Transcriptional Regulation

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

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

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

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

Targets of Activity

Actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine is able to slow prehair extension. At higher doses, a complete blockage of hair development is seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton results in the development of multiple prehairs along the apical cell periphery. The multiple hair cells are a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development, since treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton. Several other mutant genotypes produce branched or split bristles or hairs. For example, mutations in singed, chickadee and capping protein produce bristles and/or hairs that are split, bent or stunted in ways that partially resemble cytochalasin D treatment. However, the phenotypes associated with these mutations do not resemble those seen with CD treatment as closely as the phenotype associated with crinkled (e.g. there is not hair splitting in sn mutants). The recent finding that mutations in the small G-protein rho result in an inturned-like phenotype and that the expression of a dominant negative form of rac also results in multiple hair cell phenotype is interesting with regard to the interaction of the actin and microtubule cytoskeletons. Small G-proteins of the rho and rac families are thought to interact with the actin cytoskeleton, yet they produce a wing hair phenotype that is similar to what is seen with the disruption of the microtubule cytoskeleton. This could be due to both the small G-proteins and the micotubule cytoskeleton being required for localizing a common component or activity to the vicinity of the distal vertex, or to the small G-proteins affecting the structure of the microtubule cytoskeleton, or to the microtubular cytoskeleton functioning in the localization of the small G-proteins or, alternatively, these two classes of proteins could be functioning in parallel pathways that function independently to restrict prehair initiation to the distal region of the cell. The observation that the expression of a dominant negative form of rac1 causes a disruption of the microtubule array suggests the possibility that the phenotypes associate with G-protein loss could be due to their disrupting the structure/function of the microtubule cytoskeleton and not to their being part of the frizzled signaling/signal transduction pathway (Turner, 1998).

The genes spiny legs, elav and nemo are downstream of frizzled (Zheng, 1995). dishevelled is a component of the frizzled signaling pathway acting downstream of frizzled. frizzled, dishevelled, and prickle act upstream of fuzzy and inturned. These then act upstream of multiple wing hair (Krasnow, 1995). dishevelled is a segment polarity gene that mediates the transduction of a wingless signal (Krasnow, 1995).

The tissue polarity gene fuzzy (fy) has two roles in the development of Drosophila wing hairs. One is to specify the correct orientation of the hair by limiting the site of prehair initiation to the distal vertex of the wing cell. The other is to control wing cell hair number by maintaining the integrity of the cytoskeletal components that direct hair development. The requirement for fy in these processes is temperature dependent, as the amorphic fy phenotype is cold sensitive. Analysis of mosaic wings has shown that the fy gene product functions cell autonomously. The fy transcript encodes a novel four-pass transmembrane protein that shares significant homology with incompletely characterized proteins encoded by vertebrate cDNAs. The fourth putative transmembrane domain does not appear to play a significant role in tissue polarity, since it is deleted in a weak fy hypomorph. Expression of the fy transcript is developmentally regulated and peaks sharply at the time of wing cell pre-hair initiation. The extra hairs formed in fy and inturned mutants suggest a role for these genes in maintaining the integrity of cytoskeletal components required for wing hair development, as well as in hair polairty. A similar function has been proposed for Rad1, because the expression of a dominant negative form in the wing has been shown to phenocopy the multiple hair phenotype of fy and inturned (Collier, 1997)

The eye imaginal disc displays dorsal-ventral (D-V) and anterior-posterior polarity prior to the onset of differentiation, which initiates where the D-V midline intersects the posterior margin. As the wave of differentiation progresses anteriorly, additional asymmetry develops as ommatidial clusters rotate coordinately in opposite directions in the dorsal and ventral halves of the disc; this forms the equator, a line of mirror-image symmetry that coincides with the D-V midline of the disc. The currently unanswered question of how D-V pattern is established and how it relates to ommatidial rotation was addressed by assaying the expression of various asymmetric markers under conditions that lead to ectopic differentiation, such as removal of patched or wingless function. D-V patterning is found to develop gradually. wingless plays an important role in setting up this pattern. To determine if positional information associated with equatorial formation is present along the D-V axis of the disc ahead of the MF, expression of an equatorial marker (WR122, a lacZ insertion in an unknown locus) was studied in various genetic conditions that lead to ectopic neuronal differentiation. This expression is dependent on the activity of the gene frizzled, which is required for proper ommatidial rotation. Induction of patched mutant clones activates the Hedgehog pathway and leads to precocious neuronal differentiation.Ectopic ommatidia that arise in clones show that the potential to express WR122-lacZ is restricted to neurons located near the D-V midline, regardless of their position along the A-P axis of the disc. This suggests that the information necessary to restrict WR122 expression exists ahead of the MF (Heberlein, 1998).

The expression of WR122 was examined under conditions that reduce Wingless activity. A temperature sensitve wg allele was used. A reduction in Wg function during the late larval stages promotes recocious differentiation in the eye disc. This differentiation starts from the dorsal (and to a lesser degree the ventral) margin and proceeds inward, roughly perpendicular to the direction of progression of the normal differentiation front. Expression of WR122 is unrestricted among ectopic ommatidia that differentiate as a consequence of reduced Wg function. The normal expression domain of the marker is broadened toward the lateral margins. It is concluded that the expression of WR122 is inhibited by Wg in ommatidia located near the disc's margin, which restricts expression to the equatorial region. Ectopic expression of Wg is sufficient to repress WR122 expression in the more central portions of the retinal epithelium. Thus Wg functions to restrict the expression of the WR122 marker. wingless is necessary and sufficient to induce dorsal expression of the gene mirror prior to the start of differentiation and also to restrict the expression of the WR122 marker to differentiating photoreceptors near the equator. Manipulations in wingless expression shift the D-V axis of the disc as evidenced by changes in the expression domains of asymmetric markers, the position of the site of initiation and the equator, and the pattern of epithelial growth. Thus, Wg appears to coordinately regulate multiple events related to D-V patterning in the developing retina (Heberlein, 1998).

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components does not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, a combination of loss- and gain-of-function studies has been used. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun- clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. Affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Delta transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation (Weber, 2000).

Jun, as a member of the AP-1 family, is activated by many distinct extracellular stimuli and acts downstream of several signaling pathways. Besides its involvement in stress response, Jun has been implicated in the control of proliferation, apoptosis, morphogenesis and cell fate induction. In Drosophila, Jun is critical for the process of dorsal closure in embryogenesis acting downstream of the JNK module. It has also been implicated in cell fate induction downstream of Ras/ERK signaling in the eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity signaling in the eye. It is the first transcription factor implicated in Fz/planar polarity signaling. Fz signaling also requires a JNK (or related kinase) module, and thus in the eye imaginal disc Jun acts downstream of both ERK and JNK. How does Jun achieve a specific response in this context? The S/T residues that are phosphorylated in Jun are the same for both ERK and JNK. Thus, although differences in phosphorylation level and/or preference for any of the serine/threonine target residues cannot be excluded in vivo, differential phosphorylation is unlikely to create specificity. A potential mechanism for specificity might be provided by other transcription factors that cooperate with Jun in the different processes. This is supported by the observation that the sev-JunAsp (expression of a constitutively active Jun) phenotype is a composite of two events, photoreceptor recruitment and ommatidial polarity generation. These two effects can, however, be separated by the reduction of specific interacting partners. In the process of Ras/ERK signaling in photoreceptor induction Jun interacts and synergizes with the ETS domain transcription factor Pointed (Pnt). Pnt has been characterized as a target of the ERK/Rl kinase in Drosophila in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process. Removing one dose of pnt strongly suppresses the Ras/ERK-related extra photoreceptor phenotype of sev-JunAsp, whereas the polarity defects persist and thus are more prominent. This observation indicates that, in the absence of normal Pnt levels, sev-JunAsp specifically affects polarity, suggesting that the interaction with Pnt is important for its role in the ERK-mediated induction. It is likely that for its planar polarity function other specific transcription factors provide the specificity cues (Weber, 2000).

Although all components of the JNK module tested genetically interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function mutants did not show defects in planar polarity establishment, suggesting a redundant role. Even null alleles of the Drosophila homolog of JNKK hep have no effects on planar polarity (Weber, 2000).

However, expression of a dominant negative (kinase dead) isoform of Bsk interferes with planar polarity, giving rise to typical polarity phenotypes, implying that Bsk and JNK signaling are important in this process. Consistently, homozygous mutant clones of the deficiency Df(2R)flp170B that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild polarity phenotype in the eye, including the presence of symmetrical ommatidia (Weber, 2000).

What are the redundant kinases in this process? Genetic interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep and bsk, deficiencies affecting other MKKs and the Drosophila p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially) overlapping phosphorylation targets] might be responsible for the redundancy in this process. The analysis with the dominant negative (DN) Bsk isoform and the respective deficiencies suggests that the p38 kinase(s) are contributing to this redundancy, because they enhance the DN-Bsk phenotype in a manner very similar to that of the bsk deficiency. The identification of specific mutant alleles of p38a/b and double mutant analysis with bsk will be necessary to further clarify this issue (Weber, 2000).

The available results indicate that the level of JNK/p38 signaling in planar polarity establishment is important, but that the removal of a single kinase does not significantly affect this level. In support, the observation that an allelic combination of hep and puc hypomorphic alleles can give rise to planar polarity eye phenotypes suggests that the balance between negative and positive regulators of JNK and related kinases is critical. Similarly, overexpression of the negative JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to those of fz or dsh mutants. It is likely that this phosphatase negatively regulates all JNK-related kinases and thus reduces the overall signaling more than the lack of a single kinase (Weber, 2000).

In summary, these data indicate that the transcriptional events downstream of Fz in R3 specification and chirality establishment (e.g. regulation of Dl) are mediated by Jun. The factors with which Jun is redundant in the imaginal discs are not yet identified. It is possible that other members of the AP-1 family are also involved in planar polarity signaling, since they are related to Jun and could dimerize with it via the leucine-zipper motif. A potential candidate is Fos, because like Jun, Fos is required downstream of JNK in the process of dorsal closure in the embryo. Similarly, the ETS domain protein Yan acts as a negative regulator in dorsal closure and is inactivated by JNK in the process. However, these factors do not show informative planar polarity phenotypes in clones and thus their involvement in this process remains unclear. Although AP-1 and ETS family members are attractive candidates, transcription factors belonging to other families cannot be excluded in this context (Weber, 2000).

Protein Interactions

Dishevelled acts in both wingless and frizzled signaling. It is interesting to note that many frizzled proteins, including FZ2, contain a Ser/Thr-X-Val motif at their C-terminal end: this motif has been shown to interact with PDZ (or DHR) domains in a variety of proteins (Gomperts, 1996). DSH contains a PDZ domain, suggesting a direct interaction of DSH with FZ2. Unlike DFZ2, there is no cannonical PDZ consiensus binding site in the C-terminus of Frizzled (Klingensmith, 1994).

In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. A heterologous system was used to examine the interaction of Dsh with Frizzled. mRNAs encoding Fz or Frizzled2 and a fusion of Dsh to green fluorescent protein (Dsh-GFP) were synthesized in vitro and injected into Xenopus embryos at the four-cell stage. Animal caps from stage 9 embryos, dissected to reveal the blastocoelar cells, were then examined by confocal microscopy. Dsh is recruited selectively to the membrane by Fz but not Frizzled2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. When Fz is expressed simultaneously with Dsh-GFP, Dsh-GFP shows a qualitative redistribution to the membrane or cell cortex. Under these conditions, localization of Dsh-GFP to filopodia present on the blastocoelar (free) surfaces of the animal cap cells was also noticed. Staining with phalloidin (and Dsh-GFP) revealed that the filopodia contain filamentous actin. It is interesting to note that although the filopodia stain with Dsh-GFP, little or no Fz localizes there; at the cell cortex, the Fz and Dsh-GFP show imperfect colocalization. Fz staining is localized predominantly to the plasma membrane, and to a lesser extent to intracellular membranes (probably ER and/or Golgi) in these cells. This suggests that while Fz may induce localization of Dsh-GFP to the membrane and filopodia, it may do so by a mechanism other than direct binding. Frizzled2, the Wingless receptor, fails to induce membrane localization of Dsh, even in the presence of a functional Fz2 ligand (Axelrod, 1998).

Drosophila Dsh is a modular protein of unknown function that is well conserved in relation to its vertebrate homologs. Alignment of family members reveals three conserved domains. The first, a DIX domain, is similar to a domain in murine Axin, a recently described modulator of the Wnt1 pathway. The second contains a PDZ domain; PDZ domains recognize and bind short motifs at the carboxyl termini of proteins (but may bind other motifs as well). PDZ domains can also form dimers. The third domain, called DEP, is conserved among a set of proteins that have in common the ability to regulate various GTPases, including both heterotrimeric G proteins and Ras-like small GTPases. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. A single amino acid substitution in the DEP domain of Dsh is shown to confer a loss of function for PCP signaling, yet the mutant protein is functional for Wg signaling. This single amino acid substitution, coded for by the dsh1 allele, allows for translocation to the membrane, but is thought to impair the ability of Dsh1 to associate with its target at the membrane. This altered membrane interaction diminishes the ability of Dsh1 to function in PCP signaling (Axelrod, 1998).

Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. For example, the individual domains Dsh(DIX) and Dsh deleted for the PDZ domain, are each dominant negative for Wg signaling but have no effect on PCP signaling. Overexpression of Zeste white3, or an activated Arm protein, both involved in Wingless signaling, also fail to produce any effect on PCP. It is proposed that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation. This analysis suggests that Dsh has different interactions in PCP and Wg signaling and predicts an additional genetic behavior (Axelrod, 1998).

If Wg and planar polarity signaling utilize Dsh in a common fashion, then ectopic activation of one pathway should be able to cross-activate the other by promiscuously activating Dsh. In contrast, if each pathway utilizes Dsh in a distinct fashion, then ectopic activation might sequester Dsh in pathway-specific complexes, rendering it unavailable and therefore titrating the activity of the other pathway. These possibilities could best be tested under conditions in which Dsh is limiting. Overexpression of Fz causes a dominant gain-of-function PCP phenotype, and this phenotype is sensitive to the dose of dsh. Can Wg cross-activate Dsh activity for PCP signaling, or can it sequester Dsh? To investigate this question, it was first necessary to know if the Fz-overexpression phenotype would be either enhanced or suppressed by Wingless overexpression. Ectopic expression of Wg suppresses the Fz overexpression phenotype, suggesting that activation of Wg signaling may titrate the amount of Dsh available for PCP signaling. The reciprocal experiment was performed by asking if ectopic activation of the PCP pathway could interfere with Wg signaling. Because the ligand for PCP signaling is unknown, Fz was overexpressed during embryogenesis, and the cuticle phenotype analyzed. These embryos develop with lawns of denticles and are reminiscent of wg-mutant embryos, or those expressing dominant-negative Dsh constructs. The results suggest that titration can occur in this direction as well. The possibility cannot be ruled out that the titration observed in these experiments results from a promiscuous interaction between Wg and Fz, although this interaction may not occur in vivo. The observations are equally consistent with the possibility that under these conditions, activity of one pathway titrates the Dsh level available for the other (Axelrod, 1998).

Pattern formation in multicellular organisms relies on specific inductive signaling events. Many evolutionarily conserved signaling pathways are used at multiple times during development to induce tissue- and cell type-specific responses. Despite the importance of context-dependent signaling specificity, knowlege of the underlying mechanisms has remained elusive. Wnt-Frizzled (Fz) signaling pathways play recurring important roles during the development and homeostasis of vertebrates and invertebrates. Fz receptors can signal through beta-catenin-dependent and -independent pathways. In Drosophila, Fz and Fz2 are redundant receptors for Wg. In addition, Fz conveys signals through a distinct pathway to organize planar polarization of epithelial structures. The cytoplasmic sequences of Fz2 and Fz preferentially activate the beta-catenin and planar polarity cascade, respectively. Both receptors activate either pathway, but with different efficiencies. Intrinsic differences in signaling efficiency in closely related receptors might be a general mechanism for generating signaling specificity in vivo (Boutros, 2000).

Both Frizzled and Fz2 signaling pathways use Dishevelled (Dsh) as a transduction component, raising the intriguing question of how two structurally related receptors signal through a common protein into distinct effector pathways. Fz2 has a higher affinity for Wg than Fz, and removal of either Fz or Fz2 has subtle, but different, effects on the patterning of the embryonic nervous system. Moreover, only Fz is specifically required for epithelial planar polarity by signaling through a Wg-Arm-independent pathway (Boutros, 2000).

Fz overexpression during Drosophila eye development causes a gain-of-function (GOF) planar polarity phenotype. Overexpression of Fz2 in the developing wing activates Wg-Arm targets. To compare the functional equivalence of Fz and Fz2 (Fz will be referred to as Fz1) for activating either the planar polarity or Wg-Arm pathways, Fz1 and Fz2 were expressed with tissue-specific enhancers in imaginal discs during Drosophila development. Whereas Fz1 overexpression in eye and wing discs results in planar polarity phenotypes, Fz2 expression leads to planar polarity defects with only very low penetrance. Conversely, overexpression of Fz2 in wing imaginal discs leads to formation of ectopic bristles (a wg GOF phenotype), whereas Fz1 overexpression does not affect bristle formation. Thus, Fz receptors have distinct signaling abilities in imaginal discs, despite their redundant role for Wg-Arm signaling in loss-of-function (LOF) analysis (Boutros, 2000).

To assess Wnt-beta-cat signaling in a quantifiable in vitro assay, both receptors were injected into Xenopus embryos and Wnt target induction in animal cap explants was analyzed. In this heterologous assay, Fz2 induces strong activation of the Wnt-beta-cat targets Xnr-3 and Siamois (Sia), whereas Fz1 induces a much weaker response. Thus, taken together with the imaginal disc phenotypes, Fz2 is a strong activator of Wnt-beta-cat signaling, and Fz1 is a potent activator of the planar polarity pathway. However, both receptors retain a low intrinsic potential to cross-activate either pathway (Boutros, 2000).

Differential Dsh localization may determine Fz signaling specificity, whereby Fz1, but not Fz2, can induce recruitment of Dsh to the membrane in Xenopus. At normalized protein levels for Fz1 and Fz2, however, no differences were observed in their ability to recruit Dsh. Titration experiments with Fz1 and Fz2 RNA concentrations show very similar threshold levels for either receptor in Dsh membrane localization. Thus, differential Dsh recruitment is unlikely to be the mechanism by which specificity between these Fz receptors is generated (Boutros, 2000).

Fz receptors are serpentine transmembrane proteins composed of an extracellular ligand-sequestering domain (CRD), a seven-pass transmembrane segment, and a COOH-terminal cytosolic tail. To determine which domains in Fz1 and Fz2 are required for directing signaling into either pathway, chimeric and truncated receptors were constructed. These chimeric proteins were tested for their signaling potential in Drosophila imaginal disc development in wings and nota, eyes, and legs for their ability to induce either GOF Wg-Arm signaling or planar polarity phenotypes (Boutros, 2000).

Both Fz1-2 and Fz1-1-2 chimeric proteins activate a Wg-Arm target (Achaete) in the wing imaginal disc, induce ectopic marginal bristles, and show wg-associated effects in the leg. However, they have no significant effect on planar polarity signaling in the eye, the wing, or the notum. Thus, the Wg-Arm signaling outcome corresponds with the presence of the Fz2 cytoplasmic tail. In contrast, GOF planar polarity phenotypes are observed with chimeric Fz2-1 receptors in the wing, the notum, and the eye that are indistinguishable from those caused by Fz1. Both Fz2-1 and Fz2-2-1 show a (mild) dominant-negative phenotype for Wg-Arm signaling, as judged by a reduction in Ac expression, (partial) absence of marginal bristles and notches in the wing margin, and the presence of stunted legs. These data suggest that the chimeric receptors containing the high-affinity Fz2 CRD, but lacking intracellular Fz2 sequences, can sequester Wg efficiently without eliciting an efficient signal transduction response. To test this possibility directly, stabilization of Wg was analyzed in wing discs. Whereas Fz1 or Fz1-2 type chimeras have no significant effect on Wg stability, all chimeric receptors with the Fz2 CRD strongly sequester Wg. The importance of cytoplasmic sequences for efficient Wnt target activation was confirmed in Xenopus animal cap assays (exchanging cytoplasmic domains switches the signaling efficiency). All chimeric receptors are able to recruit Dsh indistinguishably from wild-type Fz1 and Fz2 (Boutros, 2000).

The observation that the Fz1-2 chimera does not dominantly interfere with planar polarity signaling in the eye and because as yet no "planar polarity" ligand has been identified raises the question of whether planar polarity signaling depends on the presumed ligand binding Fz1 CRD. This hypothesis was tested by rescue of the fz-/- polarity mutant with sev-Fz1 and chimeric transgenes. In contrast to Fz1, however, neither Fz1-2 and Fz2-1 chimeras nor Fz2 rescue the fz mutant, indicating that the Fz1 CRD, coupled to its signaling unit, is required for correct levels of activation of planar polarity signaling in the eye in vivo. Thus, although overexpression of Fz2-1 induces a GOF planar polarity phenotype, identical to that induced by Fz1, it cannot replace Fz1 in a LOF background. Although both receptor isoforms, Fz1 and Fz2-1, when overexpressed, are capable of activating planar polarity signaling and perturbing correct polarity determination, the pathway is activated to the correct level only by the Fz1 CRD (and ligand)-dependent regulation of the receptor. Because the establishment of correct ommatidial polarity results from small differences in Fz signaling levels between neighboring R3 and R4 cells, the ability to precisely respond to the ligand in a spatially and temporally controlled manner is essential. Presumably, only Fz1 is appropriately regulated through its CRD to instruct correct ommatidial polarity (Boutros, 2000).

Fz1 and Fz2 appear to have different abilities to activate Wg-Arm and planar polarity signaling in the GOF assays in imaginal discs. Whereas Fz2 induces a Wg-Arm GOF phenotype, Fz1 causes GOF planar polarity phenotypes. The chimeric receptors define the respective cytoplasmic tail (Fz2) or the cytoplasmic domains (Fz1) as largely, but not solely, responsible for mediating these differences in the GOF assays. The Wg-Arm GOF phenotype is ligand- and CRD domain-dependent, because it can only be observed close to the source of Wg. Also, Fz2 has a stronger effect than Fz1-2 or Fz1-1-2. The planar polarity ligand is, possibly, a member of the Wnt family with a different CRD binding affinity from that of Wg. The mechanism by which the ligand-CRD interaction regulates Fz signaling is unclear. The present data cannot distinguish between an activating (conformational) change, or alternatively, a constitutive signaling capacity by Fz's that is inhibited by another factor and needs to be antagonized by the ligand (similar to Smoothened/Patched signaling with Hedgehog) (Boutros, 2000).

How can one reconcile the Fz1 and Fz2 redundancy for Wg signaling in LOF analysis and the dominant-negative behavior of the Fz2-1 chimeras? Fz2 is a high-affinity Wg receptor, and fz2 transcription is down-regulated by Wg, whereas Fz1 (a low-affinity receptor) is expressed fairly uniformly. Thus, Fz2 might be the primary Wg receptor, and Fz1 substitutes only in its absence. Moreover, another Drosophila Fz family member, Fz3, acts as a negative attenuator of Wg signaling and is positively regulated by Wg, suggesting that the expression patterns of Fz2 and Fz3 shape the Wg response, whereas Fz1 does not contribute to this effect. In this context, overexpression of Fz2-1, consisting of a high-affinity Wg-binding CRD fused to a low-efficiency signaling unit, adversely affects the signaling outcome and causes a dominant-negative phenotype (Boutros, 2000).

These experiments provide a model for how signaling specificity can be achieved by closely related receptors, and they demonstrate that LOF studies, like GOF experiments, might only provide a partial answer in case of redundancies. Quantitative differences in ligand affinity and signal transduction efficiency of Fz receptors could provide overlapping and nonoverlapping functions in different cells, depending on the threshold needed to induce targets and expression levels of the various members of the receptor family. Thus, the relative ratio of the different Fz receptors on the cell surface and their degree of occupancy could be an important factor determining the signaling outcome. Additional factors such as coreceptors could influence the signaling outcome: for example, the heparan sulfate proteoglycan Dally has been identified as a coreceptor in Wg signaling. Fz1 and Fz2 signaling preferences provide an example of how quantitative differences in signaling levels can lead to redundant and specific roles for these receptors during development and evolution (Boutros, 2000).

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).

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).

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).

Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding

Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in Drosophila. This study provides mechanistic insight into Frizzled/Dishevelled signalling regulation. The ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling (Jenny, 2005).

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

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

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

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

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

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

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

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

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

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

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

Trimeric G protein-dependent Frizzled signaling in Drosophila

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

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

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

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

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

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

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

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

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

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

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

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. A direct molecular link is described between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. Patj binds the cytoplasmic tail of Fz1 and is proposed to recruit aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. During PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. A model is proposed whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active (Djiane, 2005).

The C tail of Fz receptors regulates their localization and signaling activity. A short Fz Cterm governs apical localization, which is critical for effective Fz-PCP signaling. In contrast, a long Cterm (like that of Fz2) governs baso-lateral localization, promoting β-catenin signaling and preventing PCP activity. Thus, a striking feature of all core PCP proteins, including Fz1, is their apical localization within imaginal disc cells. Fz1 colocalizes partially with several components that regulate A/B polarity such as the Crumbs/Sdt/dPatj and Baz/aPKC/Par-6 complexes within the marginal domain, even though it is also present more basally relative to these complexes (Djiane, 2005).

Detailed sequence analysis of the Fz1 Cterm has revealed the presence of two clustered conserved PKC phosphorylation sites (Ser554 and Ser560 in Fz1). Given that aPKC expression in the apical domain overlaps with Fz1, a test was performed to see if aPKC can phosphorylate the Fz1 Cterm on the two conserved PKC sites in an in vitro kinase assay. Purified human aPKC protein phosphorylates in vitro a GST::Fz1 Cterm fusion protein. Furthermore, mutations of the two PKC consensus sites (Ser to Ala) prevent aPKC-mediated Fz1 phosphorylation, confirming that these sites are targets of aPKC (Djiane, 2005).

To investigate the importance of these phosphorylation sites in vivo, flies were generated carrying UAS-inducible transgenes of Fz1 mutant derivatives with either both serines mutated to alanine (Fz1-AA), inactivating the two prospective PKC sites, or both Serines mutated to Glutamic acid (Fz1-EE), mimicking phosphorylation. These transgenes were analyzed under sevenless (sev)-Gal4 control, which is expressed specifically in R3/R4 precursor cells just posterior to the MF during PCP establishment. Overexpression of wild-type Fz1 provides too much activity and interferes with the balance of Fz1 regulation within the R3/R4 pair, resulting in ommatidia with random R3/R4 cell fate decision and chirality, as well as symmetrical R3/R3 type ommatidia. Similarly, overexpression of Fz1-AA (with both aPKC sites inactivated; sev>Fz1-AA) induces ommatidia with random chirality and symmetrical clusters. In contrast, the phosphomimetic Fz1-EE (sev>Fz1-EE) shows hardly any effect. These data suggest that aPKC-mediated Fz1 phosphorylation inhibits Fz-PCP signaling activity (Djiane, 2005).

Since apical Fz1 localization is critical for its proper PCP signaling activity (Wu, 2004), it was hypothesized that the Fz1-EE mutation could affect the localization of the receptor. To investigate this possibility, the expression of the different myc tagged Fz1 transgenes was examined in imaginal discs (under en-Gal4 or dpp-Gal4 control). No difference between the expression of either Fz1-AA or Fz1-EE with that of wild-type Fz1 was found. These mutant Fz1 isoforms were expressed at similar levels and colocalized apically with aPKC, indicating that phosphorylation of Fz1 by aPKC does not affect Fz1's localization (Djiane, 2005).

A second feature critical to Fz1 signaling activity is its ability to recruit Dsh to the membrane. Interestingly, the aPKC sites partly overlap with the region of the Fz Cterm known to bind Dsh, raising the possibility that phosphorylation by aPKC could interfere with Dsh recruitment. Thus tests were performed to see whether Dsh recruitment is affected by phosphorylation of the Fz1 aPKC sites. S2 cells, which have no endogenous Fz, were transfected with Dsh-GFP and the different Fz1 mutants. In this assay, wild-type and both mutant forms of Fz1 recruit Dsh-GFP efficiently to the membrane. Then, whether overexpression of Fz1-AA and Fz1-EE can recruit Dsh-GFP to apical membranes like wild-type Fz1 in vivo (expressed with en-Gal4 in the posterior compartment of wing discs, where there is a sharp boundary between expressing and nonexpressing cells) was examined. Both Fz1-AA and Fz1-EE behave like wild-type Fz1, sequestering Dsh to the apical cell membrane in imaginal disc cells (Djiane, 2005).

In summary, it was shown that Fz1 can be phosphorylated in vitro by aPKC. Together with the in vitro results, the in vivo analysis of a phosphomimetic Fz1 mutant suggests that aPKC phosphorylation regulates Fz1 activity negatively and that this effect is not mediated by affecting Fz1 localization or Fz1-mediated Dsh membrane recruitment (Djiane, 2005).

In light of the importance of the aPKC sites in Fz1, it was of interest to determine how aPKC is recruited to the receptor. This could be mediated by direct binding or through a bridging factor, the most likely candidates being the A/B determinants that bind aPKC. Thus a two-hybrid interaction screen was conducted using the Fz1 Cterm as bait and components of different A/B protein complexes as prey. The closely related Fz2 Cterm was included as well as the Stbm Cterm as control baits. All components of the aPKC/Par-6/Bazooka apical complex were tested. For Baz, three different fragments were used: an N-terminal fragment involved in Baz dimerization (BazA), a central fragment with three PDZ domains involved in Par-6 binding (BazB), and a C-terminal fragment that binds aPKC (BazC). Similarly, all components of the Crb/Sdt/dPatj apical complex were tested except Crb (since Crb is a transmembrane protein) and the components of the more baso-lateral Scrib/Dlg/Lgl complex were analyzed. No direct interaction was detected between the Fz1 Cterm and aPKC, but, interestingly, Patj was found to be a specific binding partner of the Fz1 Cterm. No other protein was found to interact with the Fz1 Cterm, and in turn Patj did not interact with the Fz2 or Stbm Cterms. The interaction between the Stbm Cterm and Dlg was confirmed as was the interaction of aPKC with Par-6 and BazC (Djiane, 2005)

To confirm the Patj-Fz1 interaction in vivo, coimmunoprecipitation (CoIP) experiments were performed from Drosophila S2 cell extracts transfected with GFP fusion proteins with the Fz1 or Fz2 Cterms (GFP::Fz1 and GFP::Fz2, respectively). Patj could be co-immunoprecipitated from cells transfected with GFP::Fz1 but importantly not with GFP::Fz2 or GFP alone , demonstrating that Fz1 and Patj interact in Drosophila cells. A weak interaction was found between Fz1 and endogenous Baz and aPKC, suggesting the existence of one or several multiprotein complexes among Fz1, Patj, Baz, and aPKC. In contrast, other components of A/B protein complexes, such as Par-6 or Dlg, did not CoIP with either GFP::Fz1 or GFP::Fz2 (Djiane, 2005).

To map the Patj interaction domain with the Fz1 Cterm, GST pull-down experiments were performed. Patj is a modular protein containing a N-terminal L27 domain (previously referred to as MRE), mediating its interaction with Sdt, and four PDZ domains. Consistent with the yeast two-hybrid and CoIP results, in vitro translated full-length Patj bind the GST-Fz1 Cterm protein. The fourth PDZ domain of Patj is sufficient for direct binding to the Fz1 Cterm (Djiane, 2005).

Using the CoIP approach, the residues in the Fz1 Cterm required for Patj interaction were also mapped. Whereas the full-length Fz1 Cterm interacts with Patj, removing the last three residues (Fz1ΔBS) abolishes this interaction. The removal of an internal Cterm motif, encompassing the tryptophan critical for Dsh binding, retains Fz1 ability to bind Patj albeit to a lesser extent (Keyes, 2005).

These results support a direct interaction between the apical determinant Patj and the Fz1 Cterm and suggest that Patj could provide a link between Fz1 and aPKC, since Patj was shown in vitro to bind to aPKC either directly or indirectly through Par-6. The Fz1/Patj interaction is mediated by the fourth PDZ domain of Patj, requires the last three residues of Fz1, and is largely independent of the Fz motif that mediates Dsh binding (Djiane, 2005).

Apical localization is critical for PCP protein activity and particularly for Fz1, but until now no direct link between A/B polarity and PCP establishment has been described. This study shows that the apical determinants aPKC and Patj negatively regulate Fz-PCP signaling while Bazooka antagonizes this regulation. Patj binds directly to the Fz1 cytoplasmic tail, possibly recruiting aPKC, whose phosphorylation of two serine residues within the Fz1 Cterm inhibits the activity of the receptor in cells where signaling should not occur. This reveals a direct link between A/B polarity determinants and PCP establishment (Djiane, 2005).

This work provides the first evidence for a direct molecular link between A/B polarity determinants and PCP by demonstrating that the apical determinants aPKC, Patj, and Baz regulate Fz1 activity. This regulation is independent of Fz1 recruitment to the apical membrane, however, since none of the tested A/B determinants is actively responsible for it. For instance, deleting the Patj binding site in Fz1 or replacing the Fz1 Cterm for a shortened Fz2 Cterm, which cannot bind Patj, has no effect on Fz1 apical localization (Wu, 2004), excluding Patj as a recruiting or targeting factor in Fz1 apical localization. Furthermore, Fmi apical localization is unaffected in Patj and Baz mutants. Thus, although an intact A/B polarity is a prerequisite for PCP signaling, there is no mutual dependency for localizing the Patj/aPKC and the Fz-PCP complexes to the apical side of imaginal disc cells, where they can functionally interact (Djiane, 2005).

Other studies also support the existence of a link between A/B polarity and PCP. In the mouse, Looptail (Lp), the homolog of the Drosophila PCP gene stbm/Vang, interacts genetically with mScribble, a baso-lateral determinant conserved in flies. In particular, transheterozygous Lp/mScribble mice show PCP defects in the inner ear. In Drosophila, it has also been shown that PCP factors interact with A/B determinants. Recent work in the sensory organ precursor (SOP) cells has shown that the orientation of the two opposing domains of Dlg (anterior) and Baz (posterior) is dependent on Stbm and Fz activity (Djiane, 2005).

The downregulation of aPKC and Patj in the R3/R4 cells when Fz1 signals to induce PCP is consistent with a model whereby inhibitory phosphorylation of Fz1 mediated by aPKC is occurring throughout all eye disc cells, except in those that are required for PCP establishment at the time Fz1 signaling occurs. Fz1 activity is therefore always kept low outside of the PCP signaling window, and a release of this inhibition is required for PCP signaling to take place. It is noteworthy that overexpression of Fz1 always gives a robust GOF effect without requiring additional “input,” arguing that either the receptor is constitutively active or that a ligand is always present in nonlimiting amounts. In either scenario, it would be important to control Fz1 activity to prevent signaling at the wrong time and to allow limiting signaling components, such as Dsh, to be available for canonical Wnt/Fz-β-cat signaling when PCP signaling is not needed (Wu, 2004). This is particularly true in the eye disc, where cell fate determination and PCP occur almost simultaneously within a short time window. It is thus proposed that the downregulation of aPKC/Patj in the R3/R4 precursors, at the time of PCP establishment, determines when and in which cells Fz1 is active. A detailed analysis of the expression of Fz1 and Fmi in the non- R3/R4 cells reveals that they extend more basally than aPKC and Patj. Since the precise localization of the active Fz1 is unknown, it is possible that either another mechanism inactivates Fz1 more basally or that inactivation is not needed there (Djiane, 2005).

Furthermore, these results argue that high Baz levels in R3/R4 cells promote Fz1 signaling, possibly by antagonizing the inhibitory regulation of Fz1 by aPKC. Indeed, several lines of evidence suggest an inhibitory role of Baz on the activity of an aPKC complex. (1) In Drosophila embryonic neuroblasts, aPKC phosphorylates Lgl on the apical side of the cell to inhibit its function, restricting the active Lgl to the basal domain of the cell. This is mediated through direct binding of a Par-6/aPKC complex to Lgl, which can only occur after Baz is released from the Par-6/aPKC complex, suggesting a negative role of Baz on aPKC function. (2) Direct measurements of aPKC kinase activity on an exogenous substrate reveal that addition of purified Par-3, the vertebrate Baz homolog, inhibits aPKC kinase activity, whereas Par-6 enhances it. However, whether the aPKC inhibition by Par-3 is direct or indirect remains unclear. This antagonizing role of Bazooka on the aPKC-mediated inhibition of Fz1 activity in R3/R4 cells is further evidence of the tight regulation required for PCP establishment in the eye (Djiane, 2005).

In this model, the A/B determinants are acting upstream of PCP. Consistent with this, there is no effect on either aPKC or Patj expression in cell clones mutant for PCP genes. Similarly, the initial Baz enrichment in R3/R4 precursors is Fz/PCP independent. The later enrichment of Baz in R4 is, however, dependent on PCP signaling. This could correspond to a similar situation as observed in the SOP, in which the posterior relocalization of Baz is dependent on Fz1 activity (Djiane, 2005).

How does aPKC regulate Fz-PCP activity? The aPKC-mediated phosphorylation of the Fz1 Cterm inhibits its activity without affecting its apical localization or ability to recruit Dsh. The negative regulation must therefore occur by a different mechanism. One possibility is that Fz1 phosphorylation by aPKC inhibits a PCP-specific signal transduction to Dsh. Consistent with this hypothesis, similar point mutations in the conserved PKC sites of the canonical Wnt/β-cat-dedicated Fz2 (Fz2-AA and Fz2-EE), do not affect Fz2 ability to trigger a Wnt/β-cat response when overexpressed in the wing. Another possibility is that aPKC regulates Fz1 activity by promoting its destabilization or by increasing its turnover through the recycling pathway at the apical membrane. Further investigation will be required to answer these questions (Djiane, 2005).

The selective downregulation of Patj and upregulation of Baz in R3/R4 precursors define when and where Fz1, and therefore Fz-PCP signaling, is active. This scenario represents a permissive rather than an instructive requirement of aPKC, Patj, and Baz during PCP. Fz-PCP signaling components are widely expressed but only required at specific time points and in specific subsets of cells. As no activating PCP specific ligand is known, it is possible that alternate mechanisms control their activity. This study provides evidence for a negative regulation of PCP signaling by A/B polarity determinants, unveiling new mechanisms for regulating PCP. In addition to their importance during A/B polarity, a function has been revealed for the apical determinants Patj, Baz, and aPKC in regulating PCP and evidence is provided for a molecular link between apical-basal and planar cell polarity (Djiane, 2005).

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

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

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

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

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

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

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

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

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

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

Differential stability of flamingo protein complexes underlies the establishment of planar polarity

The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. This study examined the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. Fmi was found to interact preferentially with distal-complex components, rather than with proximal components, and evidence is presented that there are different domain requirements for Fmi to associate with distal and proximal components. Distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions, and evidence is presented that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, evidence is provided that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. A model is presented in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry (Strutt, 2009).

The differing ability of overexpressed Fmi to modulate Fz:Dsh and Stbm:Pk levels at junctions could be explained by a number of mechanisms. One likely hypothesis is that Fmi may require a cofactor for a robust interaction with Stbm, and that this cofactor is limiting when Fmi is overexpressed. Alternatively, Fmi may require posttranslational modification or a conformational change to interact with Stbm, and a factor needed for this modification is limiting. The cytoplasmic C-terminal tail of Fmi is a likely region to mediate an interaction with Fz:Dsh or Stbm:Pk; therefore, a truncated form of Fmi was constructed, in which this region is either absent or replaced with GFP (Strutt, 2009).

When overexpressed in pupal wing cells, FmideltaIntra is much more efficient at recruiting Fz and Dsh to junctions than full-length Fmi, an effect similar to that caused by removal of stbm or pk. Stbm is still reduced at junctions, although less than when full-length Fmi is overexpressed. This suggests that the C-terminal intracellular domain of Fmi is dispensible for the interaction of Fmi with Fz:Dsh and, importantly, that Fz:Dsh no longer have to compete with Stbm:Pk for access to Fmi (Strutt, 2009).

Interestingly, two isoforms of Fmi have been identified, one of which contains a PDZ binding motif (PBM) at its C terminus. It is possible that loss of the PBM alone could account for the failure of overexpressed Fmi or FmideltaIntra to associate with Stbm:Pk. However, this is unlikely, because Fmi that lacks the PBM can rescue the planar polarity phenotype of fmi mutants (Strutt, 2009).

Endogenous Fmi is thought to be localized on both proximal and distal cell boundaries. This was confirmed by expressing CFP-tagged Fmi at physiological levels in clones in pupal wings, and it was observed that levels of staining appear similar at each end of the cell, consistent with the homophilic-interaction model. Notably, expression of a GFP-tagged form of FmideltaIntra results in its preferential localization to distal cell edges, where Fz and Dsh also localize (Strutt, 2009).

Interestingly, junctional localization of FmideltaIntra-EGFP is not dependent on endogenous, full-length Fmi, suggesting that this molecule is still able to participate in homophilic interactions. Hence, the ability of FmideltaIntra-EGFP to functionally rescue the polarity phenotype of fmi null mutant clones was investigated. If FmideltaIntra-EGFP interacts preferentially with the distal Fz:Dsh complex, then Stbm recruitment to junctions inside clones would be compromised. Consequently, FmideltaIntra-EGFP:Fz complexes inside the clone would preferentially interact with Fmi:Stbm outside the clone, leading to a reversal in polarity on proximal clone edges. Importantly, this prediction is upheld, and fmi clones rescued with FmideltaIntra-EGFP exhibit weak proximal polarity inversions, such that trichomes point away from the clone, and polarity proteins are recruited to the clone boundary (Strutt, 2009).

Nevertheless, Stbm localizes asymmetrically inside the clone, although not always at the correct site, whereas in a fmi null mutant it lacks any asymmetric localization. Thus, FmideltaIntra-EGFP must retain some ability to interact with Stbm. To confirm this, the ability of full-length Fmi or FmideltaIntra-EGFP to interact with Fz and Stbm in Drosophila S2 cells was analyzed. In this assay, Fmi and FmideltaIntra-EGFP are recruited to sites of cell contact, as a result of homophilic interactions between their extracellular domains. Cotransfection of Fz or Stbm with either full-length Fmi or FmideltaIntra-EGFP in Drosophila S2 cells results in the recruitment of both to sites of cell contact (Strutt, 2009).

Interestingly, if S2 cells were transfected with either Fz or Stbm and then mixed, weak recruitment is also observed to sites of cell contact, arguing that their extracellular domains can interact independently of Fmi. Nevertheless, recruitment was weaker and less frequent than when Fmi was cotransfected, suggesting that Fmi:Fmi interactions are more important than Fz:Stbm interactions in stabilizing complexes between adjacent cells (Strutt, 2009).

The data suggest that Fz:Dsh and Stbm:Pk complexes differ in their ability to associate with Fmi. Whereas endogenous levels of Fmi result in the formation of asymmetric complexes with Fz:Dsh on one side of the boundary and Stbm:Pk on the other, overexpressing Fmi favors Fz:Dsh recruitment. Furthermore, a C-terminally deleted form of Fmi preferentially localizes distally with Fz, and overexpression of this form has an even greater preference for Fz:Dsh recruitment. Thus, the C terminus of Fmi is important in promoting the interaction with Stbm:Pk. The Fmi truncation data could be explained simply by the possibility that the C terminus of Fmi contains a direct binding site for Stbm; however, this fails to explain why overexpressed full-length Fmi prefers to recruit Fz:Dsh. It is therefore proposed that the association of Fmi with Stbm:Pk requires a limiting factor that is saturated by Fmi overexpression. The most plausible hypothesis is a requirement for a cofactor for Stbm:Pk binding, but other possibilities include saturation of the machinery for a posttranslational modification or a conformational change in Fmi (Strutt, 2009).

The data also suggest that Fmi itself needs to associate with both proximal and distal components in order to be stably localized to apicolateral junctions. Although it can form homophilic dimers between adjacent cell membranes in tissue culture, in pupal wings Fmi does not localize strongly to apical junctions and presumably fails to form stable homodimers in trans. Fz on one side of the junction and Stbm:Pk on the opposite side stabilize Fmi at junctions, most likely by promoting homophilic interactions or preventing internalization. However, Fmi appears to be capable of forming complexes with either distal or proximal components alone, but these complexes (particularly the proximal complex) are apparently less stable at junctions. Taken together with overexpression experiments, this would suggest that the most stable configuration is Fz:Fmi on one side of the boundary and Fmi*:Stbm:Pk on the other (where Fmi* denotes the modified form able to preferentially associate with Stbm:Pk) (Strutt, 2009).

In order for an asymmetric complex to be stabilized across junctions, the extracellular domains must somehow 'look' different. One possibility is that the Fz and Stbm extracellular loops interact - a view supported by S2 cell data. Alternatively, the Fmi extracellular domain, when associated with either Fz or Stbm:Pk, could undergo a conformational change that promotes homophilic Fmi interactions (Strutt, 2009).

An intriguing question is why clones of cells that overexpress Fmi behave like fz loss-of-function clones. It is suggested that within the clones, excess Fmi associates with the entire available pools of both Fz and Stbm. However, there is still a pool of uncomplexed Fmi that can associate with Fmi:Fz in adjacent wild-type cells, forming the relatively stable Fmi-Fmi:Fz configuration, thus causing polarity to be reversed on distal clone boundaries. In support of this model, an identical nonautonomous effect is seen when FmideltaIntra is overexpressed, which itself interacts only poorly with Stbm but presumably can interact with Fmi:Fz in adjacent cells outside the clone (Strutt, 2009).

Interestingly, Fmi accumulates in excess at junctions in a dsh, stbm double mutant, whereas Fz does not. Thus, although Fz acts to stabilize Fmi at junctions, Fmi does not always need to associate with Fz in a stoichiometric fashion in order to be stabilized. Perhaps as long as there is some Fz associated with Fmi, this may permit local stabilization of other Fmi molecules in cis. Alternatively, this excess accumulation of Fmi might simply represent 'unstable' Fmi homodimers that are no longer being removed from junctions by the actions of Dsh and Stbm (Strutt, 2009).

The composition of the complex with which Fmi is associated appears to be critical for determining the frequency and manner by which Fmi is turned over from the plasma membrane. Most compellingly, Fmi accumulates more strongly in an enlarged endosomal compartment in Rab7TN mutant tissue when stbm and fz are absent than when they are present. Thus, it is suggested that more Fmi is resident in the endocytic pathway when it is unable to form stable asymmetric complexes. Fmi:Fz puncta have been observed that are selectively trafficked to distal cell edges. In the current experiments, these puncta colocalize with YFP-Rab4, suggesting that Fmi and Fz are recycled back to the plasma membrane by a Rab4-dependent mechanism. Furthermore, the increased intracellular and junctional levels of Fz and Fmi in dor mutant clones suggests that in addition to being recycled to the plasma membrane, a significant fraction of internalized Fmi and Fz is also sent for degradation. It is formally possible that the intracellular accumulation of Fmi and Fz seen when lysosomal trafficking is blocked by loss of Rab7 or in dor clones is due to their being sent for degradation immediately after synthesis (e.g., if damaged or misfolded); however this is unlikely because newly synthesized Fmi-ECFP appears first at junctions before been seen in puncta (Strutt, 2009).

Stbm has not been observed in large intracellular puncta, but it seems likely that it is also internalized and recycled, possibly together with Fmi, although it must do so by alternative pathways involving smaller or more rapidly recycling particles that are not visible by confocal microscopy. Indeed, the data suggest a potential role for Dsh and Stbm in regulating junctional levels of Fmi. A stbm mutant alone results in a loss of Fmi from junctions, consistent with a need for Stbm in stabilizing Fmi in asymmetric complexes. In contrast, loss of Dsh and Stbm together increases Fmi levels at junctions, suggesting a role for Stbm in internalization. It is suggested that the outcome of any interaction of Stbm with Fmi is dependent upon whether Fmi is able to form stable homodimers with Fz on the opposite cell membrane. In a wild-type situation, one could envisage that Fmi forms stable homodimers in a Fz:Fmi-Fmi*:Stbm configuration, and that both Dsh and Stbm promote internalization of any Fmi that is not in this configuration, the majority of which is subsequently recycled back to the plasma membrane. In dsh mutants, there is reduced internalization, but the effect on Fmi levels is subtle; Fz and Stbm are still present to promote Fmi homodimer formation, and Stbm still promotes internalization of any unstable Fmi. In contrast, in stbm mutants, the number of less stable Fmi complexes (associating only with Fz) is greatly increased, favoring internalization by Dsh. Finally in dsh, stbm double mutants, Fmi is again less stable (associating only with Fz), but there is no Dsh- or Stbm-mediated internalization, leading to an overall increase of Fmi at junctions (Strutt, 2009).

How do Dsh and Stbm regulate Fmi levels at junctions? Stbm contains potential interaction motifs for the endocytic adaptor AP2, but their role has not been functionally tested. In addition, in vertebrate Wnt signaling, there is evidence that Dsh interacts with the endocytic adaptor protein β-arrestin and mu2 subunit of AP2 to mediate Wnt/Fz endocytosis and downregulation of Wnt signaling. Interestingly, in planar polarity this is no evidence that Dsh directly mediates internalization of Fz, but the data rather point to Dsh promoting Fmi internalization when it is not associated with Fz. Instead, the trafficking of Fmi together with Fz into the lysosomal pathway is Dsh independent (Strutt, 2009).

In summary, it is proposed that a number of mechanisms exist by which Fmi contributes to the generation of asymmetry at the molecular level. First, the characterization of the previously inferred asymmetry in Fmi activity indicates that Fmi normally prefers to bind to Fz and requires a limiting factor for association with Stbm:Pk. Second, Fmi stability at junctions is dependent on both Fz and Stbm:Pk, with the most stable form being Fz:Fmi bound to Fmi*:Stbm. Finally, it is proposed that entry of Fmi into the endocytic trafficking pathway is decreased if it is in a stable complex, and this is regulated either by Dsh and Stbm or independently of Dsh and Stbm, depending on whether it is associated with Fz (Strutt, 2009).

An outstanding question is how these mechanisms translate into cellular asymmetry, such that in any particular cell, heterophilic polarity complexes preferentially form with Fz:Dsh at the distal junctions, rather than having heterophilic complexes in both orientations. It is thought that the acquisition of cellular asymmetry is likely to be driven by directional trafficking of Fmi:Fz, although other models, such as a mechanism for preferential stabilization of Fmi:Fz interactions at the distal cell edge, are also possible. In addition, it seems likely that an amplification mechanism would be required, although the molecular mechanisms remain to be elucidated (Strutt, 2009).

While this manuscript was in preparation, another manuscript was published, in which Fmi was proposed to mediate an asymmetric and instructive signal between proximal and distal complexes to generate asymmetry, and thus does not act merely as a scaffold for Fz:Stbm interactions across membranes. It is argued that the current data do not provide evidence for a specific signaling function of Fmi. Instead, the hypothesis is favored that the composition of the proximal and distal complexes is distinct, and that heterophilic complexes are inherently more stable than homophilic complexes. Together, removal of unstable nonasymmetric complexes through increased endocytic turnover, in concert with directional trafficking and an unknown amplification mechanism, may be sufficient to generate asymmetry without the need to invoke a specific signaling function for any components of the complexes (Strutt, 2009).

Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). This study demonstrates in Drosophila wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process (Wu, 2013).

The data indicate that Wg/dWnt4 regulate the establishment of Fz–PCP axes by modulating the Fz–Vang intercellular interactions in a graded, dosage dependent manner. Consequently they might generate different levels of Fz–Vang interactions across a Wg/dWnt4 gradient experienced by cells. This process is reiterated across the tissue, and the directionality of Fz–Vang binding is subsequently reinforced by intracellular core PCP factor interactions. The data are consistent with a model in which Wg/dWnt4 generate a Fz ‘activity'), suggesting that both of these light sensors are necessary for light avoidance behavior.' gradient models. Accordingly, PCP axes are orientated towards the Wg/dWnt4 source, which is evident in (at least) the wing and eye. The early wing PCP axis (late larval to early pupal stages) correlates well with Wg/dWnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsoventral axis towards the poles where Wg/Wnt4 are expressed. This model, relying on a Fz–Vang interaction, is also compatible with the addition of Fmi to this scenario, with intercellular (homophilic) Fmi–Fmi interactions also being required for PCP specification. As Fmi forms complexes with both Fz and Vang, the full complement of intercellular interactions includes Fz/Fmi–Fmi/Vang complexes, and these interactions would also be modulated by Wnt binding to Fz, either directly as proposed in this model or possibly by modulating the Fmi–Fmi interactions by Fmi being associated with Fz that is bound to different levels of Wg/Wnt4. In vivo, Fmi helps to enrich both Fz and Vang to the subapical junctional region, and Fmi–Fmi interactions bring Fz and Vang to close molecular proximity (Wu, 2013).

Intercellular Fmi–Fmi interactions are strong, as Fmi-expressing S2 cells form cell aggregates through homophilic Fmi interactions. The interaction between Fz and Vang is weaker, and cell–cell contacts between the two cell groups are infrequent. It was suggested that PCP signal sensing complexes include both Fmi and Fz on one cell interacting with Fmi/Vang at the surface of a neighbouring cell. Within these complexes, Fz is required for sending a polarity signal, whereas Fmi and Vang are involved in its reception, consistent with the data and model. Although it has been suggested that Fmi is capable of sensing Fz/Fmi signals in the absence of Vang, the 'Fz-sensing' capability of cells with Fmi alone (lacking Vang) is much weaker than that of cells with Vang. It will be interesting to determine if there are other PCP regulators directly involved in modifying Fmi–Fmi interactions (Wu, 2013).

How do these data relate to previous models and why was the Wg/Wnt4 requirement not observed before? Previous work attempted to address the role for the wing margin in PCP by examining either mutants affecting wing margin cells without eliminating wg/Wnt expression or in clones. Although cellular hairs near the site of wing margin loss point towards remaining wing margin areas, the effect Is considered weak. Potential effects were examined of Wnt LOF clones of Df(2L)NL, lacking wnt4, wg, wnt6 and wnt10. In contrast to the global reduction of Wg/Wnt4 through the temperature sensitive wg allele, such clones cause only mild PCP perturbations. There are several reasons why clonal loss of Wnt expression in the margin only mildly affects PCP orientation: cells can respond to Wnts from several sources/cells from remaining Wnt-expressing wing margin regions; polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than those in cells further away (at 14-17 h APF) and weak PCP reorientation in cells neighbouring wing margin clones could thus reflect the initial weak polarization in these cells; and PCP orientation changes from its initial radial polarity towards the proximodistal polarity during hinge contraction morphogenesis and associated cell flow, probably leading to significant corrections of subtle defects near the margin. Similarly, PCP orientation in cells near the margin is only very weak early (at 14-16 h APF), probably because cells close to the Wnt-producing cells are exposed to saturated Wnt levels (and not a Wnt gradient), or because the presence of other organizers (directing polarity parallel to the margin) weakens the effect of Wnts. PCP in these cells is established/corrected through more local interactions during the feedback loops among neighbouring cells (Wu, 2013).

To determine the direct role for Wg/Wnts on Fz–PCP signalling, it was examined at pupal stages, as the patterning role for canonical Wg signalling is much reduced then and PCP still correlates well with Wg/Wnt4 expression. Importantly, Wnt4 does not affect expression of patterning genes through canonical signalling at larval or pupal stages, yet Wnt4 alters PCP orientation, consistent with the model that Wnt4/Wg act directly on Fz-PCP interactions. The observation that Wnt4 requires Fz to affect neighbouring cells further supports this model. It is likely that, as well as the Wg/Wnt4 input and mechanism identified in this study, both early and late PCP axes depend on further cues, provided for instance by the parallel Ft/Ds-PCP system or other morphogenetic organizers. Strikingly, such a scenario would suggest that Wg regulates PCP directionality through both PCP systems, affecting Fz-PCP interactions directly and through canonical Wg signalling transcriptionally regulating graded fj and ds expression in eyes and wings. In summary, these data provide insight into Wnt-mediated mechanisms to directly regulate long-range Fz–PCP orientation by modulating Fz–Vang/PCP interactions during tissue morphogenesis (Wu, 2013).

The Drosophila selectin Furrowed mediates intercellular planar cell polarity interactions via Frizzled stabilization

Establishment of planar cell polarity (PCP) in a tissue requires coordination of directional signals from cell to cell. It is thought that this is mediated by the core PCP factors, which include cell-adhesion molecules. This study demonstrates that furrowed, the Drosophila selectin, is required for PCP generation. Disruption of PCP in furrowed-deficient flies results from a primary defect in Fz levels and cell adhesion. Furrowed localizes at or near apical junctions, largely colocalizing with Frizzled and Flamingo (Fmi). It physically interacts with and stabilizes Frizzled, and it mediates intercellular Frizzled-Van Gogh (Vang)/Strabismus interactions, similarly to Fmi. Furrowed does so through a homophilic cell-adhesion role that is distinct from its known carbohydrate-binding function described during vertebrate blood-cell/endothelial cell interactions. Importantly, the carbohydrate function is dispensable for PCP establishment. In vivo studies suggest that Furrowed functions partially redundantly with Fmi, mediating intercellular Frizzled-Vang interactions between neighboring cells (Chin, 2013).

The data suggest that Fw serves as a homophilic cell-adhesion molecule that physically interacts with and stabilizes Fz at membranes, facilitating Fz-Vang/Stbm intercellular interactions. It was also concluded that Fw acts in a manner similar to that proposed for Fmi and thus that Fw and Fmi may act in parallel (in a partially redundant manner) to facilitate Fz-Vang interactions (Chin, 2013).

The function of Fw appears linked to that of Fz and Fmi, but the phenotypic strength of fw LOF is weaker than fz and fmi (except for the thorax). Mechanistic studies suggest that Fw is a homophilic cell-adhesion factor and physically associates with and stabilizes Fz, promoting Fz PCP function. Similarly, the cell-adhesion factor Fmi can also associate with Fz and stabilizes it at the membrane. In vivo data suggest that fw and fmi function in parallel, partially redundantly, mediating intercellular Fz-Vang interactions as intercellular 'bridges'. It is noteworthy that the double mutant phenotype of fw or fmi is not stronger than fz itself or in most cases stronger than the fmi null phenotype (except in the thorax where fw appears the more important of the two and the fmi null phenotype is not as strong as fz). In addition, Fw might affect Fz stability in a cell-adhesion-independent manner as FwΔCCP2, with no cell-adhesion capability, still stabilizes Fz when coexpressed. Thus, the data suggest that Fw performs two separate mechanistic functions in PCP: (1) Fz stabilization via association with it (this might be of different importance in distinct tissues, e.g., more important in wing discs [thorax, wing] than eye discs), and (2) cell adhesion at junctional complexes, where it stabilizes Fz to facilitate intercellular Fz- Vang interactions (a function similar to Fmi) (Chin, 2013).

Although the CCP2 domain is critical for cell adhesion, but not its Fz interaction, as Fz needs to be stabilized at cell junction complexes, the Fw effect on Fz in the absence of the CCP2 domain has no functional consequence. On both counts, Fmi is acting in a similar manner: it promotes Fz localization to subapical junctional membrane regions and overall affects Fz levels at the membrane. fw- has stronger LOF phenotypic defects in the thorax and wing, where overexpression of Fw shows no effects; in contrast, overexpression of Fw has strong effects in the eye, where LOF displays only a weak phenotype. It is likely that Fw levels are lower in the eye (hence the strong GOF PCP effect there) and Fmi largely serves the equivalent function(s) there (Chin, 2013).

In vivo and cell culture data suggest that Fw does not directly affect other core Fz-group PCP factors. The mild enhancement of Vang GOF defects is likely due to the effect of Fw on Fz, as Fz and Vang complexes antagonize each other intracellularly. Fw does not have an apparent effect on Vang levels. Thus, it appears that the phenotypic effects of Fw are mediated via its effects on Fz. Interestingly, PCP GOF effects of Fz in the eye are not only suppressed by the complete loss of fw but are 'misdirected' toward canonical Wg-signaling GOF defects, suggesting that Fw might contribute to Fz signaling specificity between the Wnt signaling branches (Chin, 2013).

Fw is the sole selectin in the Drosophila genome. In vertebrates, selectins function as cell-adhesion molecules via their carbohydrate binding C-type lectin domain, binding to glycolipids to mediate adhesion. This type of cell adhesion is prominent between leukocytes and endothelial cells, referred to as 'rolling' under flow in blood vessels. The CCP repeats are thought to serve a structural function in this context, not mediating adhesion. In the context of PCP signaling, the CCP2 domain mediates direct homophilic adhesion between Selectin/Fw in neighboring cells. This is an unexpected result and reveals a role of selectins in cell adhesion (Chin, 2013).

The adhesive behavior of Fw is however significantly weaker than bona fide structural adhesion factors required for epithelial integrity like DE-cadherin. S2 cell-based assays suggest that Fw provides about one-fifth the strength of DE-cad adhesion (determined by cell-adhesion cluster size). Accordingly, loss of function of fw does not affect epithelial integrity. In addition to this study on fw in PCP signaling, there are two additional defects associated with fw LOF alleles: (1) overgrowth in the retina (causing a 'furrowed' appearance of the eye) and (2) a mild thickening/shortening or loss of sensory bristles. Whereas the eye and bristle structure defects depend on both the CCP2 domain (cell adhesion) and the C-type lectin domain (sugar binding?), PCP establishment does not require the C-type lectin domain, suggesting that two Fw functions can be separated. As the mild overgrowth eye phenotype eye also depends on the CCP2 motif (and possibly on cell adhesion), fw could be considered a mild tissue-specific tumor suppressor. Drosophila has an open circulation system and no blood vessels, thus it is unlikely that a glycolipid binding function, as established for vertebrate selectins, is required in flies. Selectin knockouts in the mouse or zebrafish models will provide a useful approach to address whether any of the vertebrate selectins also function in PCP signaling (Chin, 2013).

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

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