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 fz−dfz2− 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 fz−dfz2− 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 fz−dfz2− 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 fz−dfz2− 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).
The Wnt family of secreted glycoproteins mediate cell- cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the ß-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular ß-catenin. Wnt/ß-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. The transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/ß-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates (Mao, 2002).
Drosophila has neither dkk nor krm but rather an LRP6 homolog, arrow, which functions in Wnt signalling. To determine if they could inhibit Wnt signalling in the fly, Xenopus dkk1 and mouse krm2 were expressed as heterologous transgenes in Drosophila and the GAL4/UAS system was used with a scalloped (sd)-GAL4 driver to direct their expression to the wing disc. Development of the wing critically depends on Wnt signalling, and interference with wingless or components of the Wnt pathway characteristically results in loss of wing structures. Even though Dkk1 protein is produced in transgenic flies, it does not affect wing development by itself. However, coexpression of dkk1 and krm2 results in almost complete loss of wings, whereas expression of krm2 alone has no effect. These results indicate that Krm is required for inhibition of Wnt signalling by Dkk1, presumably by interacting with arrow. Indeed, Dkk1 binds to and functionally interacts with Drosophila arrow transfected in 293T cells. Furthermore, inhibition of krm1 and krm2 by antisense Morpholino oligonucleotides reveals that they are required for Wnt inhibition during embryonic head formation and interact with dkk1 in Xenopus. These results suggests a model whereby Dkk1 inhibits Wnt signalling by acting in concert with its receptor Kremen to form a ternary complex with LRP6, which is rapidly endocytosed. This eliminates the Wnt receptor from the plasma membrane, thus preventing Wnt–LRP6 interaction (Mao, 2002).
Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).
The data argue for a different regulatory mechanism of Wg signal transduction, proceeding through the inhibition of the protein Axin, rather than through the inhibition of Zw3/GSK3β. Axin has been identified in both vertebrates and invertebrates as a negative component of the pathway. Later work established Axin as a critical scaffold protein required for the assembly and function of the degradation complex. This complex functions in the destruction of Arm/β-catenin by bringing the kinase Zw3 and Arm into close proximity, leading to the phosphorylation of Arm, and thereby targeting it to the proteasome for degradation. For efficient Arm degradation, both Axin and APC must be present in the complex. How Wg input controls activity of the degradation complex has never been properly established, although most models have focused on the inhibition of the kinase Zw3. It is also unclear whether Arm degradation always plays a central role in converting Wnt input into transcriptional responses. In sea urchins and mammals, the most obvious response to Wnt signaling is a relocalization of Arm protein from the cytoplasm to the nucleus; it has been shown that both Axin and APC have a profound effect on Arm localization that cannot be explained by their interaction with Zw3 or the degradation complex alone (Tolwinski, 2003).
Evidence is presented that the Wg signal can be transmitted through a posttranslational regulation of Axin accumulation. Despite uniform transcription of Axin using the UAS/GAL4 system, Axin accumulates to different levels in different cells across each parasegment. Cells with lower steady-state levels of Axin are those exposed to Wg input, and this was strictly dependent on Wg. Loss of Wg causes excess accumulation of Axin, whereas uniform Wg expression (and therefore signaling) lowers total Axin levels. The phenomena observed in embryos parallel earlier reports showing that Axin accumulation is affected by Wnt signaling in tissue culture cells. GSK3β phosphorylation of Axin leads to its stabilization. However, the actual role that phosphorylation plays appears to be more complex, since further work contradicted this finding. In the current experiments, the phosphorylation state of Axin was not examined in cells responding to Wg (those with low Axin levels), nor in those not exposed to Wg (high Axin levels). Therefore, whether modification may inactivate Axin or whether modification leads to removal of Axin by degradation cannot be distinguished. It was found, however, that Zw3 kinase activity is not necessary for the reduction in Axin accumulation that is observed; the Axin striping pattern is maintained in embryos that lack Zw3 function. These results argue for a link between Wg signaling and Axin accumulation that is independent of the Zw3-mediated degradation complex (Tolwinski, 2003).
Although Zw3 does not appear to be required for Axin degradation, the more upstream component, Arrow, appears to be important for this mode of Wg signal transduction. The cytoplasmic domain of Arrow interacts with Axin in the yeast two-hybrid system, an interaction also identified for one of the mammalian LRPs, mLRP5, whose rapid binding of mAxin is ligand stimulated. Binding data for Arrow are largely in agreement with the mammalian study, except that no contribution is found of the Zw3 binding region of Axin in binding of Arrow bait. Interestingly, full-length Axin fails to interact significantly with the Arrow C terminus in yeast and all the Axin clones isolated in the library screen lack sequences N-terminal to position 353. This finding suggests that an inhibitory domain is present in Axin, N-terminal to the Zw3 binding domain, and that this inhibitory domain prevents Axin from binding Arrow. It is possible that the Wnt signal necessary for the mouse Axin interaction with LRP5 induces a conformational change in Axin that removes, modifies, or otherwise displaces the inhibitory domain. In contrast, Armadillo bait significantly binds both full-length and truncated Axin. These data taken together with the demonstration in Drosophila that signaling leads to loss in Axin striping and a lowered steady-state level of Axin, suggest that the Arrow/LRP5 interaction with Axin induces a change in activity and/or stability of Axin (Tolwinski, 2003).
The prevailing view on Wnt reception states that Arrow/LRP5,6 function as coreceptors together with Frizzled proteins. It is well established that Frizzled proteins bind Wnt ligands and that this interaction is essential for Wnt signal transduction. Initial work on LRP6 extended this model in suggesting that Wnt provides a bridging function in assembling a complex of Frizzleds and Arrow, at least for the particular combination mFz8/mWnt-1/mLRP6. However, biochemically, such complex formation has also not always been confirmed. In addition, the functional significance for signaling of the observed ternary complex has not been demonstrated in vivo. Therefore an experiment was designed that tested whether, in vivo, physical proximity of Arrow and Frizzled-2 is sufficient for signaling. In fact, it was found that Frizzled-2 can initiate ligand-independent signal transduction. The constitutive activity of the Fz2-Arr[intra]chimeric protein is significant, since only expression of the fusion protein but not expression of the individual components (Fz2 and Arr[intra]) activate signal transduction. It is inferred that association of Frizzled2 with the Arrow C terminus is indeed a key step in signal initiation in vivo, and that the proximity afforded here by protein fusion also occurs during normal signaling. The Fz2-Arr[intra] chimera is uncoupled from the need for ligand to trigger the intracellular signal transduction cascade. Therefore, whether the Arrow extracellular domain participates in a true “reception” complex with Fz2 in Wg binding cannot be addressed. Nevertheless, Arrow, or at least its C terminus, likely interacts intimately with Fz2 during signal initiation at the membrane. In addition, activation of the pathway by the Fz2-Arr[intra] chimera proceeds through canonical pathway components, most notably requiring Disheveled, a result consistent with the finding that Dsh functions downstream of Arrow. In cultured vertebrate cells, one report has suggested that in some circumstances, LRP6 can induce Wnt signal transduction independently of Disheveled. In contrast, the experiment of overexpressing biologically active Arrow cytoplasmic sequences in the form of the Fz2-Arr[intra] chimera revealed a strict Dsh dependence, suggesting instead an obligate role for the Dsh protein at signal initiation by Arrow, and by extension, presumably by vertebrate LRP5,6 (Tolwinski, 2003).
Though the Fz2-Arr[intra] chimera clearly signals, it is not as active as a Wg-stimulated endogenous receptor complex. Presumably, and not surprisingly, the protein fusion will present a distorted topology to cofactors required in the signal initiation complex, and therefore is not optimally configured for initiating signal transduction. This may explain why the chimera retains some measure of reliance on endogenous Arrow, as is apparent from a reduced level of signaling in its absence (Tolwinski, 2003).
In summary, Arrow and the Frizzled family of Wnt receptors function in a protein complex that triggers the intracellular signaling cascade. By binding to and causing a reduction in steady-state levels of Axin, Arrow provides a pivotal link between the receptor complex on the cell surface and the downstream events that control Arm activity. One consequence of Axin degradation may reflect its role as a scaffold for Zw3-mediated degradation of Arm. However, because zw3− embryos still respond to Wg input though they fail to degrade Arm, regulation of the degradation complex cannot be the only target of Wg signaling. A Zw3-independent branch in the Wg pathway is proposed, one that might regulate the release of Armadillo from Axin, resulting in nuclear accumulation and signaling (Tolwinski, 2003).
The maturation of cell surface receptors through the secretory pathway often requires chaperones that aid in protein folding and trafficking from one organelle to another. boca is an evolutionarily conserved gene in Drosophila melanogaster, that encodes an endoplasmic reticulum protein that is specifically required for the intracellular trafficking of members of the low-density lipoprotein family of receptors (LDLRs). Two LDLRs in flies require boca function: (1) Arrow, which is required for Wingless signal transduction, and (2) Yolkless (Yl), which is required for yolk protein uptake during oogenesis. Consequently, boca is an essential component of the Wingless pathway but is more generally required for the activities of multiple LDL receptor family members (Culi, 2003).
boca was initially isolated in a combined yeast two-hybrid and double-stranded RNA interference (RNAi) screen to search for novel developmental phenotypes in Drosophila. The sequence of a boca cDNA predicts a protein of 180 amino acids. The putative Boca protein shares ~45% identity/~65%-85% similarity with open reading frames predicted in nematode, mouse [called mesoderm development (mesd)] and human genomes, suggesting that Boca has a conserved function in metazoans. The only recognizable motifs in Boca are a putative N-terminal signal sequence and a C-terminal ER retention sequence, KDEL. Consistent with the presence of KDEL, an antibody raised against Boca detects a ubiquitously expressed cytoplasmic protein. This antibody is specific for Boca because expressing double-stranded boca RNA eliminates nearly all immunoreactivity. Boca is concentrated apically in polarized cells, such as embryonic blastoderm cells. In addition, Boca colocalizes with an ER marker protein but shows no overlap with a Golgi marker or with filamentous actin (F actin) as detected by phalloidin staining (Culi, 2003).
boca is shown to be required for the activities of at least two LDL receptor family members in flies, Yl, and Arrow. This conclusion is based on obtaining both yl and arr loss-of-function phenotypes in boca mutant flies. Specifically, using clonal analysis in imaginal discs, it was found that boca mutant cells are unable to transduce a Wg signal. Epistasis experiments with other components of the Wg pathway demonstrate that boca function is required downstream of Wg and upstream of arm. Together with the observation that boca encodes an ER protein, these experiments suggested that the defect in Wg signaling is due to a block in the maturation or processing of one of the two Wg coreceptors, Fz/Fz2 or Arr. In addition, however, females with boca mutant germlines are sterile and lay eggs that have a yolkless phenotype. This phenotype is also observed when the vitellogenin receptor, encoded by the yl gene, is nonfunctional. Since both yl and arr encode members of the LDL receptor family in flies, these results suggest that the maturation of this receptor family is specifically impaired in the boca mutant. In agreement with this suggestion, the trafficking through the secretory pathway of Arr, Yl, the human LDL receptor, and a putative Drosophila lipophorin receptor (LpR2), but not Fz2, were shown to require Boca. Thus, boca is an essential component of the Wg signaling pathway but, in addition, is more broadly required for the trafficking of multiple LDL receptor family members in Drosophila (Culi, 2003).
Interestingly, boca is required for the trafficking of Yl, Arr, LpR2, and the human LDL receptor -- four dissimilar members of this receptor family. In addition to yl, arr, and the two LpR genes, there are three other LDLR family members predicted in the Drosophila genome. It seems plausible that boca is also required for the trafficking of these other LDL receptor family members in flies. The functions mediated by these receptors are currently not known and may not have been detected by these experiments. For example, these experiments would not have identified a defect in neuronal migration or lipid homeostasis. Additional experiments will therefore be required to determine if Boca plays a role in other biological processes and to definitively assess if Boca is required for the maturation of all LDLR family members in flies (Culi, 2003).
What might Boca be doing to assist in the maturation of these receptors? The results provide some clues to this question. (1) From its sequence, Boca is predicted to be a luminal ER protein and immunostaining experiments support this prediction. Specifically, in wild-type cells Boca colocalizes with an ER marker and shows no overlap with a marker for the Golgi apparatus. (2) Immunofluorescence studies in both S2 cells and imaginal discs suggest that Boca is required for the trafficking of LDLRs through the secretory pathway. These receptors appear to remain in the ER in the absence of Boca function. The boca-dependent cell surface localization of Arr-flu was confirmed by surface biotinylation experiments in S2 cells. Taken together, these results suggest that Boca is required for the transport of LDL receptor family members from the ER to the Golgi apparatus. According to this view, boca is analogous to Star, which is required for the transport from the ER to the Golgi of Spitz, a ligand for the epidermal growth factor receptor in Drosophila. Similarly, during dorsoventral patterning of the Drosophila embryo, the ER protein Windbeutel is required for the transport of Pipe, a sulfotransferase, from the ER to the Golgi (Culi, 2003).
A third observation described here is that incorrect disulfide bonds form in both Arr-flu and Yl when Boca activity is compromised. Incorrect disulfide bond formation suggests a defect in protein folding. Thus, although its precise biochemical activity is not known, these results are consistent with the idea that Boca is a molecular chaperone that assists in the folding of LDL receptor family members in the ER. Consistent with this idea, the murine homolog of Boca, Mesd, is able to weakly interact with LRP5 and LRP6 in immunoprecipitation experiments, suggesting that Mesd/Boca may be in a complex with LDLRs. In addition, when proteins are misfolded during their progression through the secretory pathway, a quality control system blocks their exit from the ER and targets them for degradation. The results are consistent with both of these responses. In S2 cells, Arr-flu, LpR2, and human LDLR accumulate in the ER in the absence of Boca, while in vivo Arr-flu and Yl are present at lower levels and mislocalize in the boca1 mutant (Culi, 2003).
Although they share some similarities, Boca is distinct from another ER protein, receptor associated protein (RAP), that assists in the trafficking of a subset of LDL receptor family members in mammalian cells. RAP binds tightly to low-density lipoprotein receptor-related protein (LRP) and blocks its ability to bind and endocytose ligands. In contrast, Boca does not bind with high affinity to Arrow. Thus, if Boca helps LDLRs to fold, it may preferentially interact with nascent or unfolded proteins as they are translated or, alternatively, may interact with LDLRs indirectly. Another potential difference between RAP and Boca is that RAP is specifically required for the function of LRP, but not other LDLRs. In contrast, Boca is required for the trafficking of several divergent members of this family in flies. Boca and RAP are also unrelated proteins and the Drosophila genome sequence predicts the existence of an uncharacterized RAP homolog. Finally, RAP knockout mice are viable and have only reduced amounts of functional LRP whereas boca is essential for viability in the fly. As in flies, the mouse homolog of boca is also essential for viability. Thus, Boca appears to play a more essential and general role in the trafficking of LDLRs than RAP. In the future, it will be important to define a more precise biochemical function for Boca and to determine if RAP and Boca function together in the trafficking of a subset of LDL receptor family members (Culi, 2003).
The phenotypes observed in boca mutants indicate that Boca is specifically required to transduce the Wg signal and for Yl function during oogenesis. No phenotypes were obtained that would indicate a defect in the other major developmental signaling pathways in flies, including the Hedgehog, Decapentaplegic, Epidermal Growth Factor, and Fibroblast Growth Factor pathways. These genetic experiments suggest that Boca is specifically required for the activities of LDLR family members. It is unusual for a chaperone to be specifically required for several divergent members of a single family. Some chaperones, such as BiP or Hsp90, are generally required for the folding of many proteins whereas others are required to assist in the folding of a very specific subset of proteins. NinaA, for example, is critical for the trafficking of the major rhodopsin (Rh1) in flies, but is not required for the maturation of other G protein coupled receptors. In the future, it will be interesting to determine which of the features shared among LDLR family members make their trafficking through the secretory pathway dependent upon Boca. Other questions raised by these results are whether other classes of cell surface receptors have dedicated chaperones and how Boca interfaces with the more generally acting ER chaperones. Finally, although Boca appears to be present in all cells, these results raise the possibility that its regulation could be a way to modulate LDLR activity (Culi, 2003).
A large number of mutations in the human LDL receptor have been described that result in hypercholesterolemia. In addition to LDL receptor mutations, alterations in an unlinked gene that is required for LDL receptor function can also result in hypercholesterolemia. The human boca homolog maps to chromosome 15q25-q26, a region that has been linked to hypercholesterolemia in some families. Although this linkage has been disputed, the current results raise the possibility that altered versions of the human boca homolog could also contribute to hypercholesterolemia in some genetically predisposed families. In the mouse, the boca homolog corresponds to one of two genes that are deleted in the lethal mutation; mesoderm development (mesd) and additional studies demonstrate that the boca ortholog in the mouse is mesd. Interestingly, homozygous mesd mice fail to form a primitive streak and, consistent with these results, appear to have a defect in Wnt signaling (Culi, 2003).
In summary, boca encodes an evolutionarily conserved ER protein that is required for the trafficking and, therefore, activity of both Arr and Yl in Drosophila. It is postulated that boca is a molecular chaperone that is specifically dedicated to assist in the folding, trafficking, and quality control of the LDLR family (Culi, 2003).
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-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 þ-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).
A recent study has suggested that the extracellular domain of LRP might negatively regulate the signaling activity of LRP through dimerization, which can be relieved by Wnt proteins. By contrast, the current study shows that the signaling activity of LRP was markedly increased by oligomerization. The source of this discrepancy is currently unclear. However, it was found that, when overexpressed in 293 cells, full-length LRP6 is less efficiently transferred to the plasma membrane than is LRP6{Delta}N, an observation that correlates with the lower signaling activity of LRP6. In addition, upon co-expression of MESD, a chaperone of LRP, the signaling activities of LRP6 and LRP6{Delta}N become equivalent. These data suggest that the low signaling activity of full-length LRP6 is most likely due to its poor membrane localization, and strongly argue against a negative role of the extracellular domain of LRP in Wnt signaling (Cong, 2004).
Although Wnt can bind to both Frizzled and LRP, both receptors are essential for transducing the Wnt signal. It is possible that Wnt, Frizzled and LRP form one signaling complex. Alternatively, Wnt proteins might form separate complexes with Frizzled and LRP, which turn on separate signaling pathways that converge downstream (Cong, 2004).
Several lines of evidence that support the first model are provided, and the data suggest that bringing Frizzled and LRP into proximity is sufficient to trigger signaling through ß-catenin signaling. It was shown that the Wnt signaling pathway can be fully stimulated by oligomerizing Frizzled and LRP either through the intracellular region, by directly fusing the intracellular domain of LRP6 to the C terminus of human FZ5, or through the extracellular region. Furthermore, the requirement for free Wnt proteins can be bypassed by fusing Wnt to either Frizzled or LRP. These results suggest that Wnt, Frizzled and LRP form a single signaling complex, and the function of Wnt is to form a bridge between Frizzled and LRP. It is recognized, however, that Wnt-induced oligomerization of endogenous Frizzled and LRP in living cells has not been demonstrated, nor hace the physical properties of the proposed Wnt-induced oligomers been characterized (Cong, 2004).
Why is it necessary and sufficient to bring LRP and Frizzled into proximity for transducing the Wnt signal? RNA interference studies have indicated that signaling by overexpressed LRP is strictly Dvl independent, and Dvl becomes important once Wnt and Frizzled are involved. Axin is known to interact with the C terminus of LRP, and Dvl can interact with Frizzled. Presumably, once overexpressed, a high concentration of membrane LRP is able to bring endogenous Axin to the plasma membrane, based solely on its affinity with Axin, so that Axin might be inactivated or degraded. This would explain why the signaling activity of ectopically expressed LRP is Dvl independent. Under normal physiological conditions, Frizzled and Dvl might be required to translocate Axin to the membrane LRP upon Wnt signaling. Dvl might function as a molecular chaperone to deliver Axin to the Frizzled-LRP complex, based on its affinity with both Frizzled and Axin. In addition, Frizzled and Dvl might also enhance the binding affinity between LRP and Axin through promoting phosphorylation of LRP. Therefore, the Wnt-Frizzled-LRP complex might serve as a high-affinity docking site for Axin. This model is also in agreement with the recent finding that Wnt induces translocation of Axin to the membrane in a Dvl-dependent manner. Consistent with its proposed role as a shuttle, Dvl is associated with intracellular vesicles, and interacts with both actin stress fibers and microtubules (Cong, 2004).
It is proposed that Wnt stimulates the ß-catenin pathway by relocating Axin to the plasma membrane and inactivating Axin. It is still not clear whether Wnt-induced Axin membrane translocation is a prerequisite for dissociation of the ß-catenin degradation complex. Furthermore, it is unknown whether the only function of Dvl is to facilitate the transport of Axin to the plasma membrane. It is possible that Dvl also brings certain factors to Axin upon Wnt signaling and promotes inactivation of the Axin complex. Indeed, it has been suggested that in response to Wnt, Dvl can recruit Frat/GBP, a strong inhibitor of GSK3, to the Axin-GSK3-ß-catenin complex, although a requirement for Frat/GBP in Wnt signaling has not been established genetically. Furthermore, it is unclear whether inhibition of GSK3 normally plays a major role in Wnt signaling, although dominant-negative mutants of GSK3 can activate ß-catenin signaling. It has been shown that Wnt induces dephosphorylation of Axin, which might reflect inhibition of GSK3 or dissociation of the Axin-GSK3 complex. Dephosphorylated Axin appears to be less stable and binds ß-catenin less efficiently. It is currently unknown how membrane translocation of Axin is coupled to dephosphorylation and destabilization of Axin. More work will be necessary to illustrate fully the molecular mechanism by which Wnt induces the stabilization of ß-catenin (Cong, 2004).
During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly results from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).
One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).
It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).
Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).
Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).
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 TGFþ 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-þ) 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).
See the embryonic expression pattern of arr at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
The arrow gene was cloned using a lethal P-element insertion [l(2)k08131] that fails to complement arrow mutants. The longest complementary DNA (6,012 base pairs) restores smooth cuticle to alternate segments of arrnull embryos when expressed under control of Prd-GAL4, indicating rescue of Arrow function and Wg signaling (Wehrli, 2000).
Initially, Arrow messenger RNA is distributed uniformly in the embryo, owing to maternal contribution, but by stage 9 broad stripes are superimposed on this global expression. As zygotic expression takes over, Arrow mRNA stripes become more accentuated, such that by stage 13 the highest levels are posterior to the En domain, and the lowest levels are just anterior to the En domain, in the region signaled by Wg at this stage. Similarly, in leg discs arrow transcription is lowest in cells expressing Wg. This suggests that arrow transcription is negatively regulated by Wg signaling, in a manner similar to the Frizzled receptors Fz and DFz2, but unlike the signal transducers Dsh and Arm, which are both uniformly expressed. Although the significance of this transcriptional regulation is unclear, it is unlikely that it is relevant to signaling in the embryo, as global maternal arrow contribution suffices for most epidermal Wg signaling events. Polymerase chain reaction with reverse transcription (RT-PCR) analysis shows also that arrow mRNA is expressed in S2 cells, consistent with these cells being able to respond to Wg once transfected with DFz2 (Wehrli, 2000).
The Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array, with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work has shown that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave. Evidence is presented for a second and orthogonal axis of polarity: this signal can be functionally separated from the A/P axis (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). The polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps -- the activity of one signaling molecule functions to establish the graded activity of a second signal. Ectopic Wg expression results in two significant effects. (1) Clones are generated with associated polarity inversions. (2) Although significant changes in retinal polarity are associated with the clones, the distance over which the effect is exerted is restricted to from between 7 to 2 ommatidial rows. Ectopic Wg clones have two distinct features with respect to their polarity effects: (1) the aberrant polarity is asymmetrically distributed in relation to the clone (greater changes in polarity occur in polar positions relative to the center of the clone), and (2) the potency of the Wg-expressing clones to induce polarity reversals show maximial polarity-reversal effects at the equator and minimal effects at the pole (Wehrli, 1998).
Other genes downstream of wingless also appear associated with eye Eq/Pl polarity. The product of the arrow (arr) gene has been placed in the Wingless pathway based on a number of criteria:
The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).
In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein. (2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm- and arrow- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).
Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm- and arrow- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).
It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm- and arrow- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm- and arrow- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).
None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).
If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).
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date revised: 17 January 2008
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