dishevelled

DEVELOPMENTAL BIOLOGY

Embryonic

DSH protein appears in the cellular blastoderm. The cytoplasmic protein has a higher level of expression in the apical part of the cell. During gastrulation [Images], staining is uniform except for a higher density around the proctodeum. There are no variations in this uniform expression in the extended germ-band stage, even within the area of wingless expression. Dishevelled functions in larval imaginal discs as a component of the wingless pathway.

Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).

Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations – in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).

The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP – e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation – rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).

Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).

It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).

What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).

One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk¹ mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).

Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case – Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).

Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch mediating PCP and the canonical pathway playing no role in this (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier?). However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).

While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect – given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).

Wnt repulsive cue determines synaptic target specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. Single-cell microarray analysis was conducted of two neighboring muscles (M12 and M13) in Drosophila, that are innervated by distinct motor neurons, by directly isolating them from dissected embryos. A number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4, were identifed. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity (Inaki, 2007).

In each abdominal hemisegment of embryos and larvae of Drosophila, 37 motor neurons innervate 30 muscles in a highly stereotypic manner. Several candidate target recognition molecules that are expressed in different subsets of muscles have been identified, including Connectin, Fasciclin3, Semaphorin2, NetrinB, Toll, and Capricious. Genetic analysis of these molecules suggests that multiple cues expressed on the target muscles determine the target specificity in a combinatorial and overlapping manner. However, this issue has not been fully addressed; previous studies characterized only a small number of molecules that are expressed in different muscles (Inaki, 2007).

Toward more comprehensive understanding of the molecular basis of target specificity, genome-wide expression profiling was conducted of genes specifically expressed in two neighboring ventral muscles, M12 and M13, which are innervated by distinct motor neurons. M12 is innervated by RP5 and V (collectively called MN12s), whereas M13 is innervated by RP1 and RP4. Because these two muscles show similar morphology, run in parallel, and insert at adjacent muscle insertion sites, they are likely to share most functional characteristics other than neural connectivity. It was therefore reasoned that their subtractive expression profiling might lead to the identification of genes encoding target specificity (Inaki, 2007).

Individual M12s and M13s were collected from abdominal segments of dissected embryos during the stage of motor neuron targeting by aspiration with micropipettes. RNA was extracted from the samples of muscles, each containing 200 cells, and the RNA was amplified through two rounds of linear amplification. Affymetrix Drosophila genome chips were then hybridized with multiple samples of cRNA that was isolated and amplified in independent experiments. Comparing gene-expression profiles of M12 and M13, genes were selected that displayed differential expression consistently in two sets of hybridization experiments. This yielded a list of 96 genes predicted to be preferentially expressed in M12 and 77 genes predicted to be preferentially expressed in M13 (hereafter called M12 and M13 candidate genes) (Inaki, 2007).

A focus was placed on genes that encode putative membrane or secreted proteins with potential roles in target recognition. The predicted differential expression of this class of genes was verified by quantitative real-time RT-PCR analysis (qPCR). Twenty-five of the 34 genes examined gave concordant results with the array data, displaying at least 1.5-fold differential expression between the two muscles. RNA in situ hybridization further confirmed the preferential expression of knockout (ko) in M12, and of Wnt4, beat-IIIc, Sulf1, and CG6867 in M13. These results show specific expression of transcripts that encode a variety of candidate target-recognition molecules in these two muscles (Inaki, 2007).

The role of a prominent candidate gene, Wnt4, which encodes the secreted protein Wnt4 of the WNT family, was genetically analyzed. Wnt4 has been shown to function as an attractive guidance molecule that regulates dorsoventral specificity of photoreceptor-cell projection. Wnt4 is also known to regulate cell movement in the ovary. Wnt4 is expressed in ventral muscles M13 and M26. Much weaker expression is seen in other muscles, including M12. Therefore its function was studied in M12 and M13 targeting. In Wnt4 loss-of-function (LOF) mutant embryos, muscles and major motor nerves showed largely normal development. Specification of the Wnt4-expressing muscle, M13, also appears to be normal because the expression pattern of another M13-enriched gene, Toll, was indistinguishable from that of control. However, the innervation pattern of M12 and M13 was specifically altered. Staining with the anti-Fasciclin II monoclonal antibody 1D4, which visualizes all motor axons, revealed that the axon terminals on M12 were greatly reduced. Remarkably, this reduction of the synaptic endings on M12 was accompanied by the expansion of endings on M13. The number of Bruchpilot-positive putative active zones was also decreased in M12 and increased in M13 in Wnt4 mutants compared to control. These results are consistent with the idea that in Wnt4 mutants, MN12s formed smaller synaptic endings on M12 and instead arborized inappropriately on M13 (Inaki, 2007).

To determine whether the expansion of the M13 terminals in Wnt4 mutants is caused by the formation of ectopic arborization by MN12s, these neurons were specifically labeled with diI. DiI was applied on the M12 nerve endings and RP5 and/or V neurons, whose identities were verified by their axon trajectories and cell-body position, were retrogradely labeled. In late stage 16 wild-type embryos, RP5 neurons retained putative transient contacts on M13. In Wnt4 mutants, the length of the arborizations on M13 formed by RP5 neurons was significantly increased. Wild-type V neurons arborized exclusively on M12 and not along M13. In Wnt4 mutants, V neurons occasionally formed ectopic arborizations on M13, although the frequency is too low to be statistically significant. These results indicate that in Wnt4 mutants, MN12s formed larger or ectopic endings on M13, and further argue that Wnt4 is required to repel and/or restrict inappropriate arborization on M13 formed by MN12s (Inaki, 2007).

The LOF phenotypes suggest that Wnt4 functions in M13 to prevent synapse formation by MN12s. If so, ectopic Wnt4 expression in M12 may inhibit synapse formation by these neurons. To address this possibility, the Gal4-UAS system was used to induce forced expression of Wnt4 in muscles. Strong expression of Wnt4 was induced in all muscles by using the Gal4 driver 24B or E54. In this situation, MN12s often stalled at the edge of M12 and formed much smaller endings. Misexpression of Wnt4 did not cause targeting or pathfinding defects in other regions of the neuromusculature. Wnt4 therefore specifically inhibits synapse formation by MN12s. Next, expression of Wnt4 was induced only in M12 by using a more specific driver, 5053A-Gal4. Because 5053A-Gal4 induces a much higher level of Wnt4 expression in M12 than that of endogenous Wnt4 in M13, this experimental manipulation reverses the relative levels of Wnt4 expression in these muscles. In 5053A-Wnt4 embryos, the arborizations on M12 were greatly reduced in size, as was observed in 24B-Wnt4 embryos. In addition, unlike in 24B-Wnt4 embryos, the arborizations on M13 were enlarged. These results are consistent with the idea that the M12 motor neurons determine target specificity by detecting the relative levels of Wnt4 expressed by these two muscles. Taken together, LOF and gain-of-function (GOF) analyses indicate that differential expression of Wnt4 in M12 and M13 is critical for their targeting (Inaki, 2007).

Which receptor and signaling pathway in motor neurons mediate muscle-derived Wnt4 repulsion? Wnts bind to Frizzled (Fz) family receptors, and the receptor activation in turn activates the intracellular protein Dishevelled (Dsh). Wnt family proteins also bind to other classes of receptors, including Derailed/Ryk family members, which have been shown to transduce Wnt-mediated attraction or repulsion during axon guidance. Previous studies in the visual system and in the ovary have shown that Fz2 and Dsh are involved in Wnt4 signaling. Therefore whether Fz2 and Dsh are required for proper targeting in the neuromuscular system was investigated. Also the possible involvement of Derailed family members was also studied. When the function of Fz2 or Dsh was inhibited by expressing a dominant-negative form of these molecules, the same defects were observed in the targeting of M12 and M13 as observed in Wnt4 mutants. Similarly, LOF of derailed-2 (drl-2) causes the highly specific phenotype in the targeting of M12 and M13. These results suggest that Fz2, Dsh, and Drl-2 are involved in the signaling of Wnt4 repulsion in motor neurons. Whereas Fz2 is expressed in most or all neurons, drl-2 is expressed in subsets of neurons in the CNS. The specific expression of drl-2 may explain why Wnt4 is repulsive to only subsets of motor neurons (Inaki, 2007).

Systematic GOF analyses of the other candidate genes identified by the expression profiling was performed, and it was found that pan-muscle expression of five other genes, beat-IIIc, Glt, Lsp2, Sulf1, and CG6867, caused a reduction of MN12 nerve terminals similar to that seen when Wnt4 was misexpressed. All of these genes are normally expressed in M13 and thus, like Wnt4, may function as repulsive cues that inhibit synapse formation by MN12s. As in the case of Wnt4, misexpression of these five genes did not cause targeting or pathfinding defects in other regions of the neuromusculature. These results suggest a repulsive role for beat-IIIc, Glt, Lsp2, Sulf1, and CG6867 in specific aspects of target selection (Inaki, 2007).

Several molecules have previously been shown to function as attractive target cues that determine target specificity, including Netrins and Capricious in Drosophila, SYG-1 in C. elegans, and Sidekicks in vertebrates. However, little is known about the role of repulsion during target selection. During axon pathfinding, repulsive cues presented by surrounding tissues restrict the direction of the axons by deflecting or arresting their growth. Axons can also be guided by gradients of repulsive cues. Does repulsion also limit the choice of distinct target cells and thus mediate cell-to-cell specificity? Previous GOF analyses of semaphorin2 and Toll in Drosophila showed that they can inhibit synapse formation of specific motor neurons. However, whether such inhibition is essential for the selection of target cells is unknown. This study shows that Wnt4 is required for target recognition by MN12s. In wild-type, upon entering the M12/M13 target region, MN12s selectively innervate M12 with only a small putative transient contact on M13. In Wnt4 mutants, the target preference of these neurons is shifted to M13. This suggests that Wnt4 normally prevents MN12s from making large synapses on M13, and this Wnt4-mediated repulsion on M13 is required to lead these neurons to an alternative target, M12. Data from GOF analyses further support the notion that differential expression of Wnt4 in these two muscles is critical for target selection by MN12s. These results provide strong evidence that local repulsion plays a major role in target specificity (Inaki, 2007).

Microarray analysis identified a number of putative cell-surface or secreted proteins, in addition to Wnt4, that were differentially expressed between the two muscles. Furthermore, GOF analysis suggested that at least five of them may function, like Wnt4, as repulsive cues on M13. Some of them encode proteins with domains implicated in axon guidance and synapse formation; Beat-IIIc belongs to the Beat family of proteins with immunoglobulin motifs, and Glt belongs to a family of cell-surface proteins with cholinesterase domains. Identification of such a large number of potential cues in a single target cell is unprecedented and provides a valuable opportunity to study the mechanisms of target recognition. Future genetic analysis of these molecules, alone and in combination, may more clearly elucidate the mechanism of how these multiple target cues coordinate to determine target specificity (Inaki, 2007).

Larva and Pupa

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP). Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh). In this study, endogenous Dsh was visualized in the developing wing. A portion of Dsh colocalizes with Fmi, and the distributions of both proteins are interdependent. Furthermore, Fz controls the association of Dsh with cell boundaries: this association is correlated with the presence of hyperphosphorylated forms of Dsh. These results, together with studies on Fz distribution, support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that the restricted localization of this complex directs cytoskeletal reorganization only at the distal cell edge (Shimada, 2001).

Dishevelled was visualized in the developing wing by using specific antibodies. Some of the Dsh molecules are present at cell-to-cell boundaries in third-instar wing discs and in wings 2 hr after puparium formation (hr APF). By 18 hr APF, a larger fraction of Dsh molecules appear to be associated with cell boundaries, and then they are redistributed preferentially at proximal/distal (P/D), but not anterior/posterior (A/P), boundaries. This asymmetrical pattern is detectable at 24 hr APF and is most prominent at 30 hr APF, just prior to the onset of prehair outgrowth. This conversion produces a zigzag pattern on the epidermal plane: this pattern is highly reminiscent of the distribution of Flamingo (Fmi), a seven-pass transmembrane cadherin. In fact, Dsh and Fmi appeared to colocalize until 30 hr APF, and in terms of the time course, generation of the asymmetrical pattern of Dsh is indistinguishable from that of Fmi. Dsh, like Fmi, was present apically along the apicobasal axis at cell boundaries; curiously, Dsh distribution appears to be more restricted than that of Fmi. Besides Dsh molecules at cell boundaries, diffuse or punctate signals are also seen in the cytoplasm. Once prehairs emerge and initiate outgrowth at around 32–34 hr APF, the Fmi pattern starts to become depolarized and becomes almost nonpolar by 36 hr APF. In contrast, the asymmetrical Dsh distribution appears to persist, and patchy signals are found at distal cell vertexes and in outgrowing prehairs (Shimada, 2001).

Dsh functions in both the PCP signaling and the canonical Wnt (Wingless) pathway, and dsh¹ is a missense mutation that impairs the PCP signaling. The subcellular distribution of the Dsh¹ protein was monitored and it was found to remain almost entirely in the cytoplasm and is hardly detected at cell boundaries. These findings suggest that the association of Dsh with cell boundaries is a prerequisite for its role in PCP signaling. Because Dsh functions just downstream of Fz, it was expected that Fz might be necessary for the Dsh localization at cell boundaries. In fact, boundary association of Dsh is almost lost in larval wing discs or pupal wings of fz null mutants and inside fz clones. This observation is consistent with a ability of Fz, in a heterologous system, to recruit Dsh from cytoplasmic vesicles to cell boundaries (Shimada, 2001).

Fmi localization has been studied under various genetic conditions of fz that alter polarity. Dsh was examined under the same genetic conditions and it was found that Dsh, like Fmi, is redistributed to cell boundaries where there is an imbalance of Fz activity. One example was seen along borders of clones of cells homozygous for fz^R52, a strong fz allele. Another example was shown in an experiment of graded fz expression. A fz gradient expression along the anterior-posterior axis of the wing reorients hairs from high to low levels of fz expression. In these wings, ectopic Dsh and Fmi accumulation at A/P cell boundaries, instead of P/D ones, prefigures prehair outgrowth in the anterior or posterior direction. A tight coupling of Dsh and Fmi mislocalization with altered polarity is also seen in areas distal to fz^R52 clones. All of these results are consistent with the ideas that (1) an imbalance of Fz activity at boundaries is necessary and sufficient to localize Dsh and Fmi there, and (2) in the wild type, distributions of the two proteins at the P/D boundaries direct the cell to choose the distal edge for prehair development. Nevertheless, to rigorously demonstrate that the distributions of Dsh and Fmi play instructive roles in polarizing cells, one would need to design each molecule to mislocalize at A/P boundaries and then investigate how cells are reoriented (Shimada, 2001).

To investigate how colocalization of Dsh and Fmi at P/D boundaries is controlled, the subcellular distribution of one of the two was studied in the absence of the other. Fmi distribution is nonpolar in dsh¹, and the requirement of Dsh for making the Fmi pattern asymmetric was confirmed by staining dsh¹ or dsh^V26 clones for Fmi. Fmi is uniformly present at boundaries within these clones. Curiously, the intensity of Fmi signals seems to increase in the dsh mutant cells, and this increase implies the possibility that Dsh may be involved in the destabilization of Fmi. Inside the clone and at the clone border, Fmi remains at apical cell boundaries whether Dsh is colocalized or not. This is perhaps due to the intrinsic nature of Fmi as a transmembrane molecule. In contrast, the boundary localization of Dsh is dependent on Fmi, as demonstrated by the staining of fmi mutant clones for Dsh. Fmi is missing at cell boundaries inside the clones and at clone borders, and Dsh is hardly detected at those boundaries. At boundaries outside the clones, Dsh always coexists with Fmi. These results support a reciprocal dependence between Dsh and Fmi for subcellular localization until prehair formation and may imply a complex formation. Under the experimental immunoprecipitation conditions, however, evidence could not be provided for a physical association between Dsh and Fmi (Shimada, 2001).

Although it has been shown that Dsh is phosphorylated in response to Wingless in a cell culture system and in the embryo, posttranslational modification of Dsh has not been studied in the context of PCP signaling. Western blot analysis shows that a fraction of Dsh molecules in pupae is hyperphosphorylated and that those forms are hardly detectable in fz or fmi mutants. Thus, the absence of the hyperphosphorylated forms correlates with the loss of Dsh at cell boundaries and suggests that the hyperphosphorylation is either required for, or is a downstream readout of, the cell boundary localization. Besides reduction in the level of the hyperphosphorylated forms, the overall amount of Dsh also appears to decrease in fz or fmi mutants; an exception may be the most quickly migrating band, which most likely represents an unphosphorylated or poorly phosphorylated form (Shimada, 2001).

Where is Fz localized within the cell? It has been shown recently that the ubiquitous expression of Fz-GFP rescues a fz polarity defect and that Fz-GFP colocalizes with Fmi: these findings strongly suggest that endogenous Fz assembles with Dsh and Fmi at the P/D boundary. Furthermore, Fz-GFP distribution is regulated by Dsh and Fmi. Therefore, in the sense of subcellular localization, there seems to exist an interdependence between any two of Fz, Dsh, and Fmi. This triangular relationship can be summarized as follows: (1) in the absence of Fz or Fmi function, the intracellular protein Dsh cannot be attached to cell boundaries; (2) without Dsh function, boundary distributions of Fz and Fmi no longer become asymmetric along the P/D axis; and (3) Fz localization at cell boundaries is abolished by a loss of Fmi (Shimada, 2001).

Other data strongly suggest bilateral distribution of Fmi at the P/D boundary, and it is pointed out that this bilateral pattern per se does not explain how the distal cell vertex, not the proximal one, is selected for prehair formation. Importantly, Fz-GFP localization is unipolar; i.e., it is present only at the distal boundary. A distal concentration of Dsh molecules could be likely because Dsh positively relays Fz signaling, although the possibility of the bilateral localization still exists. It had been expected that one could answer this question by tracing Dsh signals in wild-type cells contacting proximal and distal borders of dsh mutant clones; what underlies this approach is the fact that dsh controls PCP in a cell-autonomous fashion. Given that Dsh is localized only at the distal cell edge and that dsh mutant cells do not affect Dsh localization in wild-type neighbors, Dsh signals could have been detected at interfaces between wild-type and mutant cells only along proximal borders of the clones. Unexpectedly, Dsh was not always localized at those cell boundaries along proximal borders; furthermore, 50% of wild-type cells in direct contact with proximal borders mispositioned Dsh at anterior/posterior boundaries. Therefore, dsh mutations appear to exert a one-cell nonautonomous effect on Dsh distribution, and this did not allow a conclusion about unipolar versus bilateral localization of Dsh. This one-cell nonautonomy could be due to misplaced Fz and Fmi molecules in dsh mutant cells, which might send an illegitimate message to wild-type neighbors. In any case, the local assembly of a tripartite signaling complex of Fz, Dsh, and Fmi in the cell most likely amplifies Fz signaling only at the distal cell vertex and induces cytoskeletal reorganization (Shimada, 2001).

It should be noted that in larval wing discs and early pupal wings, Dsh distribution is not asymmetric along the presumptive PD axis; nonetheless, it is associated with cell boundaries, and this association is dependent on Fz. Functional relevance of the cell boundary localization of Dsh has been suggested in vertebrate embryos, in which Dsh controls cell polarization in convergent extension movements. A fusion protein of a Xenopus Dsh homolog and GFP (Xdsh-GFP) is associated with boundaries in cells undergoing morphogenetic movement, but this protein remains in the cytoplasm of cells that are not undergoing such movement. It would be interesting to examine whether the boundary association of Xdsh-GFP or endogenous Xdsh, as well as distributions of Xenopus homologs of Fz and Fmi, are biased toward the direction of the cell movement (Shimada, 2001).

Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP (Axelrod, 2001).

To investigate a possible role for Dsh membrane association during Fz/PCP signaling in vivo, Dsh subcellular localization during PCP signaling was examined in the developing wing. Transgenes were produced that express a Dsh::green fluorescent protein (GFP) C-terminal fusion, driven by native dsh regulatory sequences. One or two copies of these transgenes rescue dsh^v26 null mutants to viability and produce wild-type PCP, indicating that they fully replace the function of endogenous Dsh in both Wg and PCP signaling (Axelrod, 2001).

Studies of temperature-sensitive alleles of both fz and another PCP gene, inturned, have suggested that PCP signaling is active after puparium formation (apf), and culminates just before the initiation of prehair morphogenesis (32-34 h apf). In wings, a dynamic pattern of subcellular localization of tagged Dsh protein is observed during this period. Dsh is observed predominantly in the cytoplasm of embryonic epidermis and third-instar wing discs. Some weak, perimembranous enrichment of Dsh is observed in apicolateral regions throughout third-instar wing development. At or shortly after the white prepupal stage, Dsh strongly associates with the membrane, accumulating in an apical circumferential ring, with an apparent simultaneous decrease in cytoplasmic levels. Through 18 h apf, the ring is approximately symmetric; however, by 24 h apf, and most pronounced by 30 h apf, Dsh is seen to accumulate preferentially at proximal-distal boundaries, and is depleted at anterior-posterior boundaries. Viewed en face, this produces a pattern of parallel zigzags similar to that seen for Fmi and Fz. By 32-34 h apf, Dsh is often seen in discrete patches that appear to be at the distal surface of each cell, corresponding to the site of nascent actin-rich prehair emergence. In a wild-type wing, clones of cells lacking the tagged transgene reveal that Dsh does indeed accumulate solely at the distal edge. Dsh subcellular localization therefore evolves into a pattern showing unipolar asymmetry within the plane of the epithelium, prefiguring the distal position of prehair assembly. The unipolar distribution of the PCP effector protein Dsh reflects the proximal-distal polarity vector, and strongly suggests that its distal localization is required to determine PCP (Axelrod, 2001).

To demonstrate the importance of Dsh membrane localization for Fz/PCP signaling, the dsh¹ allele was enlisted. Dsh¹ is specifically compromised in its ability to transduce the PCP, but not the Wg signal. The lesion in dsh¹ maps to its DEP domain, which is required for its membrane localization in the frog animal cap assay. A dsh¹::GFP fusion, otherwise identical to the wild-type construct, rescues dsh null mutant flies to viability, and the flies exhibit the dsh¹ mutant phenotype. In the absence of wild-type Dsh (in either a dsh¹ or a dsh^v26 null mutant background), apical membrane association of Dsh¹::GFP is severely reduced, and the Dsh¹ protein instead remains almost entirely in the cytoplasm throughout pupal development. Therefore, introducing a lesion into Dsh blocks its localization to the membrane in vivo and its ability to signal. Similarly, in the presence of wild-type Dsh, Dsh¹::GFP remains in the cytoplasm. Because wild-type Dsh does not induce the relocalization of Dsh¹::GFP, it is unlikely that Dsh acts as a multimer during PCP signaling (Axelrod, 2001).

The pattern of Dsh localization observed in pupal wings is reminiscent of that for Fmi, a seven-pass transmembrane cadherin required for PCP signaling. By 30 h apf, both are seen at the proximal-distal boundaries, though Dsh is strictly distal, whereas Fmi was proposed to be at both proximal and distal edges. Double labeling for Fmi and Dsh reveals significant colocalization in 30-h apf pupal wings, each demonstrating a zigzag pattern. However, at later times, the Dsh asymmetry persists, whereas the Fmi asymmetry decays. Transverse sections taken in wing-edge cells indicate that both are located at the most apical region of cell-cell contact, and that low levels of Dsh are also seen throughout the cytoplasm. Fmi localization depends on both Fz and Dsh, but not on Multiple wing hairs (Mwh), suggesting that Fmi functions downstream of Dsh. To study this relationship further, Dsh localization was examined in fmi mutant wings. At 30 h apf, little Dsh is associated with the membrane in fmi mutant wings. This reveals a reciprocal dependence between Dsh and Fmi for persistent membrane association, and suggests that Fmi does not simply function downstream of Dsh (Axelrod, 2001).

Fz and Fmi colocalize at proximal-distal boundaries at 30 h apf. Furthermore, the asymmetric pattern of Fz localization depends on Fmi, whereas the asymmetric pattern of Fmi localization depends on Fz. Taken together, these data are consistent with the possibility that Fz, Dsh, and Fmi function together, perhaps in a complex, during PCP signaling, with both Fz and Dsh localizing to the distal edge, and Fmi apparently localizing to both the proximal and distal edges of the cell. A mutual dependence for asymmetric localization exists between these three proteins (Axelrod, 2001).

It was asked whether Dsh localization depends on upstream signaling through the Fz/PCP pathway by examining Dsh localization in a fz mutant background. In a fz^R52 null mutant, Dsh fails to accumulate at the membrane at 30 h apf. At 2 h apf, only the weak, perimembranous, methanol-sensitive enrichment of Dsh, reminiscent of that seen in wild-type third-instar discs, remains. Absence of membrane-associated Dsh from around 2 h apf through 30 h apf indicates that both the earlier, symmetric phase of Dsh-membrane association, as well as the late, asymmetric phase, are Fz dependent. Dsh-membrane association depends not simply on the presence of Fz protein, but also on its ability to signal. Disrupting the ability to localize Dsh to the membrane, either by mutating Dsh (dsh¹) or by blocking Fz function, produces a mutant PCP phenotype. Dsh-membrane association is therefore necessary to transduce the polarity signal (Axelrod, 2001).

To determine whether Fz signaling is sufficient to produce the asymmetric localization of Dsh, its localization was examined in a Fz expression gradient that alters the polarity pattern on the wing. Consistent with previous demonstrations, graded expression of ectopic Fz in the dpp or dll expression domains reorients hairs from high to low levels of Fz expression. In these wings, asymmetric Dsh localization realigns according to the Fz gradient. Therefore, both the membrane localization of Dsh and its asymmetry are dependent on signaling through Fz. In contrast, Dsh localization is normal in a mwh mutant, consistent with previous arguments placing Dsh upstream of Mwh in the polarity signaling pathway (Axelrod, 2001).

The mechanism of the transition from a nearly symmetric to an asymmetric pattern of Dsh localization was examined. Several maneuvers to interfere with Dsh function were performed, and the effect on Dsh localization assayed. How expression of two dominant negative, truncated Dsh constructs might modify the localization of full-length (tagged) Dsh was tested. Expression of a form containing the DEP domain, but lacking the PDZ domain Dsh(DeltabPDZ) produces a polarity defect, and in pupal wings, causes a failure of Dsh::GFP to localize to the membrane. Similar results were obtained by expressing Dsh(DEP+), a form containing only the DEP domain. It is likely that the DEP domain in the truncated proteins competes with the DEP domain in Dsh::GFP (and presumably with endogenous Dsh) for membrane docking, preventing its localization. Consistent with the loss of membrane localization and the polarity defect seen in dsh¹ and fz mutants, this result indicates that membrane association is necessary for PCP signaling (Axelrod, 2001).

In contrast, expression of Dsh(DeltaDEP+), a (weaker) dominant negative construct lacking the DEP domain, also blocks polarity signaling, but in this case, Dsh::GFP (and presumably endogenous Dsh) retains its ability to localize to the membrane. In these wings, the pattern of membrane-localized Dsh no longer displays the vertically oriented zigzags seen in the wild type. This result indicates that membrane association of Dsh, although necessary, is not sufficient for PCP signaling, and that the orientation of asymmetry is important for PCP signaling (Axelrod, 2001).

Finally, advantage was taken of the observation that the Naked cuticle (Nkd) protein is known to bind the Dsh PDZ domain. Nkd is thought not to play a role in PCP signaling. However, Nkd overexpression produces a PCP phenotype by binding Dsh and interfering with its ability to function in the PCP pathway. Dsh localizes to the membrane throughout wings overexpressing Nkd in the posterior compartment. However, the transition from symmetric to asymmetric Dsh localization is abolished posteriorly. Whereas in the anterior, wild-type portion of the wing Dsh accumulation is enriched along the proximal-distal boundaries and diminishes at the anterior-posterior boundaries, in the posterior region where Nkd is overexpressed, Dsh accumulation is essentially symmetric around the cell periphery. Therefore, by binding Dsh, Nkd interferes with Dsh function, resulting in a loss of Dsh subcellular asymmetry, and producing an adult PCP phenotype. Furthermore, because specifically interfering with the ability of Dsh to signal blocks the acquisition of asymmetry, Dsh asymmetry does not simply result from passively colocalizing with asymmetrically distributed Fz. Rather, one can infer that Dsh function is required for generation of asymmetry, indicating that a feedback loop contributes to asymmetry (Axelrod, 2001).

Dsh may translocate to the membrane from an existing pool, or may be stabilized at the membrane, increasing the total cellular Dsh content. Furthermore, Dsh is a phosphoprotein, and its phosphorylation state is potentially regulated during PCP signaling. Western blot analysis was therefore used to examine Dsh protein levels and phosphorylation state in pupal discs during PCP signaling. No significant difference in total Dsh levels was observed in wild type, fz^R52, or dsh¹ wings, indicating that membrane association represents a shift in Dsh localization from the cytoplasmic to the membrane compartment. However, more than half of the Dsh protein in wild type is in a hyperphosphorylated form, whereas very little of this form exists in fz^R52 or dsh¹ mutants. The PCP signal therefore results in phosphorylation of Dsh, and phosphorylation correlates with membrane localization, suggesting it is either required for, or is a response to, localization. This result is consistent with studies in Xenopus showing that XDsh phosphorylation and membrane association correlate with activity in convergent extension, a process homologous to PCP signaling, but not axis duplication, a ß-catenin mediated process (Axelrod, 2001).

Although both Fz and DFz2 transduce the Wg signal, only Fz can serve as a receptor for PCP signaling. Analysis of chimeras points to structural differences distal to the ligand binding domains as responsible for this difference. However, the question of how Fz specifically transduces two distinct signals, both of which require Dsh function, still remains. During late third instar, Wg signals through both Fz and DFz2 to establish the proneural clusters that give rise to bristles near the D/V boundary of the wing. However, no accumulation of Dsh is observed at membranes near the D/V boundary of third-instar wing discs. Furthermore, Dsh is not observed at membranes in embryos, nor in wing discs throughout third instar. During early pupal stages, when Dsh shows the earliest Fz-dependent membrane localization, no difference is observed between cells close to Wg expressing cells and those at greater distances. Recruitment of Dsh to the membrane is therefore a specific response to the Fz/PCP signal, and does not result from the Wg signaling activity of either Fz or DFz2 (Axelrod, 2001).

The atypical cadherin Flamingo links Frizzled and Notch signaling in planar polarity establishment in the Drosophila eye

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. The atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. These results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing (Das, 2002).

To investigate the role of flamingo in eye development, fmi mutant clones were induced with the eye specific ey-FLP/FRT system. Analysis of fmi^- tissue in adult eyes shows typical PCP defects with randomized chirality, resulting in loss of mirror image symmetry. Reminiscent of fz, dsh, and stbm null alleles, fmi^- clones display defects in ommatidial chirality establishment (random chirality and symmetrical clusters) and rotation. In addition, fmi^- clones contain ~20% ommatidia lacking photoreceptors (Das, 2002).

The genetic requirement of fmi in both R3 and R4 is unique, since other PCP genes are required only in either cell (fz and dsh in R3 and stbm and N in R4), and raised the question of how Fmi relates to these genes in function and expression. Thus, the expression patterns of other PCP proteins were examined in the eye (Das, 2002).

Although Dsh is cytoplasmic, it colocalizes with Fmi at the equatorial membranes of R3 and R4. Subsequently, Dsh is found apically at membranes in R4, in a U-shaped pattern, again colocalizing with Fmi. Also, Fz, which colocalizes with Dsh in the wing, shows a similar asymmetric equatorial-polar expression pattern like that in Fmi and Dsh early in R3/R4. The later upregulation in R4 is, however, not apparent for Fz (Das, 2002).

The membrane association of Dsh is Fmi dependent because, in fmi^- cells, Dsh fails to be membrane enriched. When Fmi is overexpressed in R3/R4 (using the sev enhancer), more Dsh is localized at the membrane within these and other sev-expressing cells, indicating that excess Fmi leads to greater Dsh membrane recruitment. These data suggest that Fmi is both necessary and sufficient to induce Dsh membrane recruitment (Das, 2002).

Several pieces of evidence argue for a positive requirement of Fmi in R3. Fmi is asymmetrically distributed in response to Fz/Dsh signaling, and it is in turn required to maintain Dsh membrane localization in R3 early. Both Fz and Dsh are required in R3, and Dsh needs to be associated with the membrane for its function in R3. Since this is disturbed in fmi^- cells, Fz signaling might not function normally there in the absence of Fmi. This interdependence of Fz, Dsh, and Fmi is also supported by observations in the wing, where each component requires the presence of the other for normal localization. Thus, it is speculated that, initially, during the activation of Fz/PCP signaling, Fmi is required positively for Fz/Dsh function, prior to its inhibitory role on Fz/Dsh signaling in R4 (Das, 2002).

How could this be achieved? (1) There are the distinct requirements for dgo in R3 and stbm in R4; (2) the differences in Fmi levels in early R3/R4 versus late R4 could account for its individual functions. High levels of Fmi in R4 could lead to a formation of a different complex than that formed in R3. For example, an Fz/Dsh/Fmi/Dgo complex would promote Fz signaling, whereas, in R4, since there is significantly more Fmi, a different Fmi complex would inhibit Fz signaling by possibly sequestering Dsh from the Fz complex (Das, 2002).

Asymmetric localization of Frizzled and the determination of Notch-dependent cell fate in the Drosophila eye

During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).

Frizzled tagged with green fluorescent protein (Fz-GFP) exhibits a dynamic subcellular distribution from early stages of ommatidial differentiation. Ommatidia are born behind the furrow in rows polarized in the anteroposterior axis. In row 4, Fz-GFP is enriched on the apical membranes of the newly recruited R3/R4 pair but excluded from the region where they contact R2/R5. No Fz-GFP enrichment is apparent around R2/R5, but it does accumulate on the posterior side of R8. By row 6, Fz-GFP is no longer enriched in R3, except at the boundary with R4 and sometimes at the boundary with the anterior cone cell. Conversely, R4 still has strong accumulation around its perimeter, except where it contacts R5. This accumulation around R4 persists through row 8, but accumulation fades elsewhere. Thus, Fz-GFP is initially in a symmetric pattern in R3/R4 but rapidly resolves into an asymmetric pattern that is visible by the time ommatidial rotation occurs in row 6. Using antibodies against Dsh and Fmi, these proteins were found to colocalize with Fz and show the same dynamic distribution (Strutt, 2002).

N is also at highest levels in apical membranes of cells posterior to the furrow and in rows 4 through 6 it overlaps with Fz-GFP at the R3/R4 boundary (but shows no asymmetry). The localization of Fz-GFP (and Fmi/Dsh) to the R3/R4 boundary is therefore consistent with Fz/Dsh being able to directly modulate N activity in this location. However, if Fz/Dsh are differentially regulating N activity, a crucial requirement is that these complexes should be preferentially localized on one side of the R3/R4 boundary. Since this cannot be distinguished by light microscopy, genetic mosaics were created in which both R3/R4 had sufficient fz activity for normal signaling and fate determination, but only one of the pair carried the Fz-GFP transgene. Using this approach, it was found that Fz-GFP is more highly enriched on the R3 side of the R3/R4 boundary in row 4 and more posteriorly is found exclusively on the R3 side of the boundary. Thus, about two rows prior to ommatidial rotation, Fz-GFP is asymmetrically distributed across the R3/R4 boundary. Since studies in the wing demonstrate that Dsh adopts the identical asymmetric localization to Fz (and indeed their asymmetric localization is mutually dependent), it is inferred that Dsh is also differentially localized on the R3 side of the R3/R4 boundary (Strutt, 2002).

The polarity gene stbm is required for R4 fate: whether Stbm protein also shows an asymmetrical localization in R3/R4 was investigated using a Stbm-YFP transgene. Stbm-YFP is apically localized in cells posterior to the furrow, and, subsequently, its distribution is similar but distinct from that exhibited by Fz-GFP. In row 4, a symmetric pattern is observed, with Stbm-YFP around R3/R4, except where these cells contact R2/R5, and enriched on the posterior face of R8. This symmetric pattern is maintained until the ommatidia are already rotated in row 6 and more posteriorly. Staining then fades around R3, except where R3 contacts R4. Mosaic analysis revealed that, in contrast to Fz-GFP, Stbm-YFP is enriched on the R4 side of the R3/R4 boundary from row 4 onward, i.e., Stbm is on the opposite side of the boundary with Fz (Strutt, 2002).

Therefore Fz, Dsh, Fmi, and Stbm localize to the apical region of the R3/R4 cell boundary, where they become asymmetrically distributed prior to or concomitant with R3/R4 fate determination. Normally, Fz/Dsh are enriched on one particular side of the cell boundary, in the presumptive R3 cell. However, in mosaic ommatidia where one or other cell is mutant for polarity genes, the assembly of the asymmetrical complexes can be reversed. In all conditions examined, the polarity of Notch signaling between R3/R4 is consistent with the polarity of the asymmetric complexes, with Notch activity being lowest in the cell where Fz/Dsh accumulate. Finally, evidence is provided that the domain of N, which is known to interact directly with Dsh, is required for efficient R3/R4 fate decisions (Strutt, 2002).

Considering these results together, it is proposed that an extracellular polarity signal leads to the asymmetric assembly of a complex of planar polarity proteins at the boundary between the R3/R4 cell pair. This asymmetric complex then leads to asymmetric N activity between the cell pair and thus determines cell fate. Since no evidence is found that this regulation occurs via the proposed signaling cascade downstream of Fz/Dsh (i.e., Rho GTPases/JNK) and since manipulation of Dl transcription does not perturb polarity of Notch signaling, it is concluded that there must be an alternative pathway by which asymmetrical Fz/Dsh affects Notch activity (Strutt, 2002).

One favored mechanism for the modulation of N/Dl activity is via local interactions between N and asymmetrically localized proteins and, in particular, between the intracellular domain of N and Dsh. Four lines of evidence support the proposal that the regulation occurs at the cell cortex: (1) Fz/Dsh are in the same subcellular domain as N at the apical R3/R4 boundary during the critical stages of development when the cell fate decision is made; (2) the appearance of the asymmetric Fz/Dsh complexes is shortly prior to or concomitant with the appearance of a bias in N/Dl activity and ommatidial rotation; (3) direct interactions between N and Dsh have been previously demonstrated and proposed to be important for patterning in other tissues, and these interactions have been found to be repressive, consistent with Fz/Dsh being required in R3, where N activity is lowest and (4) deletion of the domain of N required for interactions with Dsh leads to less-efficient R3/R4 fate decisions (Strutt, 2002).

The model whereby asymmetric Fz/Dsh localization leads to downregulation of N activity on the R3 side of the R3/R4 boundary is further supported by studies in the Drosophila leg, where loss of planar polarity gene activity leads to ectopic activity of Notch. However, there are still unexplained observations: if the only role of the polarity genes is to inhibit N in R3, mutations in fmi, fz, or dsh (which result in no apical Dsh localization) should have high N activity in both R3/R4, not the reduced activity that is detected (Strutt, 2002).

This discrepancy might be explained if there are two phases to polarity gene regulation of N activity. One would be an activation/derepression of N, which would require symmetric protein localization of Fz/Dsh in R3/R4. The second would be linked to asymmetric protein localization, when Fz/Dsh would in turn become repressors of N activity in R3 (Strutt, 2002).

The asymmetric localization of Fz, Dsh, and Fmi in the eye develops in a similar manner to that seen in the pupal wing. Thus, the R3/R4 cell boundary appears analogous to the proximodistal wing cell boundaries, with the R3 side of the boundary, where Fz and Dsh are localized, being equivalent to the wing cell distal edge. Another of the polarity gene products, Stbm, is localized on the R4 side of the boundary, which is consistent with the requirement for stbm function in R4. By analogy to the wing, it is likely that Fmi is present on both sides of the R3/R4 boundary and Pk-Sple/Sple is on the R4 side (Strutt, 2002).

The adoption of the asymmetric pattern occurs in two phases. The first involves symmetric apicolateral localization of Fz, Dsh, Fmi, and Stbm in R3/R4 (and in all other cells except R2/R5); this is evident in ommatidial row 4. As in the wing, the initial apical recruitment of Fz is dependent on Fmi, and the recruitment of Dsh is in turn dependent on Fz. Subsequently, the distribution evolves rapidly into an asymmetric pattern. Adoption of asymmetry requires the function of dsh, stbm, and the LIM domain protein Prickle-Spiny-legs (Pk-Sple), and if any of these are missing, Fz distribution remains symmetric in ommatidial rows 5/6, and ommatidial rotation is delayed. It is likely that the asymmetry evolves through the same mechanisms as in the wing, where it has been proposed that an extrinsic signal leads to a small bias in Fz/Dsh signaling on either side of the cell boundary, which subsequently becomes amplified by feedback loops that lead to Fz/Dsh becoming concentrated on one side of the interface and Pk-Sple/Stbm on the other (Strutt, 2002).

One notable difference between the eye and the wing is that asymmetric Fz/Dsh distribution is eventually observed in stbm and pk-sple eye discs, but in both cases it occurs with a random bias and is delayed by about one to two ommatidial rows. This correlates well with the fact that the adult phenotypes of stbm and pk-sple exhibit a low incidence of achiral ommatidia. Conversely, in fmi, fz and dsh, negligible asymmetric protein localization occurs, and there is a relatively high proportion of 'achiral' ommatidia in the adult eye, suggesting that achirality is a result of poor asymmetric complex formation. In general, the aquisition of asymmetry also correlates with mDelta0.5 activity, particularly in pk-sple and sple mutations where its expression usually resolves into a single cell by row 10 (Strutt, 2002).

In the pupal wing, asymmetric localization of Fz/Dsh/Pk-Sple is proposed to involve a signaling feedback loop that amplifies an initially small bias in Fz/Dsh activity across the axis of each cell. In the eye, the N/Dl feedback loop was proposed to perform a similar function, amplifying an initially small difference in Fz/Dsh activity between R3/R4. With the observation that Fz/Dsh are also distributed in asymmetric complexes in the eye, it appears that both mechanisms are operating in R3/R4, although it is not clear why both would be required, since either alone should be sufficient to amplify small biases in signaling activity (Strutt, 2002).

One possible explanation is that use of both mechanisms increases the speed and robustness of the R3/R4 fate decision. A fast fate decision may be necessary because of the dynamic nature of eye patterning, in which the R3/R4 decision is only part of a complex series of events involving cell recruitment and movement to generate the final polarized ommatidium. It is also possible that a rapid decision is required because the extrinsic polarity cue is transient in nature. It is noted that the rapidity of the decision would be further enhanced if N/Dl signaling also influenced Fz/Dsh localization. While there is no direct evidence for this, it could explain the eventual, randomly polarized, asymmetric protein localization seen in stbm and pk-sple backgrounds in the eye. In this case, the inability of Fz/Dsh to efficiently localize asymmetrically in the absence of Stbm/Pk-Sple might lead to N/Dl making a stochastic decision that then leads to Fz/Dsh asymmetry. Conversely, in the pupal wing, where N/Dl are not active in planar polarity decisions, Stbm/Pk-sple activity would be absolutely required, since their absence would not be compensated for by the N/Dl feedback loop (Strutt, 2002).

A number of lines of evidence have previously suggested that Rho/Rac GTPases and the JNK cascade are required for ommatidial polarity decisions and, in particular, the R3/R4 fate decision. These include the following: overexpression of Fz or Dsh in the eye gives a polarity phenotype that is dominantly suppressed by RhoA, bsk, hep, and Djun; RhoA clones or expression of dominant-active/negative RhoA or Rac1 gives ommatidial polarity phenotypes; overexpression of dominant-active/negative JNK pathway components and human Jun elicits ommatidial polarity defects, and expression of a Dl enhancer trap is altered by overexpression of either fz or dsh or by activated human Jun, Hep, RhoA, or Rac1. These observations led to the hypothesis that higher levels of Fz/Dsh signaling in R3 result in higher activation of Dl transcription in R3 via a Rho GTPase/JNK cascade, biasing the N/Dl feedback loop to produce high N in R4 (Strutt, 2002).

Taken together, the phenotypic evidence from loss-of-function studies does not support a primary role for Rho GTPases/JNK cascades in the R3/R4 fate decision. But the weight of genetic evidence does support a secondary role for some of the proposed pathway components, possibly in the augmentation of polarity decisions driven largely by asymmetric localization of polarity proteins and direct repression of N activity. In addition, the observation that RhoA mutations result largely in defects in ommatidial rotation supports the hypothesis that RhoA acts downstream of the planar polarity genes in regulating this aspect of ommatidial polarity (Strutt, 2002).

The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila

Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. This study established a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. DAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, it was demonstrated that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of DAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that DAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, it is proposed that the Wnt5/PCP navigation system is linked by DAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way (Gombos, 2015).

This study has shown that DAAM plays an important role in the regulation of axonal growth and guidance of the Drosophila MB neurons. Several lines of evidence suggest that DAAM acts in concert with Wnt5 and the core PCP proteins to ensure correct targeting of the KC axons. DAAM functions downstream of Dsh and Rac1, and its ability to promote actin assembly is absolutely required for neural development in the MB. These data suggest a simple model in which axon guidance cues, such as Wnt5, signal through the PCP pathway to activate DAAM to control actin filament formation in the neuronal growth cone. Thus, PCP signaling appears to be linked to cytoskeleton regulation in a direct way, and these results provide compelling experimental evidence suggesting that, at least in neuronal cells, the major cellular target of PCP signaling is the actin cytoskeleton (Gombos, 2015).

Formins are highly potent actin assembly factors that are under tight regulation in vivo. The major mechanism of controlling the activity of the Diaphanous-related formin (DRF) subfamily involves an intramolecular autoinhibitory interaction between the N-terminal diaphanous inhibitory domain (DID) and the C-terminal Diaphanous autoinhibitory domain (DAD). This inhibition can be relieved upon binding of an activated Rho family GTPase that interacts with the GBD (GTP-ase binding domain)/DID region and also by proteins that bind to the DAD domain. Consistently, this study found that the Rac1 GTPase and the DAD domain binding Dsh protein both play role in DAAM activation in MB neurons. With this regard, it is notable that, despite that dsh¹ is considered a PCP-null allele, the DAAM^Ex1, dsh¹ double hemizygous mutants exhibit a stronger MB phenotype than dsh¹ mutants alone, suggesting that DAAM must receive Dsh-independent regulatory inputs for which Rac1 is a prime candidate. Although previous work indicated that Rho GTPases might function downstream of Dsh in a linear pathway), the data suggest that Dsh and Rac1 act in parallel pathways in the MB. As the impairment of GTPase binding severely, but not completely, abolishes DAAM activity, it is concluded that Rac1 is likely to have a stronger contribution to DAAM activation in vivo; nonetheless, the simultaneous binding of Dsh appears to be required for full activation (Gombos, 2015).

Presumably, the most remarkable feature of the PCP system relies in its ability to create subcellular asymmetries. Therefore, it is a tempting idea that, upon guidance signaling, the PCP proteins are involved in the generation of molecular asymmetries within axonal growth cones, yet recent attempts failed to reveal such polarized distributions in MB neurons. Interestingly, however, it was shown that Fz and Vang display a differential requirement during development of the MBs, with Fz predominantly acting in the dorsal lobes and Vang predominantly acting in the medial lobes (MLs). This study found that, in contrast to Fz and Vang, DAAM plays a crucial role in both lobes of the MBs. Additionally, it was demonstrated that Fz promotes the formation of membrane-associated Dsh-DAAM complexes in S2 cells. This result, together with genetic data, suggests that DAAM acts as the downstream effector of a Fz/Dsh module, which is required for the correct growth and guidance of the dorsal MB axon branches (Gombos, 2015).

In addition to their potential connection to Fz signaling in the dorsal lobe, DAAM and Dsh were linked to Vang- and Wnt5-dependent ML development as well. Wnt5 and Vang have an identical effect on ML development when overexpressed, and this GOF phenotype can be suppressed by the same set of mutations (DAAM, dsh, Rac1). In particular, the putative PCP-null dsh¹ allele and heterozygosity for Rac1 cause an almost equally strong, yet partial, suppression with regard to the ML fusion phenotype. This is best explained by assuming that Wnt5 and Vang signal both in a Dsh-dependent and in a Dsh-independent, but Rac-dependent, manner. With regard to DAAM, this study has shown that DAAM nearly completely suppresses the GOF of Wnt5 and Vang, and Dsh and Rac1 both contribute to DAAM activation. Collectively, these data suggest a model in which Wnt5 and Vang promote β lobe extension by signaling to Dsh and Rac1 that will activate DAAM in parallel to each other. The colocalization of Vang and DAAM, observed in S2 cells, indicates that they may bind each other directly, which would be in good accordance with genetic data suggesting a close functional link between DAAM and Vang during β lobe development. However, formins are not known to bind Vang proteins; therefore, an indirect interaction, mediated by Rac1, which has recently been shown to be bound and redistributed by Vangl2 in epithelial cell lines, appears a more likely possibility (Gombos, 2015).

As discussed above, and contrary to Vang, Fz does not appear to be required for ML development, or if anything, it might play an opposite role, as loss of fz leads to ML fusion in 16.1% of the lobes. This is a surprising observation at first glance as Wnt proteins are thought to activate members of the Fz receptor family, but former analysis of Wnt5 signaling during MB development also failed to reveal a Fz requirement in the β lobes. Instead, Wnt5 has been linked to other type of Wnt receptors, the Ryk/Derailed atypical tyrosine kinase receptors, which are known to be involved in axonal guidance in flies and vertebrates. In light of these results, it will be of future interest to analyze the Wnt5-Vang connection in the MB in more details and identify the Wnt5 receptor in this context (Gombos, 2015).

Consistent with the lack of lobe-specific requirement for dsh and DAAM, the current studies revealed that Dsh, DAAM, and Rac1 are used as common effector elements of a dorsal lobe-specific Fz-dependent signal and a Vang-dependent ML-specific signal. It follows that Dsh and DAAM are likely to take part in two types of PCP complexes. Although, in vitro, Dsh has the ability to interact with both Fz and Vang, the conclusion that Dsh functions downstream of Vang in the β lobes is markedly different from the classical PCP regulatory context in which the Fz/Dsh and Vang/Pk complexes have opposing effects. Thus, this result, together with the Wnt5-Vang data, substantiates the earlier findings that the PCP system operates at least partly differently in neurons than during tissue polarity signaling (Gombos, 2015).

During PCP signaling, the vertebrate DAAM orthologs control convergence and extension movements, polarized cell movements during vertebrate gastrulatio. In contrast, DAAM is dispensable for classical planar polarity establishment in flies, suggesting that the tissue polarity function of DAAM might be restricted only to vertebrates. Despite the lack of direct function in establishing tissue polarization, this study provides evidence that DAAM is linked to the PCP pathway in another important regulatory context, notably directed neuronal development in the adult brain. Consistent with the results, recent studies revealed that PCP signaling and DAAM regulate neural development in planarians and in Xenopus embryos. Given that the vertebrate PCP proteins are known to be involved in multiple aspects of CNS development, and the vertebrate DAAM orthologs are strongly expressed in the CNS, it is conceivable that the PCP/DAAM module represents a highly conserved regulatory system that is used to regulate various aspects of neuronal development throughout evolution (Gombos, 2015).

Effects of Mutation or Deletion

The finding that Wingless (WG) and Decapentaplegic (DPP) suppress each others transcription provides a mechanism for creating developmental territories in fields of cells. What is the mechanism for that antagonism? The dishevelled and shaggy genes encode intracellular proteins generally thought of as downstream of WG signaling. The effects of changing either DSH or SGG activity were investigated on both cell fate and wg and dpp expression. At the level of cell fate in discs, DSH antagonizes SGG activity. At the level of gene expression, SGG positively regulates dpp expression and negatively regulates wg expression while DSH activity suppresses dpp expression and promotes wg expression. Sharp borders of gene expression correlating precisely with clone boundaries suggest that the effects of DSH and SGG on transcription of wg and dpp are not mediated by secreted factors but rather act through intracellular effectors. The interactions described here suggest a model for the antagonism between WG and DPP that is mediated via SGG. The model incorporates autoactivation and lateral inhibition, which are properties required for the production of stable patterns. In the Dorsal part of the leg disc, DPP signalling predominates; DPP together with SGG inhibit wg expression and the consequencent lack of inhibition of SGG promotes further dpp expression. In the ventral part of the disc, WG signaling predominates and WG acts through DSH to inhibit SGG activity thus removing the activator of dpp (SGG) and promotes its own expression by removing the combinatorial inhibition of SGG and DPP. The regulatory interactions described exhibit extensive ability to organize new pattern in response to manipulation or injury (Heslip, 1997).

In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway (van den Heuvel, 1993). dsh and porc act upstream of shaggy/zw3, and arm acts downstream of shaggy (Siegfried, 1994).

A wingless transgene, controlled by a heat-shock promoter, was used for genetic epistasis experiments. Wingless acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed, and cuticle differentiation (Noordermeer, 1994).

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

The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response of Hh/Wg/Dpp target genes such as optomotor-blind and dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This repression is mediated by the activity of Dll and dac. One prerequisite for appendage development is the inactivation of the exd/hth genes (Azpiazu, 2000 and references therein).

htx is originally expressed uniformly in the wing imaginal disc but, during development, its activity is restricted to the cells that form the thorax and the hinge, where the wing blade attaches to the thorax, and it is eliminated in the wing pouch, which forms the wing blade. Repression of hth in the wing pouch is a prerequisite for wing development; forcing hth expression prevents growth of the wing blade. Both the Dpp and the Wg pathways are involved in hth repression. Cells unable to process the Dpp signal (lacking thick veins or Mothers against Dpp activity) or the Wg signal (lacking dishevelled function) express hth in the wing pouch. vestigial has been identified as a Wg and Dpp response factor that is involved in hth control. In contrast to its repressing role in the wing pouch, wg upregulates hth expression in the hinge; teashirt is a positive regulator of hth in the hinge. tsh plays a role specifying hinge structures, possibly in co-operation with hth (Azpiazu, 2000).

In the second instar wing disc, the Hth product accumulates uniformly in the thoracic and appendage regions of the disc, but throughout the third larval period hth expression is downregulated and, by the late third instar, Hth only appears in the presumptive regions of the thorax and the wing hinge. The central part of the disc, which gives rise to the wing pouch, shows no hth expression. The repression of hth function is important for wing development, because if hth activity is forced in the wing pouch, the wing does not form. A similar observation has been made in the leg disc; hth or exd expression in the distal part results in a truncated appendage in which all the distal components are missing. In the leg, the subdivision between distal and proximal regions results from the antagonism between Hh signaling and exd/hth function. Hh response genes such as Dll and dac are instrumental in repressing hth (Azpiazu, 2000 and references therein).

The downregulation of hth in the wing pouch is a consequence of the activity of the Dpp and the Wg signaling pathways. In cells in which the response to the Dpp signal is prevented, as in tkv or Mad mutant cells, hth is expressed at high levels. Similarly, dsh minus cells, in which the transduction of Wg is blocked, show ectopic hth activity and consequently nuclear exd expression. These results also indicate that hth is latently active in the wing cells and has to be repressed by the continuous activity of the Dpp and Wg signals. The inability of cell clones to proliferate, cells in which the Dpp or the Wg pathways have been totally eliminated, may be due to high levels of hth expression. The Dpp and Wg pathways repress hth expression independently. This is illustrated by the experiments inducing dsh mutant clones: ectopic hth expression is only observed in clones located away from the AP border. This suggests that the high levels of Dpp expression near the AP border are sufficient to impede hth expression despite the removal of the control by Wg (Azpiazu, 2000).

The mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. The arrow gene function is essential in cells receiving Wingless input. arrow acts upstream of Dishevelled and encodes a single-pass transmembrane protein; this indicates that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, a new and conserved function is suggested for the LRP subfamily in Wingless/Wnt signal reception. The position of arrow in intracellular Wingless siganl transduction cascade was examined. Smooth cuticle is restored to arr^null embryos in alternate segments when Dsh is expressed using Prd-GAL4, indicating rescue of Wg signal transduction. This contrasts with the overexpression of Wg, which has no effect in arr^null embryos. These data suggest that Arrow acts downstream of Wg but upstream of Dsh, because signaling, once activated by Dsh, no longer requires Arrow. It remains possible that Arrow might normally act as a scaffold and concentrate Dsh to an appropriate subcellular location; in this model, flooding the cell with Dsh simply bypasses the requirement for Arrow. In either case, Arrow is unlikely to act in a pathway parallel to Wg signal transduction since (1) the arrow mutant phenotype is rescued by Dsh, a canonical Wg signal transducer, and (2) loss of arrow function blocks signaling even when excess Wg is presented. In addition, the fact that the arrow phenotype is not suppressed by excess Wg distinguishes arrow from the genes involved in proteoglycan-assisted presentation of the Wg ligand. Each of the mutants affecting this step, sugarless, sulfateless and dally, is substantially suppressed by providing excess Wg ligand, showing that glycosaminoglycans are not essential for reception of the signal but only increase the efficiency of ligand presentation to the receptor. Together, these data argue that Arrow is absolutely essential for Wg to signal, and are consistent with a role for Arrow in reception rather than presentation of signal (Wehrli, 2000).

Cross-talk between Notch and Wingless pathways in bristle development

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called N^Mcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, N^Mcd1 and N^Mcd5 (collectively N^Mcd1/5), were chosen for further analysis. In clones for N^Mcd1 and N^Mcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the N^Mcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of N^Mcd is enhanced when N⁺ is lowered and is partially suppressed when N⁺ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. N^Ax alleles exhibit a similar genetic behavior and a similar phenotype to the N^Mcd alleles. However, several differences distinguish N^Ax from N^Mcd. The N^Ax mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, N^Mcd affects only microchaetae. Furthermore, the remaining microchaetae of the N^Mcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, N^Ax/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the N^Mcd/+ flies appear as those of wild-type flies. In this study of the N^Mcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

Since clones of N^Mcd cells lack microchaetae, the development of their precursors was examined during pupal stages by means of neural-specific markers. The loss of microchaetae observed in N^Mcd1/5 is due to the loss of neural cells, as visualized by stainings using the neural-specific antibody 22C10, and to the loss of their precursors, as detected with the reporter neu^A101. Since the proneural Ac activity is known to promote the development of the microchaetae precursors, Ac expression was examined in the N^Mcd mutants. The loss of microchaetae precursors is associated with a severe decrease in Ac expression (Raiman, 2001).

The N^Mcd phenotype is unlikely to be due to a lack of differentiation of the outer elements of the sensory organs, since 'escaped' microchaetae have a normal morphology. Thus, these results indicate that the N^Mcdmutations disrupt the early establishment of neural precursors rather than the late lineage that permits the differentiation of the sensory bristle (Raiman, 2001).

Different lines of work have suggested that the existence of Notch-signaling events are independent of the mechanism of lateral inhibition. Some of these experiments suggest that the adaptor protein Deltex (Dx) might be involved in some of these events (Raiman, 2001).

Dx is a cytoplasmic protein that regulates Notch through binding to the ankyrin repeats. Loss-of-function alleles of dx display an excess of microchaetae, whereas overexpression of Dx inhibits neurogenesis. It has been suggested that Dx is involved in a signal transduction event downstream of Notch. Loss-of-function dx alleles behave as dominant suppressors of all the N^Mcd alleles , and N^Mcd1/5 dx^-clones display a fairly normal microchaetae pattern. The Dx effector, therefore, might represent an essential regulator of the antineural activity revealed by the N^Mcd receptors (Raiman, 2001).

In contrast, Shaggy, the Drosophila glycogen synthase kinase 3 (GSK3) is a central element in Wingless signal transduction and behaves genetically as a downstream element of the Notch pathway. Mutations in Sgg suppress the effects of N^Mcd mutants, like mutations in Dx. Altogether, these results indicate that both Dx and Sgg might be involved in the Notch-signaling event that is distinct from lateral inhibition (Raiman, 2001).

Since Achaete/Scute expression is required for the establishment of the neural fate, the novel Notch pathway revealed by the N^Mcd mutants must be repressed during wild-type neural development. One candidate to exert this repression is Dishevelled (Dsh), a component of the Wingless-signaling cascade, which has been shown to bind Notch and block some of its activities. Using a yeast two-hybrid assay, it has been found that Dsh does bind to the C-terminal 114 amino acids of the NICD that are absent in the truncated receptors. Therefore, the Dx-dependent repressive effect of the N^Mcd receptors appears as the consequence of the loss of the Dsh binding site (Raiman, 2001).

Therefore, Notch associates in vitro with Dsh through its C-terminal 114 amino acids. In order to test the functional significance of this C-terminal domain of Notch in vivo, the effect of overexpressed Dsh on the development of microchaetae was examined either in wild-type or in N^Mcd8 flies lacking the Dsh binding site. Flies carrying four copies of a hsp70-Dsh transgene were analyzed. One 15-min heat pulse (37°C) at the onset of pupariation leads to an increase of 5.8% of the number of microchaetae in a wild-type background. In contrast, the pulse has no effect on N^Mcd8 flies. These experiments suggest that Dsh binds the 114 amino acid C terminus of Notch in vivo to antagonize the Dx-dependent signaling of the receptor. The effects of overexpressed Dsh were examined in Notch mutant-carrying lesions in the extracellular EGF repeats (nd³; spl;Ax^9B2; Ax^E2). In each case, an increase in the number of microchaetae was observed after heat treatment (Raiman, 2001).

Dsh and Dx display antagonistic activities. Overexpressed Dx inhibits neurogenesis, whereas overexpressed Dsh increases the number of microchaetae in wild-type flies. Furthermore, this latter excess of microchaetae is accentuated when the dosage of Dx is lowered (Raiman, 2001).

Potentially, Dsh could exert its repressing effect by modulating the proteasome-dependent proteolysis of Notch or the phosphorylation state versus cytoplasmic/nuclear distribution of the NICD. Interestingly, Dsh contains two proline-rich sequences, PPLP and PPXY, putative binding sites for Su(dx), a cytoplasmic ubiquitin ligase involved in ubiquitinylation/turnover of proteins. When binding to Notch, Dsh could serve as a docking protein for Su(Dx) and could regulate the activity of Dx in targeting the proteasome activity to the C terminus of Notch (Raiman, 2001).

How the Dx-dependent transduction is achieved in the cells is poorly understood. One could speculate that the repressing activity of Dsh may also rely on a direct effect on the Dx-dependent signaling. Thus, Dsh and Dx antagonistically regulate a common target, JNK (JUN N-terminal kinase), and Sgg antagonizes JNK-dependent activation of the JUN transcription factor. dJUN might therefore represent an element mediating the antineural activity of Dx (Raiman, 2001).

The Dx-dependent antineural activity of Notch is regulated by elements of the Wingless-signaling cascade, e.g., the cytoplasmic protein Dsh or the kinase Sgg. Overexpression of Dsh generates extrasensory organs in wild-type flies and fails to elicite ectopic bristles in the N^Mcdmutants lacking the Dsh binding site. The kinase Sgg is negatively regulated by Dsh in the Wingless-signaling cascade. Dsh and Sgg have opposite effects on the Dx-dependent Notch pathway. Loss-of-function alleles of sgg lead to a constitutive derepression of Wingless signaling and elicit the same number of ectopic bristles in wild-type and N^Mcd mutant flies (Raiman, 2001).

This analysis of the N^Mcd mutants supports the idea that Dsh, an effector of the Wingless pathway, directly interacts with Notch in wild-type flies in order to maintain the neural potential. Dsh antagonizes the cytoplasmic activity of Dx and then represses the antineural Dx-dependent function of Notch. In wild-type flies, crosstalks between Wingless and Notch allow stimulation of the ac/sc expression in the equivalent cells of the proneural clusters until a given threshold. It has been reported that Su(H) functions as the core of a molecular switch, acting as a repressor of Notch target genes in the absence of nuclear NICD. Thus, prior to the onset of lateral signaling, the repressive activity of Su(H) is compatible with the activation of ac/sc by the Wingless-dependent pathway. When a given level is reached, ac/sc can activate the Dl gene, and cells can compete with each other for the choice of the neural precursor via lateral signaling. At this stage, the Wg and the Su(H)-dependent Notch signalings have opposite effects on the expression of ac/sc. ac/sc is repressed in the inhibited cells, suggesting that the Su(H)-dependent Notch signaling overrides the Wingless pathway (Raiman, 2001).

Though the N^Mcd5 allele shares the same loss-of-microchaetae phenotype as other N^Mcd and affects the same developmental pathway, the N^Mcd5 mutant receptor carries a single point mutation, leading to the C739Y substitution that disrupts the 18^th EGF repeat of the extracellular domain, whereas the other N^Mcdalleles encode truncated receptors lacking the C terminus of the intracellular domain. Experiments with N^Mcd5 suggest that the region of the 18^th EGF is instrumental for the regulation of alternative Notch signaling. The extracellular EGF domain is known to physically bind Wingless. Further experiments are necessary to determine whether the N^Mcd5 lesion in the 18^th EGF repeat specifically alters the binding of Wingless, Fringe, or other unknown effector(s) (Raiman, 2001).

The present study of N^Mcd alleles demonstrates that a Deltex-mediated function of Notch represses the proneural activity during establishment of the neural precursors of the thoracic microchaetae. This repressive activity precedes and is distinct from that which mediates lateral inhibition and is constitutively active in N^Mcd mutants. The N^Mcd alleles encode truncated receptors that lack the binding domain of the repressor Dishevelled, which is involved in functional interactions between Notch and Wingless signalings. The results suggest a model in which Dishevelled is used to alleviate this initial repressive function of Notch in wild-type development, thereby permitting lateral inhibition to generate the regularly spaced sensory microchaetae. In the absence of ligands or effectors, the repressive function of the Dx-dependent activity of Notch could therefore maintain the cells in an uncommited state. In the presence of effectors like Dsh (Wingless signaling) that repress this antineural activity, cells become competent for further choice between two alternative fates (lateral inhibition). It is proposed that Notch acts during development either as a repressor preventing cell differentiation or as a receptor involved in the choice of cell fate during lateral signaling. This dual function is likely to be regulated in a ligand-dependent manner by crosstalk between the Notch and Wingless pathways. It will be important to find out the different components of this new Dx-dependent repressive cascade of Notch (Raiman, 2001).

Two prominent characteristics of epithelial cells, apical-basal polarity and a highly ordered cytoskeleton, depend on the existence of precisely localized protein complexes associated with the apical plasma membrane and on a separate machinery that regulates the spatial order of actin assembly. ERM (ezrin, radixin, moesin) proteins have been proposed to link transmembrane proteins to the actin cytoskeleton in the apical domain, suggesting a structural role in epithelial cells, and they have been implicated in signalling pathways. The sole Drosophila ERM protein Moesin functions to promote cortical actin assembly and apical-basal polarity. As a result, cells lacking Moesin lose epithelial characteristics and adopt invasive migratory behaviour. These data demonstrate that Moesin facilitates epithelial morphology not by providing an essential structural function, but rather by antagonizing activity of the small GTPase Rho. Thus, Moesin functions in maintaining epithelial integrity by regulating cell-signalling events that affect actin organization and polarity. Furthermore, these results show that there is negative feedback between ERM activation and activity of the Rho pathway (Speck, 2003).

To test the relationship of Moesin with the Rho pathway, whether a reduction in Moe function would suppress a phenotype associated with downregulation of Rho1 was investigated. Rho1 has been shown to function downstream of dishevelled (dsh) in the planar cell polarity (PCP) pathway that regulates the polarity and number of hairs generated by each wing blade cell. Each wild-type wing cell produces a single hair; however, multiple wing hairs result when Rho pathway function is impaired. In flies that are mutant for the dsh1 allele (which inactivates the PCP but not the wingless-signalling function of dishevelled), double-hair-producing cells occurred at a frequency of 6.3%. Removal of a single dose of Moe suppresses the number of cells with double hairs in dsh¹ mutants to 1.2%, suggesting that Rho pathway function is upregulated in response to reduction in Moesin activity (Speck, 2003).

Dishevelled and tissue polarity

see Dishevelled Effects of Mutation part 2/2

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