced in a gradient throughout most of the wing pouch. In lipophorin-RNAi discs, the Distalless gradient is abnormally narrow. This suggests that lipophorin knockdown specifically perturbs long-range Wingless signalling (Panakova, 2005).

Conclusions: This study establishes the principle that lipid-linked proteins of the exoplasmic face of the membrane associate with lipoproteins. These include many gpi-linked proteins with diverse functions, as well as the lipid-linked morphogens Wingless and Hedgehog. The mechanism allowing long-range dispersal of lipid-linked proteins is not yet understood. The finding that these proteins exist in both membrane-associated and lipoprotein-associated forms suggests reversible binding to lipoprotein particles as a plausible mechanism for intercellular transfer, and the consequences of lowering lipoprotein levels in Drosophila larvae supports this idea (Panakova, 2005).

Lipophorin knockdown narrows the range of both Wingless and Hedgehog signalling. Hedgehog accumulates to an abnormally high level in cells near the source of production and long-range signalling is inhibited; short-range target genes, however, are expressed normally. These data suggest that Hedgehog does not move as far when lipophorin levels are low. The range over which Hedgehog moves is normally restricted by Patched-mediated endocytosis. In discs from lipophorin RNAi larvae, accumulated Hedgehog co-localizes with Patched in endosomes, suggesting that it is more efficiently sequestered by Patched. How might lipophorin antagonize Patched-mediated sequestration and promote long-range movement (Panakova, 2005)?

The data are consistent with the idea that lipophorin is continuously needed for movement, rather than required only for the release of morphogens. If lipophorin were important only for Hedgehog secretion, lipophorin RNAi would be expected to decrease the amount of Hedgehog found in receiving tissue; this seems not to be the case. Furthermore, altered Hedgehog trafficking in receiving tissue is consistent with a model in which lipophorin is required at each step of intercellular transfer. The idea is favored that reversible association of Hedgehog with lipophorin particles facilitates its transfer from the plasma membrane of one cell to that of the next. This model predicts that lowering lipophorin levels should increase the length of time that Hedgehog spends in the plasma membrane before becoming associated with lipophorin. This would slow its rate of transfer and increase the probability of Patched endocytosing Hedgehog before it moved to the next cell. Hedgehog would then signal efficiently in the short range, but be so efficiently sequestered by Patched that very little protein would travel far enough to activate long-range target genes. These predictions are completely consistent with the current observations (Panakova, 2005).

This model differs significantly from the original concept of argosome function. It was initially speculated that argosomes were exosome-like particles with an intact membrane bilayer, and that lipid-linked morphogens needed to be assembled on these particles to be secreted by producing cells. Instead, it was found that argosomes are exogenously derived lipoproteins that facilitate the movement of morphogens through the epithelium. Many questions remain as to how morphogens become associated with argosomes, and how the spread and cell-interactions of these particles are regulated. Clearly, heparan sulphate proteoglycans are essential for the movement of Hedgehog and Wingless into receiving tissue. Because heparan sulphate binds to vertebrate lipoprotein particles, one might speculate that heparan sulphate proteoglygans (HSPGs) facilitate morphogen movement through lipoprotein binding. Conversely, many gpi-linked proteins, including the HSPG's Dally and Dally-like, are found on lipoprotein particles themselves. These associated proteins have the potential to modulate the cellular affinities or trafficking properties of lipoproteins and the morphogens they carry (Panakova, 2005).

The data suggest that lipophorin particles not only mediate intercellular transfer of Hedgehog, but may also be endocytosed together with the morphogen. Interestingly, LDL-receptor-related proteins Arrow and Megalin have demonstrated roles in Wingless signalling and Hedgehog endocytosis, respectively. It is intriguing to speculate that these receptors might be important for interaction with the lipoprotein-associated form of the morphogen (Panakova, 2005).

Cholesterol has the potential to modulate the activity of the Hedgehog pathway at many different points. Whether changes in the level of cellular cholesterol normally play a role in regulating the activity of the pathway is unclear. This study shows that Hedgehog interacts with the particle that delivers sterol to cells. This observation raises the possibility that internalization of Hedgehog is linked to sterol uptake, and suggests new mechanisms to link nutrition, growth and signalling during development (Panakova, 2005).

Directional transport of extracellular Wingless

Continued: wingless Protein Interactions part 2/3 | part 3/3

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation |Targets of Activity | Developmental Biology | Effects of Mutation | References

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