The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg) in tissue culture cells. While previous results indicate that loss of function for fz results in tissue polarity defects, the loss-of-function effects of Dfz2 are not known. The requirements of fz and Dfz2 during neurogenesis have now been examined. Both Fz and DFz2 function in Wg signaling, and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg. While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is significantly enhanced in embryos lacking both. Since the penetrance of the CNS phenotypes is not complete in double mutants, additional components that allow some degree of Wg signaling must exist in vivo (Bhat, 1998).

In the ventral nerve cord of the Drosophila embryo, wg is expressed in row 5 cells within a segment. It is nonautonomously required for the formation and specification of row 4 neuroblasts as well as for the formation of a few neuroblasts in row 5 and most neuroblasts in row 6. Among those neuroblasts that are affected in wg mutants, NB4-2, a row 4 neuroblast that gives rise to the RP2/sib lineage, has been one of the most studied neuroblasts in the CNS. The RP2 and its sibling cell are formed from the first GMC of NB4-2 (this GMC is known as GMC-1 or GMC4-2a). In this lineage, wg is required for both the formation and specification of this neuroblast. The elimination of maternal and zygotic fz gene products causes loss of NB4-2->GMC-1->RP2/sib lineage and failure in the formation of row 6 neuroblasts in the ventral nerve cord of the Drosophila embryo (Bhat, 1998).

It has been argued that Fz might not function in the Wg signaling during wing and eye development, and that in these tissues, while DFz2 functions to receive the Wg signal, Fz receives the signal from some other Wnt. If DFz2 is solely responsible for receiving the Wg signal in the CNS, elimination of Dfz2 should have eliminated the NB4-2 lineage in a manner similar to loss of function for wg. Given that the wg-like CNS defects in Dfz2 embryos are only partially penetrant, as is the case with fz mutants, the simplest explanation is that there is a genetic redundancy between fz and Dfz2 and both function in the transduction of the Wg signal. The observation that the penetrance of the RP2/sib lineage phenotype is significantly enhanced in embryos lacking both fz and Dfz2 activities certainly reinforces this view. These results are also consistent with the observation that during epidermal patterning, while the intracellular localization of Arm is not significantly affected in embryos missing Dfz2, it is nearly lost in embryos missing both the activities (Bhat, 1998 and references).

Drosophila Ric-8 regulates Gαi cortical localization to promote Gαi-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of G&alpha:i depends on Partner of Inscuteable (Pins). This study establishes that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Gαi, regulates cortical localization of the subunits G&alpha: and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells. This demonstrates, first, that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and, second, that a pI-specific activity tilts the spindle in the absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI-specific activity for two reasons. First, Fz signalling is still active in the pins and Gαi mutants as the spindle was correctly oriented along the antero-posterior axis in these mutants. Second, Fz accumulates at the posterior apical cortex of pI cells and this accumulation of Fz is maintained in Gαi pI cells. It is therefore envisaged that, although orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. fz,pins double mutants were analyzed to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium. Therefore, in the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells, indicating that Fz may also tilt the spindle in wild-type cells along their apico-basal axis. To test this, spindle orientation was analyzed in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells. Together, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric-8a/Pins/Gαi-dependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arises from this balance. Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex. The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (David, 2005).

In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR-Gαi complex, which is the homologue of the Pins-Gαi complex. These data demonstrate that, in the dividing pI cell, Ric-8a is required for asymmetric localization of Pins, Baz and Numb and for mitotic-spindle positioning. It is proposed that these activities of Ric-8a depend on an unexpected function of Ric-8a: localizing Gαi and G<β13F at the plasma membrane. This study of ric-8a also revealed that, in the pI cell, ric-8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype that was observed in ric-8a pI cells could be accounted for by some persistence of the Ric-8a protein in somatic clones. Alternatively, an intriguing possibility is that ric-8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling. ric-8a loss of function would thereby affect both the Fz- and Gαi-dependent activities exerted on the spindle, resulting in a milder apico-basal tilt (David, 2005).

Importantly, developmental processes ranging from gastrulation, neural-tube closure, neurogenesis and retina formation to asymmetric segregation of cell-fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral, etc) and parallel to the plane of the epithelium. This study shows that, in dividing pI cells, these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the antero-posterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz- and Gαi-dependent activities are likely to act through forces pulling on astral microtubules. Fz and heterotrimeric G signalling are implicated in mitotic-spindle positioning during both symmetric and asymmetric cell division. The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to understanding of the mechanisms that control mitotic-spindle positioning (David, 2005).

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. 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).

Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains

The core planar polarity proteins localize asymmetrically to the adherens junctions of epithelial cells, where they have been hypothesized to assemble into intercellular complexes. This study shows that the core proteins are preferentially distributed to discrete membrane subdomains ('puncta'), where they form asymmetric contacts between neighboring cells. Using an antibody internalization assay and fluorescence recovery after photobleaching in prepupal and pupal wings, the turnover of two key core proteins, Flamingo and Frizzled, was investigated, and it was found that the localization of both within puncta is highly stable. Furthermore, the transmembrane core proteins, Flamingo, Frizzled, and Strabismus, are necessary for stable localization of core proteins to junctions, whereas the cytoplasmic core proteins are required for their concentration into puncta. Thus, this study defines the distinct roles of specific core proteins in the formation of asymmetric contacts between cells, which is a key event in the generation of coordinated cellular asymmetry (Strutt, 2011).

Since the first report over a decade ago of the asymmetric subcellular localization of Fmi in the Drosophila pupal wing, the mechanisms underlying the distribution of the core polarity proteins have been extensively investigated. A growing number of models have been presented to describe how the core proteins might achieve asymmetric localization, with a common feature being the general assumption that the core proteins assemble together into a stable asymmetric intercellular complex. However, the existence of such a complex is largely inferred from the distributions of the proteins, and the actual roles of individual proteins in the formation, stabilization, and subcellular distribution of such complexes are poorly understood (Strutt, 2011).

This study shows that a fundamental organizing principle for core protein asymmetry is their distribution into discrete plasma membrane subdomains in the apicolateral junctions, which are referred to as 'puncta.' Using the independent methodologies of antibody internalization and FRAP, this study demonstrates that the populations of Fmi and Fz in puncta are highly persistent, supporting the view that the core polarity proteins do indeed form stable asymmetric complexes, and that these complexes are preferentially clustered together in puncta (Strutt, 2011).

The data lead to make several inferences about the formation of such asymmetric complexes. It was previously observed that in the absence of Stbm, an asymmetric Fz-Fmi:Fmi complex was preferentially formed between neighboring cells. The results suggest that this Fz-Fmi:Fmi complex is the primary building block for the core protein complex. In the absence of Fmi, Fz does not localize to junctions, and in the absence of Fz, Fmi is also poorly localized to junctions and subject to endocytic turnover. Importantly, loss of other core proteins (Stbm, Pk, Dsh) has less or no effect on Fmi endocytosis, and similarly does not eliminate the stable fraction of Fz, indicating that Fz and Fmi stably localize to junctions in the absence of these factors. Nevertheless, although Stbm does not preferentially form an asymmetric complex with Fmi in the absence of Fz, its ability to further stabilize Fmi at junctions in the presence of Fz indicates an important secondary role in formation of the asymmetric complex (Strutt, 2011).

Although the cytoplasmic core proteins do not appear to play any role in the formation of stable complexes, they do promote the 'clustering' of such complexes into puncta. This is consistent with previous data suggesting that the cytoplasmic factors are not required for intercellular communication but that they have an intracellular function in generating asymmetry. This absence of a requirement for the cytoplasmic factors in polarized intercellular communication, and the ability of Fz and Fmi to form asymmetric complexes in the absence of Stbm both suggest that protein complexes are already asymmetric in the absence of clustering (Strutt, 2011).

Several lines of data suggest that puncta are functionally important for generation of cellular asymmetry. First, they are the major sites of asymmetric localization of the core proteins. Second, their size, and the degree to which they contain a stable faction of Fz, varies over time and correlates with the degree of cellular asymmetry observed. Third, core polarity gene mutations that affect cellular asymmetry to different extents have a corresponding effect on the size of the stable fraction of Fz in puncta (Strutt, 2011).

The mechanism by which asymmetric complexes are clustered into puncta is unknown. The simplest model is that cytoplasmic factors act as 'glue' to hold complexes of the same orientation together and reduce their rates of lateral diffusion in the membrane. The alternative hypothesis that the cytoplasmic factors promote clustering by reducing rates of endocytic turnover is inconsistent with the observation that the overall stable fraction is not altered in the absence of cytoplasmic core protein function. The preference for clustering complexes of the same polarity may also be promoted by inhibitory interactions between proximal and distal complex components (Strutt, 2011).

A key question is how such clustering might lead to the establishment of cellular asymmetry. One possibility is a process of self-organization involving local self-enhancement and longer-range inhibition. If planar polarity represents such a self-organizing process, clustering of asymmetric complexes into puncta is likely to provide local enhancement, whereas formation of intrinsically asymmetric complexes between cells may effectively provide longer-range subcellular inhibition that prevents all the clusters within a cell having the same orientation. In support of such self-organization in the pupal wing, it is noted that induction of Fz, Fmi, or Stbm expression as late as 24 hr APF can lead to locally organized cellular polarity within a few hours that is not oriented on the PD axis and as such is unlikely to be specified by long-range patterning cues (Strutt, 2011).

Overall, a model is proposed in which molecular asymmetry is initially established by formation of Fz-Fmi:Fmi complexes that are intrinsically stable and in which Fmi endocytosis is attenuated. Entry of Stbm into the complex further promotes Fmi localization to junctions. The cytoplasmic components Dsh, Pk, and Dgo can then be recruited into the complex but do not increase its stability. Instead, they are required for clustering of asymmetric complexes of common polarity into junctional puncta, which are sites of local asymmetry. Through a self-organization process, which would normally be globally biased by an upstream patterning cue, locally organized puncta adopt an asymmetric distribution within the cell, linking the polarity of neighboring cells (Strutt, 2011).

Time-lapse experiments indicate that individual puncta are stable for several hours. Nevertheless, in the Drosophila wing, morphogenetic changes such as wing eversion, hinge contraction, and junctional remodeling necessitate some rearrangement of junctions, and this appears to be accompanied by reduced puncta size and loss of cellular asymmetry. Interestingly, although during junctional remodeling (at 20 hr APF), brighter regions are still visible in the junctions, FRAP experiments reveal that these regions are no longer enriched for the stable fraction of Fz. This suggests that the membrane subdomains in which puncta form may be persistent, but the mechanisms that promote accumulation of asymmetric complexes in puncta are not active. This may allow the remodeling of planar polarity, following morphogenetic changes (Strutt, 2011).

Transient asymmetric localization of polarity proteins is also seen in more dynamic systems, for example in vertebrate gastrulation, where their distribution is also highly punctate. It is possible that in cells that are undergoing movement and changing their contacts, local organization of polarity proteins into puncta allows more rapid reestablishment of polarized interactions between neighboring cells (Strutt, 2011).

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of 'upstream' planar cell polarity proteins

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins, inturned, fuzzy and fritz, accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, this study has found that when over-expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. It was further shown that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. It was also found that over-expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group (Wang, 2014).

Larva and Pupa

Immunostaining of pupal wings shows that the FZ protein is evenly distributed throughout the wing (Park, 1994). This suggests that FZ protein is not a graded morphogen. FZ proteins localize to the apical region of pupal cells (Park, 1994).

Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the 'equator'), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, it is shown that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium (Tomlinson, 1999).

The focus for the chirality choice maps to the presumptive R3/R4 photoreceptor pair; it maps specifically to the R3 cell. Although these two cells lie adjacent in the ommatidial precluster, fate-mapping experiments, together with histological analysis, indicate that they are initially distant from one another, separated along the equatorial axis by at least the remaining three cells that will enter the precluster. During normal development, the member of the pair that is closest to the equator invariably chooses to develop as an R3 cell, while the remaining member develops as an R4 cell. This asymmetric cell fate choice determines the chirality of the ommatidium. The R3/R4 cell fate decision is governed by the relative difference in activity of Frizzled (Fz) protein in the two cells comprising the presumptive R3/R4 pair. The cell with higher Fz activity becomes R3, while the remaining cell becomes R4. Since fz encodes a serpentine receptor-like protein, Fz is a candidate receptor for a factor X, consistent with the notion that the presumptive R3/R4 pair decodes the factor X gradient into a relative difference in Fz activity between the two cells. The difference in Fz activity between the two members of the presumptive R3/R4 pair biases a process of lateral specification between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl). In essence, the cell with higher Fz activity appears to be better at sending the Dl signal while the cell with lower Fz activity is better at receiving the signal. This difference is probably then amplified by a feedback mechanism in which receptor activation blocks ligand production in the same cell, while the loss of receptor activity leads to enhanced ligand production in the other cell. As a consequence, the N transduction pathway is fully induced in one cell but silenced in the other. The resulting disparity in N signal transduction is both necessary and sufficient to specify the reciprocal R3 and R4 cell fates and to determine the chirality of the ommatidia (Tomlinson, 1999).

Two models are presented for how Fz activity within the R3/R4 pair might bias the N-Dl interaction. In the first model, a scalar model, factor X activity positively regulates Fz activity, whereas in the second it negatively regulates Fz activity. A convention of factor X activity has been adopted; factor X is considered as being high at the equator and low at the poles. However, the results can be equally well explained if factor X has the opposite distribution, high at the poles and low towards the equator. In this scenario the positive and negative influences of factor X on Fz activity in the two models will be reversed. A difference in the levels of Fz activity between the two cells of the R3/R4 pair determines which cell will become R3 and which will become R4. Because the presumptive R3 cell lies closer to the equator than the presumptive R4, it will detect higher levels of factor X activity and consequentially will have a higher level of Fz activity. The higher levels of Fz activation then bias the subsequent N-Dl interaction so that the cell with greater Fz activity becomes a dedicated Dl signaling cell, while its partner, with less Fz activity, becomes a dedicated Dl receiving cell. For example, the level of Fz activity in each cell could govern the activity or level of expression of a component of the N-Dl signaling mechanism, such as N itself or Dl. Even a small difference in signaling capacity between the two cells would then bias the N-Dl system of feed-back regulation causing the cell with initially higher N transducing capacity to become a dedicated Dl receiving cell (and hence R4), while the remaining cell becomes a dedicated Dl sending cell (R3). Experimentally induced changes in Fz activity, which reverse the relative difference in Fz activity between the presumptive R3 and R4 cells, might cause a corresponding reversal in direction of N-Dl signaling and the R3/R4 cell fate decision. An issue raised by this model is whether the presumptive R3 and R4 cells have the capacity to meter accurately what are likely to be small differences in their absolute levels of factor X. Consider that up to 15 ommatidia can form along the equatorial-polar axis of each half of the eye, with the presumptive R3 and R4 cells located next to each other in each cluster and separated by several cell diameters from their counterparts in neighboring clusters. More than 75 cells are arrayed in each half of the eye along the Eq/Pl axis and any two neighboring cells at any position within that array would need to faithfully decode the factor X gradient. However, the results of fate-mapping analysis and histological studies suggest that the presumptive R3 and R4 cells are initially located at a distance from one another, separated along the Eq/Pl axis by the remaining three cells of the precluster (the presumptive R2, R8 and R5 cells). This raises the possibility that the presumptive R3 and R4 may meter factor X abundance when they are located at opposite ends of these cell lines before they come to lie next to each other. The physical separation between the two cells at this time would allow them to sample a broader segment of the factor X gradient, much as the separation between the two tips of the forked tongue of a snake facilitates the detection of odor gradients (Tomlinson, 1999).

In a second model, the vectoral model, each cell in the retina, and hence both the presumptive R3 and R4 cells, detects the gradient of factor X activity across its diameter and decodes it into a steeper gradient of Fz activity within the cell. Specifically, it is proposed that the factor X activity might negatively regulate Fz such that Fz activity is highest within each cell on its polar side and lowest on its equatorial side. Within the precluster, the presumptive R3 cell presents its polar face to the equatorial face of the presumptive R4 cell. The differential Fz activities across the two faces then bias the subsequent N-Dl interaction between the two cells. In this case, it would be argued that the bias is likely to be mediated through the direct and local modulation of a component of the N-Dl signaling apparatus, e.g. a post-translational modification of N or Dl activity along the surface of one of the two cells, where they abut. When Fz is overexpressed in the presumptive R4 cell, Fz activity is enhanced throughout the cell, including at the surface comprising the equatorial face where it abuts the polar face of the R3 cell. As a consequence, any influence of the polarizing factor X gradient would be subsumed, reversing the N-Dl signaling predisposition that would otherwise occur. Conversely, when Fz activity in the presumptive R3 cell is abolished, the polar face of the R3 cell would now have lower Fz activity than the adjacent, equatorial face of the R4 cell. As in the overexpression case, this change would reverse the direction of N-Dl signaling and hence reverse the reciprocal cell-fate choices made by the two cells (Tomlinson, 1999).

A key difference between the two models is that the scalar model requires cells to meter the absolute concentration of factor X via the level of activation of Fz protein, whereas the vectorial model requires them to detect and then amplify a relative difference of factor X concentration across the cell's diameter. Another difference is that factor X is predicted to have opposite effects on Fz activity, activating Fz in the scalar model and inhibiting Fz in the vectorial model. Hence, it is difficult to envisage how both mechanisms could work in concert, and as a consequence, they are viewed as mutually exclusive (Tomlinson, 1999).

One way in which a cell might amplify a difference in factor X abundance across its diameter would be to polarize the distribution of Fz itself in response to the ligand. Although the mechanisms are not yet clear, there are several precedents for the ability of cells to respond to shallow gradients of extracellular signals by polarizing the distribution of receptors and cytoskeletal components (e.g. during shmooing of yeast cells in response to mating pheromone, and the extension of pseudopods in Dictyostelium in response to cAMP). It is notable that Fz activity gradients have been implicated in the control of cell polarity in other epithelial tissues in the fly, particularly the wing. However, there is, at present, no indication that N signaling is involved in the establishment of cell polarity in this context. It is therefore suggested that Fz signaling generally mediates the establishment of cell polarity without requiring an N-Dl feedback amplification step. Hence, the involvement of N-Dl signaling in establishing ommatidial chirality may reflect a special attribute of this system, perhaps to allow the polarity of just two cells, the presumptive R3/R4 pair, to be used as a cue to control the pattern of a much larger ensemble of cells, the ommatidium (Tomlinson, 1999).

When Fz activity is absent throughout eye development, the Fz-dependent bias should be eliminated and each cell of the presumptive R3/R4 pair should have an equal chance of becoming either the signaling cell (R3) or the receiving cell (R4). Under these conditions, the choice of which cell becomes R3 and which becomes R4 would be determined by a stochastic variation, which gives one of the two cells a slight advantage that is then amplified by the N-Dl feedback mechanism. This explains why ommatidia in fz mutants 'choose' their chirality randomly when both cells have the same N gene dosage, but non-randomly when there is a 3:2 differential in N gene dosage. However, in fz minus eyes, approximately one third of the ommatidia are symmetrical, indicating that the R3/R4 distinction has not been resolved. One possible explanation is that the interaction between the R3 and R4 cells may be limited to only a few hours and this may be too short to ensure that a stochastic variation will arise and be amplified by the N pathway in all fz minus ommatidia. Timing may not be as critical in wild-type ommatidia, because the bias from Fz signaling is sufficient to ensure an appropriate resolution during this relatively brief interval (Tomlinson, 1999).

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. The asymmetric divisions that give rise to these cells have been visualized in developing pupae using green fluorescent protein fusion proteins. Spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorients to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, it is proposed that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila (Roegiers, 2001).

The dynamics of Pon-GFP localization and the coordination of spindle positioning and Pon-GFP crescent localization were examined in frizzled (fz) mutant pupae. The Frizzled protein is required for maintaining planar polarity during SOP divisions and thus the proper orientation of sensory bristles on the thorax. During the pI division in wild-type pupae, Pon-GFP forms an anterior crescent and is segregated to the anterior pIIb cell after mitosis. The divisions of the pI cells on the pupal notum are asynchronous, but the A-P orientation of the divisions is invariant. In fzr54 mutant pupae, the pI division occurs with an orientation that is random with respect to the A-P axis. Moreover, in time-lapse confocal images of pI divisions in fz mutants, the Pon-GFP crescent had drifted between 45° and 90° from its initial position by late anaphase in four out of ten SOPs. This result suggests that Fz may have a role in stabilizing the positioning as well as orienting the Pon crescent during mitosis (Roegiers, 2001).

The coordination of Pon crescent with spindle orientation was examined in the fz mutant background. Despite the mispositioning of the crescent relative to the A-P axis, the dynamics of spindle orientation are similar to those of the wild type. In most cases (90%), the mitotic spindle positions itself perpendicular to the Pon-GFP crescent, regardless of the relationship of the crescent to the A-P axis. The spindle has been observed to seesaw during the accumulation of Pon-GFP to one side of the cell; however, in about one-tenth of cases, the mitotic spindle is positioned parallel to the Pon-GFP crescent, but appeared to have both spindle poles anchored to the cortex. The spindles also have an abnormal curved morphology throughout mitosis. Notably, at telophase these cells appear to segregate the bulk of the Pon-GFP to one of the two daughter cells, despite the abnormal position of the spindle during metaphase and anaphase (Roegiers, 2001).

Owing to this rescue of the asymmetric localization of Pon at anaphase, cell-fate transformations have never been reported in fz mutants, and no loss of external sensory structures have been observed that would indicate a mis-segregation of Numb protein in the pI division. In SOPs co-expressing Pon-GFP and Tau-GFP in a wild-type background, spindles are always oriented perpendicular to the Pon-GFP crescent at anaphase. The orientation of subsequent divisions within the SOP lineage does not depend on Fz. This analysis reveals that in 14 out of 18 SOPs, the division pattern of the SOP lineage occur in the same orientation relative to pI as in the wild type, despite the randomization of the pI division with respect to the A-P axis. In the 4 out of the 18 remaining SOPs, the divisions were asymmetric, but slight deviations from the wild-type orientation were observed, such as the pIIa division occurring at a 45° angle from the pI division. Together, these results indicate that Fz participates in establishing the correct orientation and stabilization of the position of the Pon-GFP crescent in the pI cell, and that it may participate in the coordination of spindle orientation and crescent formation (Roegiers, 2001).

These results suggest that there are two fundamental types of asymmetric divisions in the developing Drosophila nervous system. During Drosophila development these two types of divisions are reiterated in different tissues at different times to generate cell-fate diversity. The divisions of the sensory organ precursor cell provide a unique system for studying different types of asymmetric cell divisions within the same lineage and how they might be coordinated. The orientations of the divisions are tightly regulated: two divisions occur along the A-P axis, and two divisions occur in the apical-basal orientation. In the pI division, which occurs along the A-P axis, the spindle is symmetric and reorients to align perpendicular to the crescent of Pon-GFP, and fz is important for the proper orientation of the crescent and appears to contribute to the coordination of spindle orientation and crescent positioning. In contrast, the spindle in the pIIb cell orients along the apical-basal axis and exhibits a strong size asymmetry. insc, a gene of central importance in coordinating spindle orientation and crescent formation in embryonic neuroblast divisions, also has an important role in orienting the mitotic spindle in the pIIb cell. These findings provide strong evidence that the pIIb division is a neuroblast-like division. It will be interesting to know whether other genes known to be involved in controlling the asymmetric divisions of neuroblasts, such as bazooka or partner of inscuteable, are also required for the pIIb division. The results may reveal general mechanisms for generating cell-fate diversity in Drosophila as well as in other species (Roegiers, 2001).

Asymmetric localization of Frizzled and the establishment of cell polarity in the Drosophila wing

The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. During cell polarization, the Frizzled receptor (visualized using Frizzled-Green fluorescent protein) is localized to the distal cell edge, probably resulting in asymmetric Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across the axis of a cell (Strutt, 2001).

To understand the asymmetric distribution of Fz-GFP, the distribution was studied in flies mutant for other genes involved in trichome polarity establishment. In clones of cells lacking starry night (stan) function, both the apical and PD localization of Fz-GFP is completely abolished. However, in cells lacking dsh function, in which Fz signal transduction is compromised, Fz-GFP apical localization is preserved, but there is no proximodistal (PD) localization, with a splotchy irregular distribution being seen instead. The same phenotype is observed for mutations in the prickle-spiny-legs (pkpk-sple) and Van Gogh (Vang) genes. This would be consistent with the trichome polarity phenotypes of these mutations being due to a failure of Fz localization (Strutt, 2001).

Genetic data indicate that the polarity genes in, fy, and mwh act downstream of Fz/Dsh, inhibiting trichome formation where Fz is not active. In agreement with this, mutations in these loci do not alter Fz-GFP distribution despite trichome polarity being disrupted (Strutt, 2001).

The localization of Stan has also been reported to be disrupted in cells lacking dsh function but not in those lacking mwh. Therefore, whether Fz-GFP and Stan remain colocalized in different mutant backgrounds was tested. In clones of cells lacking dsh function, it was found that both Fz-GFP and Stan remain predominantly apical, and although the distribution of both appears diffuse, they nevertheless show broad colocalization. Similarly, in an in background, both Fz-GFP and Stan remain apical and colocalized to the PD cell boundaries (Strutt, 2001).

It is concluded that one important function of Stan is to localize Fz apically in the cell during polarity establishment. Stan may also play a role in localizing and/or anchoring Fz at the distal cell edge. However, in the absence of fz autonomous polarity signaling activity, neither Fz-GFP nor Stan is localized to PD boundaries. This leads to the speculation that the Fz receptor is responsible for receiving an extracellular polarity signal, and that the interpretation of this signal drives the localization of Fz to the distal cell edge and Stan to the distal and proximal cell edges, Stan thus acting downstream of Fz. Nevertheless, the data would equally well support Fz acting downstream of Stan; the codependence of the localization of both proteins would support this possibility, as would the observation that Stan has homology to G protein-coupled receptors. Thus far, no ligand has been identified for the Fz receptor in polarity signaling, and it is conceivable that in fact Fz might be activated by association with another transmembrane receptor, a role for which Stan is clearly a candidate (Strutt, 2001).

Both Fz-GFP and Stan localization are also downstream of fz nonautonomous signaling activity. One of the functions of Stan is to measure differences in fz activity between adjacent cells, as Stan accumulates on the boundary between fz+ and fz- cells. These findings have been extended to show that Stan in fact accumulates on the boundaries between cells with different levels of fz nonautonomous signaling activity. This observation is consistent with there being a long-range gradient of fz nonautonomous signaling activity across the pupal wing, with each cell having different levels of fz nonautonomous activity relative to its neighbors (Strutt, 2001).

An intriguing observation is that in the cells bordering a clone deficient in only fz autonomous signaling, Stan appears to show a preference for localizing on the cell boundaries lying perpendicular to the clone boundary. Thus, in cells bordering the proximal and distal clone edges, Stan is sometimes seen lying on the lateral cell boundaries rather than the PD cell boundaries. Similarly, the same is true for Fz-GFP localization on the boundary of stan clones. This phenomenon seems to suggest that if Stan or Fz-GFP cannot localize on one of the PD boundaries of a cell, they show a preference for not localizing on the opposite PD boundary. This, in turn, opens up the possibility that there is an intracellular communication mechanism that couples Stan/Fz-GFP accumulation on one cell boundary to that on the opposite cell boundary. Interestingly, such a mechanism would enable the propagation of a wave of Stan/Fz-GFP polarization across the wing, starting from a single row of polarized cells at one edge. Obviously, such a mechanism for propagation of polarity would obviate the need for an external ligand gradient and would also argue against the existence of a long-range gradient of fz nonautonomous activity. However, long-range gradient models and cell-cell communication models for the propagation of polarity need not be mutually exclusive, and both may operate side by side (Strutt, 2001).

Taking these observations together, the following model is put forward for Fz function in the polarization of single cells in the developing wing. Initially, unlocalized Fz is required for the long-range propagation of a polarity signal. Fz is then recruited apically in a Stan-dependent manner and becomes stably localized at the distal cell edge in a process requiring Fz signaling and the activities of Stan, Dsh, Pkpk-sple, and Vang. This Fz localization then restricts the site of trichome initiation to the distal cell vertex. It is possible that Fz signaling activates Stan molecules to bind both to Fz (in the same cell) and to Stan molecules in the adjacent cell, and so anchors Fz at the distal edge of the cell. Localization of Fz may lead to further increased Fz signaling (possibly through the effects of receptor clustering), which could, in turn, recruit more Stan and Fz. Over time, increased activity of clustered Fz receptors at the distal cell edge would lead to the majority of the Fz in the cell being recruited to this location. In heterologous systems, Fz activity leads to recruitment of Dsh to the cell membrane, so it is likely that Dsh is also present at the distal cell boundary. A precedent for Fz-dependent localization of a cytoplasmic protein during planar polarity establishment is provided by the observation that the Numb protein requires Fz activity for correct asymmetric subcellular localization during sense organ precursor cell divisions (Strutt, 2001).

The stable PD localization of Fz also requires Pk-Sple and Vang activity, with their loss having a similar effect on Fz-GFP localization as loss of Dsh activity. It is possible that, like Dsh, they are required for the transduction of the Fz signal, or they may be involved in the function of Dsh itself. Interestingly, Vang activity on only one side of the PD boundary is sufficient for Fz-GFP localization to occur. Further investigations of the biochemical activities of these proteins will be required to fully elucidate their roles in planar polarity establishment (Strutt, 2001).

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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. 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 (D. Strutt, 2002).

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 dshv26 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).

Fz and Fmi colocalize at proximal-distal boundaries at 30 h apf. Furthermore, the asymmetric pattern of Fz localization depends on Flamingo (Fmi), whereas the asymmetric pattern of Fmi localization depends on Fz. 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 fzR52 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).

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, fzR52, 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 fzR52 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).

PCP aspects of the fmi phenotype are apparent from the earliest stage in the five-cell precluster. All markers reflecting the arrangement and rotation of ommatidial preclusters (Spalt: R3/R4; Bar: R1/6; svp-lacZ: R3/R4 and R1/R6) show typical polarity defects in fmi- tissue, with a random selection of the direction of rotation and abnormal rotation degrees. Thus, PCP defects are the primary phenotypic features of fmi clones, confirming its critical role in ommatidial polarity establishment (Das, 2002).

During eye disc patterning, Fmi protein is localized apically in all cells anterior to the morphogenetic furrow (MF), within the MF, and in a few rows of developing ommatidia posterior to the MF. Subsequently, Fmi is detected in differentiating photoreceptor cells in perinuclear areas and growing axons, possibly reflecting a late function of fmi in photoreceptor differentiation (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).

It was asked how Fmi localization is regulated in the R3/R4 pair. In the wing, Fmi, Fz, Dsh, and Dgo are codependent for their localization. This interdependence is only partially observed in the eye. Within fz- or dsh- eye tissue, Fmi is not polarized within the early R3/R4 pair and subsequently not found enriched in R4. Strikingly, borders of fz- clones display three specific features: (1) Fmi gets enriched at cell boundaries between fz+ and fz- cells; (2) this is only observed within the region of the five-cell precluster, where Fz signaling takes place, and (3) only when the fz-/fz+ boundaries are perpendicular to the equatorial-polar axis. This enrichment is reminiscent of the Fmi asymmetry seen in R3 and R4 and suggests that differences in Fz-signaling levels (but not absolute protein amounts) between neighboring cells lead to Fmi accumulation at the respective membranes (Das, 2002).

Thus, Fmi localizes to the membranes between cells that have different Fz-signaling levels in the axis of signaling. This also provides additional evidence to support the hypothesis that there is a significant difference in Fz-signaling levels between the R3 and R4 precursors (Das, 2002).

This study shows that Fmi is also important for the R3/R4 fate decisions and polarity establishment. Unlike other PCP genes fmi is required in both cells of the R3/R4 pair. Fmi protein is dynamically expressed within the R3/R4 pair, being first enriched at equatorial cell borders of R3 and R4 and subsequently detected in R4. The data suggest that Fmi has multiple roles in polarity establishment. The early R3/R4 equatorial enrichment is Fz dependent and results from Fz activity differences in this axis. The later R4-specific function is to downregulate Dl in R4, by antagonizing Fz signaling directly or indirectly (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).

Cell interactions and planar polarity in the abdominal epidermis of Drosophila

The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. Four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi) were examined in this study, and what happens when these genes are either removed or overexpressed in clones of cells was examined. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, a model is presented that builds on an earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. It is conjectured that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient (Lawrence, 2004).

Evidence is discussed that cells compare their scalar readout of the level of X with that of their neighbors and set their own readout toward an average of these. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information, and that Vang assists in the receipt of this information. The comparison between neighbors is crucial, because it gives the vector that orients hairs: these hairs point toward the neighbor cell that has the lowest level of Fz activity (Lawrence, 2004).

Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins studied, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view -- it is suggested that these localisations may be more a consequence than a cause of planar polarity (Lawrence, 2004).

There are a number of simple systems in which isolated cells orient to a polarising signal. These include the localized outgrowth, or 'schmooing' of yeast in response to mating pheromone and directed migration of Dictyostelium cells up a gradient of cyclic AMP. Small differences (as little as 1%-5%) in receptor activation across single cells are sufficient to polarise them, a response that, in yeast and elsewhere probably depends on localised exocytosis. It is not known whether the polarisation of single, isolated cells is a model for planar polarity of cells in an epithelium, but it is likely that they share at least some of the mechanisms (Lawrence, 2004).

It has been proposed that, in the abdomen of Drosophila, morphogen gradients (Hh in the A compartment and Wg in the P compartment) organise a secondary gradient ('X'); the vector of X specifying the polarity of each cell. Although the composition of X is unknown, at least three proteins, Fj, Ds and Ft, are implicated. All three may be expressed, or be active, in bell-shaped distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries. Ds and Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in the Golgi. Ds and Ft are integrated into the membrane, suggesting that the X gradient itself may not be diffusible but instead might depend on information transfer from cell to cell (Lawrence, 2004).

How does Hh set up the X gradient? Although changing the real or perceived level of Hh does affect polarity, many clones (for example clones that lack Smo, an essential component of Hh reception) show there is no simple correlation between Hh concentration and polarity. For instance, large smo- clones in the center of the A compartment are polarised normally, even though they are blind to Hh. Also, while smo- clones in some regions of the A compartment do affect polarity, both mutant and wild-type cells are repolarised. Both these observations argue for some transfer of information about polarity between cells, a process that would be at least partly Hh independent. This paper explores this process and is concerned with four genes (stan, fz, Vang and pk) that probably act downstream of ds, ft and fj (Lawrence, 2004).

Perhaps normal cells could transfer information from one to another (this might be particularly important for nascent cells following mitosis) to help keep the readout of X as a smooth gradient? To do this they might make a comparison of their neighbors and modify this readout of X toward an average of those neighbors. X might be read by a receptor molecule and the results point to Fz being the most likely candidate. The results indicate that the comparison itself requires the cadherin Stan. Thus, a cell would need to read and compare (using Stan) the levels of X (recorded in the activity of Fz) in neighboring cells. Then, in a way analogous to how a Dictyostelium amoeba reads the vector of a cAMP gradient, a cell would determine its polarity from the vector of Fz activity. The results suggest that Vang also acts in this step, helping cells to sense the level of Fz activity in neighboring cells (Lawrence, 2004).

Some of the results are discussed in terms of the model (Lawrence, 2004). Clones that lack, or overexpress Fz cause local and consistent repolarisations of cells that extend from within the clone and affect normal wild-type cells outside it. Because simply removing the fz gene from all cells randomizes polarity in the ventral pleura, it is self-evident that these organised polarity reversals must result from an interaction between the clone and the surrounding cells. It has been argued that Stan and Fz act in this process, but how? Note that stan and fz are the only mutants that have randomised hairs in the pleura, and the results indicate that neither Stan nor Fz can function properly without the other. Averaging might depend on the capacity of Stan to form homophilic dimers as bridges between neighboring cells, with such Stan:Stan dimers serving as a conduit for information about the relative level of Fz activity in each cell. However, with respect to non-autonomy, the results with the two genes differ:

  1. stan- cells cannot be repolarised by, and cannot repolarise, neighboring cells. This shows that Stan is essential in both neighboring cells for the transfer of information between them. Without Stan, the cells cannot compare and cannot therefore determine any vector. However, a stan+ cell, even if it is adjacent to a stan- cell, can be polarised normally; having Stan it should be able to read the levels of all neighboring cells except the stan- one and, having Fz, it should be able to set its own level (Lawrence, 2004).

  2. But, fz- cells, unlike stan- cells, can repolarise neighboring wild-type cells. Also, a fz- cell, again unlike a stan- cell, can itself be repolarised. The results also show that to be repolarised, or to polarise a neighbor, a fz- cell must be adjacent to a stan+ fz+ cell. Such a fz- cell will accumulate Stan in that membrane which abuts the stan+ neighbor so it should be able to read the level of the neighboring wild-type cell and be polarised accordingly. Consider a fz- cell at the outer edge of a fz- clone: lacking Fz, its activity level would be zero, but this level would be communicated by Stan to the neighboring cells. Wild-type cells outside would obviously have a higher level (than zero). The result would be that the two cells abutting the interface, the fz- cell inside, and a fz+ cell outside, would both make hairs that point into the center of the clone. The nextmost interior cell would not be polarised, since all its neighbors would be cells with level zero. In contrast, the next most exterior cell would be repolarised, since its scalar level would be brought down by the averaging process (Lawrence, 2004).

How far does the non-autonomy spread into wild-type cells? This process can be stimulated. According to the model this range would depend on the value of a single adjustable parameter, a that relates to how much a cell's scalar is read from X. At one extreme for this parameter (a=0), when the scalar of a cell depends only on X, a wild-type cell just posterior to a clone of fz- cells would reset its scalar as it was before; there could be no averaging and only that cell and its fz- neighbor will be repolarised. Thus the non-autonomy would be limited to one cell. At the other extreme (a=1), any local disturbance produced by a clone would decay rapidly because of averaging, and the repolarisation will tend to be lost altogether. In between these extremes, the non-autonomy spreads more than one cell, but over diverse values for this parameter, the range is near the amount usually observed (2-4 cell diameters) (Lawrence, 2004).

It has been observed that fz- clones have effects over longer range in backgrounds such as ds- where the X gradient might be flatter than normal. Similarly, cells are normally polarised in large smo- clones in the middle of the A compartment, where, because there can be no input from Hh, the X gradient could also be flat. Both these results are consistent with the model, because the range affected by averaging will increase (Lawrence, 2004).

Many of the proteins required for normal cell polarity, including Fz, Dsh, Dgo, Pk, Vang and Stan are found to be asymmetrically localised in the proximodistal axis of wing cells. This localisation is restricted to a brief period of just a few hours shortly before the wing hairs grow out, but, nevertheless it is assumed to be mechanistically important to planar polarity. For example, non-autonomy could be explained if localised proteins were components of one or more molecular complexes that propagate polarity from cell to cell. In support of this, note that loss of any of these proteins, including the removal of both Pk and Sple, prevents the asymmetric localisation of the others (Lawrence, 2004).

But the results do not seem to fit with such a mechanism, mainly because they provide evidence that polarity can propagate into cells that lack, or fail to localise all of these proteins. In particular, pk- cells are normally polarised throughout the P compartment and can be repolarised in both compartments by sharp discontinuities in Fz activity even in the pleura (where polarity is randomized in fz- and stan- animals). At a minimum, these findings challenge the hypothesis that Pk itself is an essential component of a feedback amplification mechanism responsible for polarising cells. Furthermore, if it is assumed that the observed failure of Fz, Dsh, Vang and Stan to localize in pk- wing cells reflects a general property, these results also challenge the idea that Fz, Dsh, Pk, Vang, Diego and Stan must be able to accumulate asymmetrically in order for cells to detect, and be polarised by, the X gradient, or by disparities in Fz activity. Indeed, Adler (2002) has already hinted that there is no convincing evidence that the asymmetric localisation of these proteins actually functions in planar polarity: 'the preferential accumulation [of proteins] along the...edges of wing cells is a process that intuitively seems likely to be part of a core system...but perhaps it is not and if not...this would leave rather little in the core' (Lawrence, 2004).

Are wing cells polarised only briefly just prior to the hair outgrowth? The reason for raising this possibility is that the proteins are apparently only asymmetrically localised at that time. If this localisation were not causal, as it is now suggested, it could be that the cells are polarised for all or most of development -- again arguing that the ephemeral localisation of the proteins is more a consequence than a cause of polarisation (Lawrence, 2004).

Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. This study shows that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, it is proposed that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways (Wu, 2004).

Frizzled chimeras described below are denoted by three numbers, the first being the source of the N-terminal Wnt-interacting cysteine-rich domain (CRD), the second being the source of the remaining proximal extracellular domain and 7 transmembrane region and loop region (collectively referred to as 7-TM), and the third refering to the source of the intracellular C-tail (Wu, 2004).

It was asked whether the apical localization of Fz is required for PCP signaling? The Fz1-1-2 chimera, which is distributed ubiquitously within the apical-basolateral membrane, only partially rescues the fz- eye phenotype, and it can also cause defects related to canonical Wg/Arm signaling. In contrast, apically localized Fz1-1-2S ('S' for 'short') fully rescues the fz- phenotype and has no additional effects. The Fz1-1-2 chimera also shows much weaker PCP phenotypes in the GOF assay. Taken together, these results suggest that a reduction in the apical localization of Fz leads to a reduction in PCP signaling activity. However, about 80% of the chirality defects in fz- eyes are rescued by tub-fz1-1-2, and in the wing tub-fz1-1-2 rescues the fz- mutant to a similar extent as tub-fz1-1-1 and tub-fz1-1-2S, suggesting that Fz1-1-2 contains substantial PCP signaling activity (Wu, 2004).

Because both GOF and loss-of-function studies indicate that the Fz1 7-TM region is critical for Fz1 function, Fz1-1-2 is expected to have Fz/PCP signaling activity, although with altered subcellular distribution. Thus, the remaining PCP signaling activity of Fz1-1-2 seen is probably due to the presence of some of this protein in apical regions. It is difficult to determine how much of Fz1-1-2 is actually localized to this membrane region. Since the immunohistochemical staining indicates that it is not excluded apically, it is assumed that Fz1-1-2 has enough apical localization to participate when PCP signaling is initiated. It has been suggested that wing cell orientation does not depend on absolute Fz levels, but instead depends on relative Fz/PCP activity differences in a Fz activity gradient across a field. Thus, although the absolute activity of Fz1-1-2 is reduced (based on weaker GOF phenotypes and weaker rescue of fz- in the eye), the relative difference might be sufficient for the partial rescue (Wu, 2004).

In this context, it is worth noting that tub-fz1-1-2 rescues the fz- phenotype better in the wing than in the eye, whereas there is no apparent difference in rescue activity between the eye and the wing for tub-fz1-1-1 or tub-fz1-1-2S. The difference could be due to the observed nonautonomous PCP signaling effects in the wing, where neighboring cells affect each other's planar polarization. Fz1-1-2 may allow some wing cells to adopt the correct orientation, which then in turn influences many of the remaining wing cells to also orient themselves correctly through nonautonomous interactions (Wu, 2004).

It has been shown that Fz1 localization is affected in flamingo (fmi) mutant clones at about 30 h APF, leading to the proposal that Fmi recruits Fz1 into apical junctions. However, this study found that Fz1 is localized normally in fmi null mutant clones earlier in the third instar wing disc. What causes the difference between these two observations? PCP signaling in the wing is thought to act in two phases (one 6-24 h APF and the second 24-32 h APF [, and it results in the distal enrichment and maintenance of Fz1. Since Fz1/PCP signaling is modulated by Fmi, Fmi-dependent changes in Fz1 localization likely result from effects on PCP signaling activity. At the same time, Fmi localization is also dependent on Fz1 activity and becomes also less apically localized in fz- tissue at 30-36 h APF, suggesting that the regulation of apical localization between Fz1 and Fmi is complicated and mutual at these late stages (Wu, 2004).

This study has shown that initial apical localization of Fz1, preceding both stages of PCP signaling, is not fmi dependent. This result suggests that Fmi and Fz1 get recruited to apical junctions independently. During later stages, Fmi and Fz1 then affect each other's localization through PCP signaling. At this point, it remains unclear which molecules initially recruit Fz1 into the apical junctional region (Wu, 2004).

Secreted Wg mainly binds to Fz2 at basolateral membrane regions of the wing epithelium, indirectly suggesting that canonical signaling occurs in the basolateral membrane compartment. The current experiments show that overexpression of Fz1-1-1 or Fz2-1-1 leads to a cell-autonomous loss of wing margin bristles and associated tissue, suggesting that these molecules act like dominant negatives, inhibiting Wnt/β-cat signaling. As these molecules are enriched apically and sequester Dsh there, Fz-Dsh complexes at apical junctions may be largely inactive for canonical Wnt signaling. This result suggests that canonical Wnt signaling and PCP signaling occur in different subcellular compartments. Basolateral Wnt/β-cat signaling is also suggested by the fact that (1) secreted Wg binds to Fz2 at the basolateral membrane and that (2) apical Wg secretion and signaling could lead to mis-specification in disc folds and cells in the peripodial membrane (Wu, 2004).

Both Fz1 and Fz2 are capable of canonical Wnt/β-cat signaling. Consistently, different Fz1/2 chimeras, including related versions of Fz2-1-1 and Fz2-2-1, are capable of rescuing the fz, fz2 double mutant phenotype. However, when Fz1-1-1, Fz2-1-1, or Fz2-2-1 is expressed at high levels, Dsh accumulates at apical junctions, thus decreasing cytosolic Dsh levels. Since the chimeric receptors can rescue the fz, fz2 double mutant when expressed at low levels (under the control of the tub promoter), the relative level of each receptor together with its subcellular localization appear critical for the signaling outcome (Wu, 2004).

In conclusion, this study has shown that subcellular localization contributes to Fz signaling specificity. The data indicate that the localization of Fz1 at apical junctions promotes Fz/PCP signaling, whereas this localization can inhibit canonical Wnt/β-cat signaling. The localization is mediated through sequences in the C-tail (Wu, 2004).

Mathematical modeling of planar cell polarity to understand domineering nonautonomy

Planar cell polarity (PCP) signaling generates subcellular asymmetry along an axis orthogonal to the epithelial apical-basal axis. Through a poorly understood mechanism, cell clones that have mutations in some PCP signaling components, including some, but not all, alleles of the receptor frizzled, cause polarity disruptions of neighboring wild-type cells, a phenomenon referred to as domineering nonautonomy. A contact-dependent signaling hypothesis, derived from experimental results, is shown by reaction-diffusion, partial differential equation modeling and simulation to fully reproduce PCP phenotypes, including domineering nonautonomy, in the Drosophila wing. This work suggests that Fz does not require a Wnt ligand in PCP signaling but that its activity is regulated by interactions between neighboring cells and differential levels of the cytoplasmic mediators Pk and Dsh. The sufficiency of this model and the experimental validation of model predictions reveal how specific protein-protein interactions produce autonomy or domineering nonautonomy (Amonlirdviman, 2005).

As the understanding of cellular regulatory networks grows, system behaviors resulting from feedback effects have proven sufficiently complex so as to preclude intuitive understanding. The challenge now is to show that enough of a network is understood to explain such behaviors. Using mathematical modeling, the sufficiency of a proposed biological model is shown and its properties studied, to demonstrate that it can explain complex PCP phenotypes and provide insight into the system dynamics that govern them (Amonlirdviman, 2005).

Many epithelia are polarized along an axis orthogonal to the apical-basal axis. On the Drosophila adult cuticle, each hexagonally packed cell elaborates an actin-rich hair that develops from the distal vertex and points distally. Genetic analyses have identified a group of PCP proteins whose activities are required to correctly polarize these arrays. The domineering nonautonomy adjacent to cell clones mutant for some, but not other, PCP genes has not yet been adequately explained. For example, in the Drosophila wing, Van Gogh/strabismus (Vang; encoding a four-pass transmembrane protein) clones disrupt polarity proximal to the mutant tissue, whereas null frizzled (fz; encoding a seven-pass transmembrane protein) alleles disrupt polarity distal to the clone. Models to explain this phenomenon have often invoked diffusible factors, referred to as factor X or Z, because they have not yet been identified. It is proposed instead that the observed behaviors of known PCP proteins are sufficient to explain domineering nonautonomy (Amonlirdviman, 2005).

Fz and other PCP signaling components accumulate selectively on the distal or proximal side of wing cells. Evidence has been provided that these proteins function in a feedback loop that amplifies an asymmetry cue, which converts uniform distributions of PCP proteins into highly polarized distributions. The proposed feedback mechanism depends on several functional relationships. Fz recruits Dishevelled (Dsh; a cytoplasmic protein) to the cell membrane. In addition, Fz promotes the recruitment of Prickle-spinylegs (Pk; a LIM domain protein) and Vang to the cell membrane of the adjacent cell. Feedback is provided by the ability of Pk (and Vang) to cell-autonomously block Fz-dependent recruitment of Dsh. This feedback loop functions strictly locally, between adjacent cells. Global directionality is imposed through the agency of the novel transmembrane protein Four-jointed and the cadherins Dachsous and Fat (Ft). Widerborst, a regulatory subunit of protein phosphatase 2A, accumulates asymmetrically within each cell and is required to bias the feedback loop. Although the mechanism by which Ft biases the direction of the feedback loop is unknown, one possibility is that Ft may direct Widerborst distribution (Amonlirdviman, 2005).

However, it is not readily apparent that this biological model does not readily explain the complex patterns observed in fields of cells containing mutant clones, and it has been argued that it cannot account for some of the observed phenotypes. Indeed, progress in understanding PCP signaling has been hampered by an inability to deduce, given a particular signaling network hypothesis, definitive links between molecular genetic interventions and tissue patterning effects. For example, although it is apparent that removing Dsh or Fz would disrupt the feedback loop, it is not obvious how the feedback loop in adjacent wild-type cells responds, such that dsh mutant clones behave autonomously, whereas for most fz alleles, mutant clones behave nonautonomously. Interestingly, though, for some fz alleles, mutant clones produce an almost cell-autonomous phenotype. As another example, Pk overexpression promotes the asymmetric accumulation of Dsh and Fz, despite the role of Pk in the feedback loop as an inhibitor of Dsh membrane recruitment (Amonlirdviman, 2005).

A mathematical model has been developed based on the described feedback loop and an initial asymmetry input representing the global directional cue. Although mathematical modeling cannot prove the correctness of the underlying biological model, the ability of the mathematical model to capture the known behaviors of the system proves the feasibility of the biological model, provides testable hypotheses, and yields insight into the factors contributing to autonomy and nonautonomy (Amonlirdviman, 2005).

The features of the biological feedback loop model have been represented as a mathematical reaction-diffusion model that describes the concentrations of Dsh, Fz, Vang, and Pk throughout a network of cells. Although the mechanisms that underlie the local feedback loop are not fully understood, the essential logic of this feedback loop is preserved by representing these interactions as binding to form protein complexes (Amonlirdviman, 2005).

Inhibition of Dsh membrane recruitment by Pk and Vang is represented in the mathematical model as an increase in the backward reaction rate of reactions in which Dsh binds Fz (or Fz complexes) by a factor dependent on the local concentration of Pk and Vang. The specific mechanism for the introduction of the directional bias into the feedback loop network is not known. Two forms of a global biasing signal were therefore implemented, and the results using either of these models were similar. The resulting mathematical model consists of a system of 10 nonlinear partial differential equations. With a given set of model parameters, an array of cells could then be simulated, and the resulting hair pattern assigned on the basis of the final distribution of Dsh (Amonlirdviman, 2005).

The model parameters, including the initial protein concentrations, reaction rates, and diffusion constants, were not known, and so these parameters were identified by constraining them to result in specific qualitative features of the hair pattern phenotypes. A sensitivity analysis showed that the model results are not highly sensitive to the precise parameter values and suggests that the conclusions regarding the feasibility of the model are valid for considerable ranges of parameters (Amonlirdviman, 2005).

In simulated wild-type cells, Dsh and Fz localize to the distal membrane, and Vang and Pk localize to the proximal membrane, as is seen in vivo. Simulated clones of cells lacking fz function disrupt polarity in wild-type cells distal to the clones, whereas simulated clones lacking Vang function disrupt polarity on the proximal side of the clones. Simulated clones lacking dsh function result in the disruption of polarity within the mutant cells, but only show a mild effect outside of the clones. The nearly, though not fully cell autonomous, phenotype is similar to that which is observed experimentally. Clones lacking all pk function show only a subtle phenotype. Examination of protein distributions shows that the results are highly concordant with published observations. Similarly, simulated overexpression clones produce results closely mimicking observed experimental results. In simulations and in wings, relatively small clones lacking a global biasing signal show no phenotype, demonstrating that not all cells need to respond to the global directional signal for the feedback loop to cooperatively align all of the cells (Amonlirdviman, 2005).

Previously, it was found that Pk overexpression in the posterior wing domain enhances the accumulation of Fz and Dsh at cell boundaries, despite the observed ability of Pk and Vang to block Dsh recruitment. Consistent with these results, Dsh and Fz are seen in a simulation of this experiment to accumulate to higher levels in the region overexpressing pk than in the wild-type region, and they accumulate perpendicular to the wildtype orientation near the anterior-posterior boundary (Amonlirdviman, 2005).

The results suggested a mechanistic explanation for the difference between autonomous and nonautonomous fz alleles. Because the nearly autonomous fz alleles (fzJ22 and fzF31) have phenotypes similar to dsh clones, it is hypothesized that these alleles may be selectively deficient in complexing with Dsh, but normal in their ability to complex with Vang. Simulations of clones in which the interaction was disrupted between Dsh and Fz by reducing the corresponding forward reaction rates produced nearly cell autonomous polarity phenotypes (Amonlirdviman, 2005).

This hypothesis makes two easily testable predictions. (1) Fz autonomous proteins should be present in the membrane and should recruit Vang to the adjacent membrane, whereas Fz nonautonomous protein should not recruit Vang. It has previously been shown that GFP-tagged FzJ22, expressed in a wild-type background, is present at the apical cell cortex, but remains symmetrically distributed, a distribution in accordance with the simulation of this condition. Examining this further, it was found that in cells adjoining clones of the autonomous fzF31 allele, Vang is recruited to the boundary between wild-type and mutant cells, whereas substantially less Vang is recruited to those boundaries in cells adjoining clones of the nonautonomous fzR52 allele. Thus, Fzautonomous proteins recruit Vang to the opposing cell surface, whereas nonautonomous alleles do not. (2) Autonomous Fz proteins should fail to recruit Dsh. Indeed, it was found that both are substantially impaired in Dsh recruitment, though somewhat less impaired than the very strong, nonautonomous fzR52 allele. Thus, strong fz alleles, many of which fail to accumulate Fz protein, display no or severely impaired interaction with Dsh and Vang, whereas autonomous alleles have impaired interaction with Dsh, but retain substantial ability to recruit Vang to the adjacent membrane. Notably, simulated overexpression of Fz with impaired Dsh interaction also produced the correct polarity disruption in cells proximal to the clones (Amonlirdviman, 2005).

The Dsh1 protein produces nearly autonomous clones, and it carries a mutation in its DEP domain, which is required for membrane localization; autonomous fz alleles bear point mutations in the first cytoplasmic loop, suggesting these mutations may affect the same interaction. A low affinity interaction between the Dsh PDZ domain and a sequence in the cytoplasmic tail of Fz has been demonstrated. These data suggest that sequences in the Dsh DEP domain, and in the Fz first intracellular loop, are also important for Dsh membrane association. Thus, a regulated, bipartite, high affinity association of Dsh with Fz may be selectively disrupted in fzautonomous alleles (Amonlirdviman, 2005).

The ability of the mathematical model to simultaneously reproduce all of the most characteristic PCP phenotypes demonstrates the feasibility of the underlying biological model as a PCP signaling mechanism. Further, the mathematical model demonstrates how the overall scheme of the model -- a local feedback loop between adjacent cells amplifying an initial asymmetry -- can explain the autonomous and nonautonomous behavior of PCP mutant clones. Alternative models invoking diffusible factors have not been supported by the identification of such factors, and the contact-dependent intercellular signaling model more readily accounts for the slight nonautonomy of dsh and fzautonomous clones than do the diffusible factor models (Amonlirdviman, 2005).

The ability of the mathematical model to make predictions and provide a detailed picture of PCP signaling is limited by the lack of complete biological understanding. Although the validity of quantitative model predictions is subject to its assumptions and the set of features used in parameter identification, the model has allowed a direct connection of a biological model to the complex behaviors it is hypothesized to explain and to explore the implications of variations in the model (Amonlirdviman, 2005).

Polarized transport of Frizzled along the planar microtubule arrays in Drosophila wing epithelium

Cells in a variety of developmental contexts sense extracellular cues that are given locally on their surfaces, and subsequently amplify the initial signal to achieve cell polarization. Drosophila wing cells acquire planar polarity along the proximal-distal (P-D) axis, in which the amplification of the presumptive cue involves assembly of a multiprotein complex that spans distal and proximal boundaries of adjacent cells. This study pursues the mechanisms that place one of the components, Frizzled (Fz), at the distal side. Intracellular particles of GFP-tagged Fz move preferentially toward distal boundaries before Fz::GFP and other components are tightly localized at the P/D cortex. Arrays of microtubules (MTs) are approximately oriented along the P-D axis and these MTs contribute to the formation of the cortical complex. Furthermore, there appears to be a bias in the P-D MTs, with slightly more plus ends oriented distally. The hypothesis of polarized vesicular trafficking of Fz is discussed (Shimada, 2006).

It is proposed that Flamingo- and Fz::GFP-containing vesicles are transported preferentially toward the distal cell cortex along P-D-oriented MTs. It has been considered that a cassette of transmembrane proteins (Four-jointed, Dachsous, and Fat) functions upstream of Fz and confers an initial small asymmetry of Fz activity across the cell. This small imbalance is amplified through formation of the multiprotein complex across the P/D boundary, reinforces Fz signaling, and eventually leads to specification of prehair formation at the distal cell end. The polarized transport of Fz that was observed may reflect one of the outputs of the upstream cassette to set an initial bias of Fz activity and/or an intermediate step of the Fz signaling feedback amplification (Shimada, 2006).

Immuno-EM studies revealed intracellular vesicles that were associated with Fz::GFP or Flamingo (Fmi) on the AJ plane. The number of these vesicles per cell was an order of magnitude higher than that of Fz::GFP particles visualized by confocal microscopy. Only a subpopulation of the vesicles, one containing large numbers of Fz::GFP molecules, may have been detected by confocal microscopic observations. Although both the multiprotein complex of the cortical PCP signaling components and the DE-cadherin-catenin adhesion complex are located at AJ in an almost overlapping manner, this study supports the possibility that components of these two complexes were separated into distinct vesicles at their exit from the trans-Golgi network (TGN). Sorting of Fz::GFP, Fmi, and possibly Dsh as well from DE-cadherin should also take place when molecules on the plasma membrane are incorporated into recycling endosomes. Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for sorting apical and basolateral plasma membrane proteins. Wing epidermal cells appear to have a machinery to subdivide molecules that are targeted to AJ (Shimada, 2006).

The results of double staining for Fz::GFP and Fmi indicated that the majority of Fmi-containing vesicles are transported distally, not to the proximal boundary. Then how are Fmi, Stbm/Vang, and Pk distributed to the proximal cortex (Shimada, 2006)?

Fmi, Stbm/Vang, and Pk have already been distributed rather uniformly at the cell cortex in imaginal and early pupal epithelia before the polarized transport starts, and these molecules may be allowed to diffuse laterally within the plasma membrane. Fmi, which is transported to the distal boundary together with Fz and Dsh, can lock a counterpart on the proximal membrane of the adjacent cell through its homophilic binding property. Formation of this Fmi-Fmi bridge across the P/D boundary would anchor the Fz-Dsh complex at the distal cortex, increasing local activity and the copy number of Fz-Dsh. This slight input may initiate recruitment of Stbm/Vang-Pk on the opposing proximal cortex by means of mutual exclusion between Dsh and Pk, and by means of hypothetical ectodomain interaction between Fz and Stbm/Vang. Then the nascent asymmetric complex amplifies the imbalance of Fz activity by unknown mechanisms. This amplification could be involved in either facilitating loading Fz-Dsh and Fmi into vesicles at their exit from the TGN, accelerating the transport of the vesicles to the distal cortex, and/or restricting diffusion of Fz-Dsh and Stbm/Vang-Pk out of the distal and proximal membrane domains, respectively (Shimada, 2006).

In setting up the initial bias, the polarized transport may not necessarily require Stbm and Pk, whereas the subsequent amplification presumably depends on the formation of the cortical complex. This hypothesis is consistent with the observation that bright Fz::GFP particles were hardly seen in the dsh, fmi, or pk mutant backgrounds. It also explains why FzP278L::YFP particles did not move distally, because FzP278L is postulated to lose its interaction with Dsh and most FzP278L::YFP particles did not contain Fmi (Shimada, 2006).

Besides noncentrosomal MTs along the apicobasal axis, P-D-oriented MTs were unequivocally visualized in the AJ plane, and the important role of this MT array in localizing the cortical PCP proteins and the distal-oriented movement of Fz::GFP particles was shown. Assuming the importance of the P-D MT alignment, how is the Fz::GFP particle transported or recycled back along the MT track in terms of vesicular trafficking? The particles showed staggered trajectories when followed at 1 or 10 s intervals, and this back-and-forth motion of the Fz::GFP particle can be interpreted as follows: (1) individual Fz::GFP particles bind multiple motors of different classes, and (2) activities of the motors of opposing directionality are coordinated. This bidirectional transport has been shown for organelle trafficking; despite such back-and-forth motion, cargos can still achieve net transport on longer time scales. Another, not mutually exclusive, possibility could be that the particle/cargo motor complex may repeat cycles of running, falling off the MT track, and reengaging. Whether specific members of MT motors contribute to the Fz::GFP transport or not awaits further genetic as well as biochemical characterization of the particle (Shimada, 2006).

Given that either of the above possibilities of motor-driven motions is the case, why did the Fz::GFP particle move predominantly toward distal cell boundaries when followed at 1 min intervals? Attempts were made to trace the origin of this asymmetry to the polarity of individual P-D MTs and it was found that the wing cell had slightly more + end-distal MTs than + end-proximal ones. Nevertheless, it is an open question as to whether such a small difference can be causally related to the distally oriented transport and whether the subtle difference in MT polarity can be one of the critical parameters in operating the cortical feedback loop (Shimada, 2006).

How are noncentrosomal MTs aligned approximately along the P-D axis at the level of adherens junction? P-D-oriented MTs were still observed in the absence of Fz function, and this strongly suggests that generation of the P-D MT array is controlled by a mechanism upstream of, or separate from, the cortical complex. One clue for this mechanism involving MTs may be Widerborst (Wdb), a B' regulatory subunit of PP2A. It would be necessary to investigate how exactly MTs are disorganized when Wdb function is abrogated. Another clue may be a recent report that the presence of apical MTs is related to Dpp signaling activity, although a cytoskeletal function for Dpp remains to be shown (Shimada, 2006).

A large gap remains in cell biological understanding of the global cue that is considered to be provided by the cassette of transmembrane proteins (Fj, Ds, and Ft). It should be pointed out that each of Fj, Ds, and Ft controls the ratio of proximal-distal to anterior-posterior growth of appendages and that Ds and Ft have been recently shown to control the shape of the growing organs by regulating orientations of cell divisions in imaginal discs. This supports the hypothesis that Ds and Ft can orient the mitotic spindle, and hence MT organization, during wing morphogenesis. It would be intriguing to investigate whether the MT orientation and polarity observed in this study are also under the control of the upstream cassette, and to pursue a molecular connection between tissue shape and polarity (Shimada, 2006).

Frizzled/PCP-dependent asymmetric Neuralized expression determines R3/R4 Fates in the Drosophila eye

Planar cell polarity (PCP) is a common feature in many epithelia, reflected in cellular organization within the plane of an epithelium. In the Drosophila eye, Frizzled (Fz)/PCP signaling induces cell-fate specification of the R3/R4 photoreceptors through regulation of Notch activation in R4. Except for Dl upregulation in R3, the mechanism of how Fz/PCP signaling regulates Notch in this context is not understood. The E3-ubiquitin ligase Neuralized (Neur), required for Dl-N signaling, is asymmetrically expressed within the R3/R4 pair. It is required in R3, where it is also upregulated in a Fz/PCP-dependent manner. As is the case for Dl, N activity in R4 further represses neur expression, thus, reinforcing the asymmetry. Neur asymmetry is show to be instructive in correct R3/R4 specification. These data indicate that Fz/PCP-dependent Neur expression in R3 ensures the proper directionality of Dl-N signaling during R3/R4 specification (del Alamo, 2006).

PCP establishment in the eye depends on the specification of photoreceptors R3 and R4 in two steps. First, Fz signaling occurs at higher levels in R3, and second, as a consequence, Dl signaling is directed from R3 to the R4 precursor, where N specifies R4 fate. This study shows that neur is required for proper Dl-N signaling directionality in the R3/R4 pair. In the absence of neur, defects occur in R3/R4 cell-fate specification and PCP. Importantly, neur expression is upregulated in R3 in a Fz/PCP-dependent manner. Finally, this study shows that the asymmetry in neur expression is required for PCP specification (del Alamo, 2006).

Neur is an E3-ubiquitin ligase known to enhance Dl signaling in a variety of Dl-N mediated processes, including lateral inhibition or lateral specification events (e.g., pIIb to pIIa specification in sensory organ development). This study shows that neur is required for lateral specification in R3 for Dl to signal to R4. Analysis of R3/R4 Dl mosaics revealed that the Dl mutant cell always acquires R4 fate, while the wt cell acquires R3 fate. This is consistent with neur analysis showing that in 94.2% of the cases, ommatidia mutant only in R3 showed a PCP defect, indicating that neur is required only in R3, the signal-sending cell (del Alamo, 2006).

There is, nevertheless, a difference between the PCP phenotypes of Dl and neur mosaic ommatidia: Dl mosaics show reversed polarity (chirality flips) when R3 is mutant, while the equivalent neurIF65 mosaics show mostly a symmetric phenotype (89.5% of ommatidia displaying chirality defects). It is likely that the cold-sensitive neurIF65 allele is not null and it is not clear if remaining Dl activity is present in the absence of Neur, accounting for the difference (del Alamo, 2006).

Mib1, another E3-ubiquitin ligase-regulating signaling by Dl and Serrate (Ser, the other N ligand in flies), has no effect on PCP specification. These results are in agreement with data showing that Neur and Mib1 have complementary functions. Taken together, the data indicate that neur but not mib1 is required for R3/R4 specification (del Alamo, 2006).

Previous studies suggested that neur has a permissive role in Dl-N signaling. In lateral inhibition processes, neur is expressed in proneural clusters, whereas in asymmetric cell division, Neur is selectively inherited by one of the daughter cells. In either case, Neur makes the cell in which it is expressed competent for Dl signaling. In the eye, Dl is enriched in R3 as a result of Fz signaling, and this study provides evidence that Neur is enriched in R3 and that this enrichment is also regulated by Fz/PCP signaling. While neur is initially expressed in both cells, the data indicate that Fz/PCP-dependent R3 upregulation of neur is necessary and sufficient for Dl signaling directionality. Since Neur affects Dl activity posttranslationally, Dl is still upregulated in R3 when Neur is misexpressed. This implies that the elimination of the difference in Neur levels between the R3/R4 precursors affects the direction of Dl-N signaling. These data indicate that the Neur expression asymmetry, mediated by Fz/PCP signaling, is instructive for R3/R4 specification (del Alamo, 2006).

The phenotypes resulting from Neur misexpression are relatively mild. Only when both Dl and Neur are coexpressed, chirality defects are induced, suggesting that differential expression of both factors in the R3/R4 cell pair is instructive for cell fate. Furthermore, other factors could also be present in R3/R4 precursors to ensure robustness of the cell-fate decision. These observations suggest a complex network of molecular interactions between Fz/PCP and Notch signaling (del Alamo, 2006).

The Frizzled planar cell polarity signaling pathway controls Drosophila wing topography

The Drosophila wing is a primary model system for studying the genetic control of epithelial Planar Cell Polarity (PCP). Each wing epithelial cell produces a distally pointing hair under the control of the Frizzled (Fz) PCP signaling pathway. Fz PCP signaling also controls the formation and orientation of ridges on the adult wing membrane. Ridge formation requires hexagonal cell packing, consistent with published data showing that Fz PCP signaling promotes hexagonal packing in developing wing epithelia. In contrast to hair polarity, ridge orientation differs across the wing and is primarily anteroposterior (A-P) in the anterior and proximodistal (P-D) in the posterior. Evidence is presented that A-P ridge specification is genetically distinct from P-D ridge organization and occurs later in wing development. A two-phase model is proposed for PCP specification in the wing. P-D ridges are specified in an Early PCP Phase and both A-P ridges and distally pointing hairs in a Late PCP Phase. The data suggest that isoforms of the Fz PCP pathway protein Prickle are differentially required for the two PCP Phases, with the Spiny-legs isoform primarily active in the Early PCP Phase and the Prickle isoform in the Late PCP Phase (Doyle, 2008).

If the Fz PCP pathway polarizes wing cells for both hair polarity and ridge orientation, why is there a different relationship between hair polarity and ridge orientation in the anterior wing compared to the posterior wing? A possible explanation is suggested by the wing phenotypes associated with the hypomorphic dsh1 and the isoform-specific pkpk alleles. In each case, P-D ridges still form in the posterior wing, but anterior ridge orientation is also P-D rather than the normal A-P. Hair polarity is abnormal in both the anterior and posterior wing. The dsh1 allele has been shown to be permissive for an early phase of Fz PCP pathway activity, but restrictive for a late phase of Fz PCP activity immediately preceding wing hair initiation. Similarly, the Pk protein isoform, which is inactivated by the pkpk30 mutation, is required only for the late period of Fz PCP activity. This suggests a two-phase mechanism for PCP specification in the wing. In an Early PCP Phase, P-D ridges are specified in both the anterior and posterior wing. In a Late PCP Phase, ridges in the anterior wing are reorganized to an A-P orientation and P-D hair polarity is specified. This temporal separation of A-P and P-D ridge specification could explain how the Fz PCP signaling pathway can organize ridges in two different orientations in the same wing (Doyle, 2008).

The model is supported by the results from over-expressing the Sple protein isoform at different timepoints during pupal wing development. Ubiquitous over-expression of Sple during wing development causes specific changes in ridge orientation and hair polarity in both the anterior and posterior wing. Inducing Sple over-expression prior to 15 h a.p.f. similarly affects ridge orientation and hair polarity in both the anterior and posterior wing. However, Sple induction after 15 h a.p.f. alters anterior ridge orientation from A-P to P-D, but does not affect P-D ridge formation in the posterior wing. Hair polarity is still abnormal in both the anterior and posterior wing. This is a similar phenotype to the dsh1and pkpk alleles. Assuming that the ability of Sple over-expression to disrupt ridge and hair specification coincides with the normal timing of these developmental events, then this experiment supports the proposal that there is an Early PCP Phase in which P-D ridges are specified and a Late PCP Phase when A-P ridges and P-D hairs are organized (Doyle, 2008).

The presence of P-D ridges in the anterior of dsh1 and pkpk mutant wings, as well as when the Sple isoform is over-expressed at after the Early PCP Phase (induction after 15 h a.p.f.), imply that P-D ridges in the anterior wing are the default if cell polarization in the Late PCP Phase fails. This, in turn, suggests that P-D ridges are specified in both anterior and posterior wing in the Early PCP Phase. The reorganization of pupal wing epithelial cells to hexagonal packing, a process that is thought to be associated with ridge development, is known to occur at the same stage in both the anterior and posterior wing. This remodeling of cell packing begins at 18 h a.p.f., immediately after the early period of Fz PCP signaling and it is at this time that a deficiency in hexagonal packing is first observed in a pkpk-sple mutant. These observations support the idea that ridges are specified in both the anterior and posterior wing during the Early PCP Phase. Furthermore, the ability of Sple over-expression during the Early PCP Phase (induction before 15 h a.p.f.) to alter ridge orientation in both the anterior and posterior wing, also suggests ridges are organized in both regions at this time (Doyle, 2008).

The early phase of Fz PCP signaling has been localized to 16-18 h a.p.f. At first glance, this appears to differ from the results of the Sple induction experiment which identified induction at 15 h a.p.f. as the transition point between the Early and Late PCP Phases. However, in the experiment there must be a delay between the heat-shock induction of Gal4 expression and the accumulation of sufficient Sple protein to disrupt development. It is estimated that the length of this delay is 3-4 h as Sple induction after 28 h a.p.f. failed to affect hair initiation at around 32 h a.p.f. Assuming a similar time is required for Sple accumulation earlier in pupal development, this places the end of the Early PCP Phase at around 18 h a.p.f., which agrees with published data (Doyle, 2008).

The Pk and Sple protein isoforms appear to have complementary functions in wing development. The pkpk mutant phenotype suggests that the Pk isoform is required for both A-P ridge orientation in the anterior wing and for hair polarity in both the anterior and posterior wing. In contrast, the Sple isoform is primarily required for P-D ridge formation in the posterior wing. Although, since the P-D ridges present in the pkpk mutant anterior wing are lost in a pkpk-sple mutant, it also appears that the Sple isoform is required for P-D ridge formation in the anterior wing. These specific and complementary roles suggest that Sple is primarily active in the Early PCP Phase and Pk in the Late PCP Phase. A role for the Pk isoform in the Late PCP Phase is supported by published data showing Pk is primarily active during a late phase of Fz PCP pathway activity and by the ability of Sple over-expression after the Early PCP Phase (induction at 15 h a.p.f.) to closely phenocopy a pkpk mutant (Doyle, 2008).

Over-expression of the Sple isoform has two separable effects during pupal wing development. During the Early PCP Phase (induction prior to 15 h a.p.f.), Sple over-expression causes a specific change in ridge orientation in both the anterior and posterior wing. It is proposed that this is due to the abundance of Sple isoform in the Early PCP Phase modifying the normal activity of Sple in organizing ridges across the wing. The reason for the specific change in ridge orientation is unclear, but since a similar phenotype is seen with uniform heat-shock induction as with both C765-gal4 and MS1096-gal4 wing drivers, it is unlikely to depend upon a specific Sple expression pattern. Over-expression of Sple in the Late PCP Phase (induction after 15 h a.p.f.) phenocopies a pkpk mutant, probably by displacing Pk from the Fz PCP pathway. In contrast, over-expression of the Pk isoform during pupal wing development affects both ridge formation and hair polarity, but does not produce consistent or easily interpretable phenotypes (Doyle, 2008).

If the Fz PCP pathway specifies hair polarity and ridge orientation simultaneously, it is expected that there will be a fixed relationship between hair and ridge orientation. The model suggests that ridges and hairs are only normally specified together in the anterior wing in the Late PCP Phase, where there is an approximately orthogonal relationship between hair polarity and ridge orientation. Therefore, it is thought that this orthogonal relationship is the normal outcome when ridges and hairs are organized by the same polarization event. It follows that whenever hair polarity and ridge orientation appear orthogonal on either a normal or mutant wing, this is indicative that both were organized by the same polarization event (Doyle, 2008).

An orthogonal relationship between hairs and ridges is seen across the entire pkpk mutant wing. According to this model, cell polarization occurs in the Early PCP Phase in a pkpk mutant and P-D ridges are specified. However, in the absence of the Pk isoform, cell polarization in the Late PCP Phase fails. Therefore, hairs will orient with respect to cell polarization in the Early PCP Phase and so are perpendicular to the P-D ridges specified during that Phase. A similar phenotype is seen when the Sple isoform is over-expressed after the Early PCP Phase (induction later than 15 h a.p.f.). In this case, it is thought that the abundance of Sple displaces the Pk isoform from the Fz PCP pathway again causing cell polarization in the Late PCP Phase to fail. When the Sple isoform is over-expressed during the Early PCP Phase (induction earlier than 15 h a.p.f.), ridge orientation is abnormal in both anterior and posterior wing. The abundance of Sple also causes a failure of polarization in the Late PCP Phase and so hairs are oriented perpendicular to the aberrant ridge orientation specified in the Early PCP Phase i.e. approximately reversed compared to wild-type (Doyle, 2008).

The variation of ridge orientation across the adult wing membrane presumably relates to wing function. It is well established that ability of the wing to deform specifically is vital for insect flight and that physical features of the wing (e.g. veins) provide the appropriate rigidity and flexibility. A ridged structure should make the wing membrane deformable parallel to the ridges, but resistant to folding perpendicular to the ridges. The A-P ridges in the Drosophila anterior wing run perpendicular to the local wing veins suggesting a rigid anterior structure, while the posterior ridges are approximately parallel to most local veins suggesting a more flexible wing structure. This conformation is the expectation for 'Type C' insect wings that are characteristic of true flies and have been described as having 'a well-supported leading edge and a soft flexible trailing edge'. This need for this varying flexibility across the Drosophila wing may have determined the specific orientation of ridges in different wing regions (Doyle, 2008).

Asymmetric homotypic interactions of the atypical cadherin Flamingo mediate intercellular polarity signaling

Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold or instructively as a signal is unclear. This study provides evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. It is proposed that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, bind each other to create cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry (Chen, 2008).

Nonclassical cadherins generally exhibit weak homophilic binding in vitro, raising the possibility that they regulate signaling rather than adhesion. Moreover, after cell-cell recognition, cadherins are thought to function either homophilically and symmetrically or heterophilically and asymmetrically between cells. This study shows that the atypical cadherin Fmi acts homophilically to communicate PCP signals between neighboring cells, yet its action is asymmetric, serving to link the accumulation of Fz on one cell boundary with Vang on the adjacent cell boundary, and vice versa. These data lead to the proposal of a model in which Fmi exists in two functional forms on opposite sides of intercellular borders, one of which selectively and cell-autonomously interacts with Fz (F-Fmi), and the other with Vang (V-Fmi). The native form of Fmi is V-Fmi, but upon interaction with Fz, V-Fmi is converted to F-Fmi. It is inferred that Fmi homodimers consist preferentially of opposite forms, thereby producing asymmetric function of the complex. By virtue of this mechanism, Fmi-mediated intercellular signaling communicates information about PCP protein asymmetry between neighboring cells (Chen, 2008).

How might Fmi achieve its homophilic yet asymmetric function? Although two splice forms exist, a single form can fulfill both V- and F-Fmi functions. A second possibility is that different stoichiometries of Fmi interact on opposite sides of the boundary -- for example, cis-dimers of Fmi might behave as V-Fmi whereas monomers function as F-Fmi. However, a reproducible proximal-distal difference in levels of tagged Fmi have not been detected when expressed in a mosaic pattern. A third possibility is that posttranslational regulation results in two distinct forms of Fmi that are selectively recruited or retained, directly or indirectly, by Fz or Vang. A fourth model is that V-Fmi and F-Fmi are alternate conformers or modified forms of Fmi where conversion of V-Fmi to F-Fmi depends on interaction with Fz. Although it is not possible to distinguish between the latter three possibilities, the finding that Fz and Fmi directly interact favors models in which Fz physically alters the properties of V-Fmi, thereby inducing the F-Fmi form. Extensive evidence shows that interacting proteins can modify the activity of cadherins. Of note, Xenopus Fz7, which mediates convergent extension during gastrulation, has been reported to directly bind a protocadherin through its extracellular domain. Detailed molecular and structural studies will be required to determine the precise nature of V-Fmi and F-Fmi and how they interact with Fz and Vang (Chen, 2008).

During PCP signaling, cells each receive a signal that orients polarization. Cells then consolidate this information by amplifying the asymmetry in a process that involves communicating and aligning polarity with surrounding cells. By signaling to a neighbor that a given cell boundary is enriched for either Fz or Vang, asymmetric Fmi homodimers transmit this information bidirectionally between cells. In the wild-type, amplification through feedback control is required to produce sharp differences between Fz and Vang levels on adjacent cell surfaces. Pk, Dgo, and Dsh are required for this amplification, though they are not required for intercellular signaling per se. The mechanism by which this amplification occurs is unknown, but the result is a mutual exclusion of Fz and Vang from a given region of the cell surface. Fmi therefore serves to link the action of feedback loops in neighboring cells, assuring a coordinated polarization (Chen, 2008).

It has been proposed that asymmetric placement of core PCP proteins is itself the signal that controls morphological polarization. However, an alternative model has been proposed in which the absolute, scalar value of Fz activity within each cell varies in a gradient across the tissue in response to an unidentified ligand. Scalar Fz levels are proposed to be refined by an averaging process between neighboring cells. According to this view, asymmetric PCP protein localization is only an epiphenomenon and is not required for function. It is suggested that several observations are inconsistent with this model. (1) The model predicts that the extent of domineering nonautonomy near fz mutant clones should vary according to position in the Fz activity gradient. However, its extent was reported to be equal throughout the abdomen, and no proximal-distal difference is evident in the fly wing. (2) FzΔCRD rescues polarity in fz null mutant flies despite deletion of the CRD, leaving little protein to which an extracellular ligand might bind (Chen, 2008).

In essence, the scalar Fz model argues for a Fz gradient across the tissue, whereas the asymmetric protein localization model invokes gradients of core PCP protein localization or activity within each cell but not across the tissue. As an additional test to distinguish between these two models, small wild-type islands of ~20 cells in size surrounded by fmi mutant cells were exsmained. These wild-type cells are prevented from communicating with and receive no repolarizing signal from the surrounding fmi mutant cells. The scalar Fz model predicts that these small wild-type islands should still be directly responsive to the proposed morphogen gradient and should therefore generate a normal Fz activity slope, resulting in normal polarity. In contrast, because the asymmetric PCP protein model posits only subcellular gradients of PCP protein activity but no tissue level gradient of Fz or other core PCP protein activity, each cell's tendency to align polarity with its neighbors could lead to other patterns of local alignment. Consistent with this latter possibility, prehairs in many of these islands exhibited PCP defects and formed swirling patterns. Because there is no discontinuity as one follows the polarity of cells in these islands, the scalar Fz activity model cannot accommodate this result without invoking an Escher's staircase of infinitely rising Fz levels. In contrast, the asymmetric protein model easily explains this result by organizing proximal and distal PCP protein domains in a spoke-like pattern between the cells of the swirl, as is indeed observed. In light of the evidence presented in this study that Fmi homodimers can instructively generate asymmetric Fz and Vang localization and locally align the polarity of neighboring cells, a model is favored in which instructive protein localization mediated by Fmi homodimers is itself the signal that transmits PCP information between cells. It is thought that this is the only known example of a cadherin homodimer providing dissimilar signals across intercellular boundaries (Chen, 2008).

Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation

Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. This study shows that in response to planar cell polarity (PCP) signals, Ras-related protein (Rala) modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. This study discovered a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, it was shown that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation (Cho, 2011).

The results presented support the model for Ral function in which Ral transcription is upregulated in response to Fz activation. As Fz is activated more in the equatorial cell than the polar cell, Ral is enriched in the equatorial cell. Ral activity represses ligand-independent Notch activation, and thus biases the equatorial cell to become R3. One way in which ligand-independent Notch activation occurs is an accident when normal Notch trafficking is disrupted. Notch receptor undergoes endocytosis and endosomal trafficking continually and mutations that block trafficking of late endosomes to the lysosome block Notch degradation and result in endosomal accumulation of Notch and ligand-independent activation. One possibility is that the endosomal environment promotes production of Nicd by Presenilin cleavage. Ligand-independent Notch activation may also occur normally in the lysosomal membrane. Ral GTPase activity might block ligand-independent Notch activation by regulating Notch trafficking to the lysosome, or by inhibiting another process, such as endosomal acidification, Nicd production or nuclear translocation. The punctate appearance of Ral protein suggests the possibility that Ral may play a role in endosomal trafficking. Although further experiments are required to determine the precise mechanism of Ral function, this study has shown that Ral, a protein that prevents ligand-independent Notch activation, is a target for regulation during pattern formation. Fz/PCP signaling upregulates Ral expression to ensure that ligand-independent Notch activation does not tip the scales in favor of pre-R3 becoming the signal receiving cell. Moreover, it was shown that prevention of ligand-independent Notch activation is not simply a constitutive process, but one that is modulated to ensure specific developmental outcomes (Cho, 2011).

Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). In this study, using an in vivo assay for function, it was shown that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, it was shown that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. It is deduced that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. It is proposed that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction (Struhl, 2012).

In Drosophila and other animals, including vertebrates, there appear to be at least two conserved genetic systems responsible for planar cell polarity (PCP); this study is concerned with the Stan system. In Drosophila, epithelial cells become polarised by a multicellular gradient of Fz activity. To read this gradient, the Stan system builds intercellular bridges of Stan-Stan homodimers that allow neighbouring cells to compare their levels of Fz activity. Under this hypothesis, Fz and Stan are essential components, as without Fz there is nothing to compare and without Stan there is no means to make comparisons. The Stan system also depends on a third protein, Vang, which appears to act in a complementary way to Fz. This study has dissected the function of these proteins by confronting adjacent cells of different fz, Vang and stan genotypes and assaying the effects on PCP. The main finding is that, even in the absence of Vang, Fz can function to polarise cells if it is present in at least one of the two abutting cells. By contrast, Vang has no detectable function when Fz is absent. Based on these and on other results, it follows that, at the core of the Stan system, intercellular bridges form between Stan on its own and Stan complexed with Fz (StanFz), and these act to polarise cells on both sides. It is concluded that Vang acts as an auxiliary component, helping Stan bridge with StanFz. Furthermore, it is posited that the numbers and disposition of asymmetric Stan<<StanFz bridges linking each cell with its neighbours are the consequence of the Fz activity gradient and serve to polarise the cell (Struhl, 2012).

A model has been built for how bridges between Stan and StanFz might determine the polarity of a cell (see The Stan system in PCP - a model). In the absence of Vang, expression of Fz in a sending cell can bias the polarity of a receiving cell that lacks Fz. Previous results indicate that within the receiving cell, Stan should accumulate only on the surface that faces the sending cell - because it is the only interface where it can form bridges with StanFz - and it is now proposed that it is this localised accumulation of Stan that biases the Vang- fz- receiving cell to make hairs on the other side, pointing away from the sending cell. A parsimonious hypothesis is that the apical membrane of each cell would have an unpolarised propensity to form hairs, and that an excess of Stan on one side locally inhibits this propensity, directing the production of hairs to the opposite side where there is least Stan. The response by a Vang- fz- cell eloquently suggests that the local accumulation of Stan bridged to StanFz in neighbouring cells is the main, and possibly the only, intracellular transducer of Stan system PCP (Struhl, 2012).

Next consider the finding that Vang functions in receiving cells to help Stan interact productively with StanFz in sending cells. In the key experiment Vang is added to just the Vang- fz- receiving cell: this cell is now more strongly polarised by StanFz signal coming from the sending cell. Thus, Vang can act in the same cell as Stan to help it receive incoming StanFz signal. The model also explains why the polarising effect of the Fz-expressing cell propagates only one cell into the fz- surround, even when Vang activity is restored to the receiving cells - as Stan-Stan bridges do not form, and/or do not function, between neighbouring cells that lack Fz (Struhl, 2012).

Last, consider the finding that cells lacking Fz can polarise cells devoid of Vang. In this case, only Stan<<StanFz and StanV<<StanFz bridges can form between the two cells, and as a consequence, only the StanFz form of Stan will accumulate on the surface of the Vang- receiving cell where it abuts the fz- sending cell. It is conjectured that Fz, when in a complex with Stan, acts to inhibit the normal action of Stan to block hair outgrowth. Therefore, the only place within the Vang- receiving cell where Stan can accumulate and block hair formation is on the far side, where it can form intercellular bridges with StanFz in the next Vang- cell. Accordingly, the receiving cell would be directed to make a hair on the near side, where it abuts the fz- sending cell. This reasoning also explains why the polarising effect of fz- sending cells on Vang- receiving cells appears to be limited mostly to the adjacent Vang- cell; because Stan<<StanFz and StanFz>>Stan bridges should form and/or function poorly between this cell and the next Vang- cell. Nevertheless, some imbalance between these two kinds of bridges probably does spread further than one cell; indeed fz- sending cells can polarise receiving cells up to two rows away in Vang- pupal wings (Struhl, 2012).

All the many other experiments fit with the simple model, in which Stan accumulates at the cell surface only where it can form intercellular bridges with StanFz, and each cell is polarised by differences in the amounts of Stan that accumulate along each of its interfaces with adjacent cells. Vang is not essential for these bridges, but by acting on Stan it helps them form and/or makes them more effective (Struhl, 2012).

How do wild-type cells acquire different numbers and dispositions of asymmetric bridges on opposite sides of the cell? In the Drosophila abdomen, in the anterior compartment of each segment, it has been argued that the Hh morphogen gradient drives a gradient of Fz activity. The slope of the vector of the Fz gradient would then be read by each cell via a comparison of the amount of Stan in its membranes. Within each cell, most Stan will accumulate on the cell surface that abuts the neighbour with most Fz activity, whereas most StanFz will accumulate on the opposite surface, where it confronts the neighbour with least Fz activity. This differential would then be amplified by feedback interactions both between and within cells. The result in each cell is a steep asymmetry in Stan activity that represses hair formation on one side, while allowing it at the other, directing all cells to make hairs that point 'down' the Fz gradient. The model differs in various and simplifying ways from the several and overlapping hypotheses published before. It makes Stan, rather than Fz, the main mediator of PCP, with differences in Fz activity between cells serving to regulate the local accumulation and transducing activity of Stan within cells (Struhl, 2012).

A central premise of this model is that morphogen gradients do not act directly on each cell to polarise Fz activity, but rather indirectly, by first specifying stepwise differences in Fz activity between adjacent cells. Such an indirect mechanism is favored for two reasons. First, PCP in much of the abdominal epidermis is organised by Hh, which is transduced primarily by its effects on the transcription factor Cubitus interruptus (Ci). It is difficult to understand how graded extracellular Hh could act directly - without cell interactions and only through the regulation of transcription - to polarise Fz activity within each cell. In addition, previous studies used temperature to drive tissue-wide gradients of transcription of a fz transgene under the control of a heat shock promoter; these studies nicely establish that cell-by-cell differences in Fz activity generated by transcriptional regulation are sufficient to polarise cells. Second, it has been previously shown that the polarising action of Hh depends on the Stan system. Specifically, cells in which the Hh transduction pathway is autonomously activated by the removal of the negative regulator Patched require Stan to polarise neighbouring cells. That result adds to evidence that graded Hh creates differences in Fz activity between cells - presumably via transcriptional regulation - that lead to asymmetries in Fz and Stan activities within cells. The target gene could be either fz itself or any other gene whose activity might bias the formation of Stan<<StanFz versus StanFz>>Stan bridges (Struhl, 2012).

Two previously developed staining experiments provide further support for this model with respect to Stan<<StanFz bridges. First, when Vang- clones are made in fz- flies (generating patches of Vang- fz- cells within fz- territory), a situation in which no Stan<<StanFz bridges can form, there is no accumulation of Stan near or at the border between the clone and the surround - and indeed this study now finds no polarisation of the fz- cells across the clone border. Second, and by contrast, when fz- clones are made in Vang- flies (generating patches of Vang- fz- cells within Vang- territory) Stan accumulates strongly along cell interfaces at the clone borders. Moreover, it is depleted from the cytoplasm of those cells of a clone that abut that border, indicating that Stan in Vang- fz- cells is accumulating at the apicolateral cell membrane where it can form stable intercellular Stan<<StanFz bridges. Previously, there was no evidence that this localisation of Stan within such Vang- fz- cells would polarise them. However, this study now shows that the Vang- fz- cells are polarised by their Fz-expressing neighbours and, also that the effect is reciprocal, their Fz-expressing neighbours are polarised in the same direction (Struhl, 2012).

The molecular mechanisms by which Fz and Vang control the formation and activity of Stan bridges remain unknown. Consistent with a direct action of Fz on Stan, both in vivo and in vitro studies suggest a physical interaction between the two proteins. Thus, Fz might act in a StanFz complex to regulate both the bridging and transducing activities of Stan. There is no comparable evidence in Drosophila for direct interactions between Vang and Stan. However, their mammalian counterparts have been shown to interact with each other (Devenport, 2008). But Drosophila Vang does interact directly with Pk, while a different Pk-related protein, Espinas, appears to interact directly with Stan during Drosophila neuronal development. Hence, Vang and Pk might form a cis-complex with Stan in epidermal cells, allowing Vang to act directly on Stan and help it form intercellular bridges with StanFz. Intriguingly, there is some evidence that Vang in one cell can interact directly with Fz in adjacent cells. Such an interaction might enhance the capacity of Stan to bridge with StanFz by providing an additional binding surface between the two forms of Stan. Alternatively, Vang might affect the formation or stability of Stan<<StanFz bridges indirectly, consistent with evidence implicating it in the trafficking of proteins and lipids to the cell surface. For example, evidence has been presented that any Stan or StanFz on the cell surface that is not engaged in Stan<<StanFz bridges is rapidly endocytosed and recycled to other sites on the cell surface. Vang activity could bias this process in favour of Stan, thereby enhancing its capacity to form bridges with StanFz (Struhl, 2012).

The results point to parallels between the Stan and Ds/Ft systems of PCP. First, both systems depend on the formation of asymmetric intercellular bridges between two distinct protocadherin-like molecules. For the Ds/Ft system, these are the Ds and Ft proteins themselves; for the Stan system, it is argued that these are two forms of Stan, either alone or in complex with Fz (StanFz). Second, morphogens may organise both systems by driving the graded transcription of target genes to create opposing gradients of bridging molecules. For the Ds/Ft system, at least two such target genes have been identified: ds itself and four-jointed (fj), a modulator of Ds/Ft interactions. For the Stan system, the existence of at least one such target gene induced by Hh is inferred. Third, for both systems, the two kinds of asymmetric bridges become distributed unequally on opposite faces of each cell, providing the information necessary to point all cells in the same direction. Thus for the Ds/Ft system, it is proposed that different amounts of Ds-Ft heterodimers would be distributed asymmetrically in the cell and this has been recently observed. Similarly, for the Stan system, there is plenty of evidence showing that Stan, Fz and Vang are unequally distributed within each cell. Finally, both systems have self-propagating properties: sharp disparities in Stan, Vang or Fz activity repolarise neighbouring cells over several cell diameters, even in the absence of the Ds/Ft syste, and the same is true of sharp disparities in Ds or Ft activity in the absence of the Stan system. Thus, the Stan and Ds/Ft systems may share a common logic that links morphogen gradients via the oriented assembly of asymmetric molecular bridges and feedback amplification, to cell polarisation (Struhl, 2012).

The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2

Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. This study reports a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and evidence is presented that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, it was shown that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. This study thus reveals a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements (Warrington, 2013).

Looking in three different tissues, this study found that core planar polarity pathway activity promotes E-cad turnover from junctions, most likely via local recruitment and regulation of RhoGEF2 and RhoA activity. In general terms, it is believed that local assembly or disassembly of adherens junctions through trafficking of E-cad is likely to be important for polarised tissue rearrangement; however, few specific contexts in which this occurs have been identified (Warrington, 2013).

One process in which regulation of E-cad turnover is strongly linked to cell intercalation is elongation of branches in the Drosophila embryonic tracheal system. Loss of core pathway function and also reduction of RhoGEF2 and RhoA activity give a similar phenotype to blocking endocytosis in this tissue, resulting in increases in both overall levels of and the stable fraction of E-cad at junctions, and a delay in cell intercalation. Consistent with the increase in E-cad levels being the cause of the intercalation defect in core pathway backgrounds, this phenotype can be suppressed by lowering E-cad gene dosage. It is speculated that core planar polarity proteins might transiently show polarised distribution or activity in this context, thus selectively weakening junctions and allowing cells to slide over one another. However, consistent with previous studies, this study failed to detect such asymmetry. The possibility cannot therefore be ruled out that core pathway activity is uniform within cells in this tissue, and only plays a role in general modulation of E-cad trafficking (Warrington, 2013).

In the pupal wing, the core pathway has already been linked to regulation of E-cad trafficking, and evidence has been presented that this promotes junctional remodelling that gives rise to a regular hexagonal arrangement of the cells. The exact mechanism by which the core pathway modulates E-cad trafficking was not defined, although the observation that Sec5 is recruited to proximodistal junctions suggested that there might be a role for local exocytosis of E-cad. Looking at a stage shortly after junctional remodelling, when the core proteins are strongly asymmetrically distributed, planar-polarised localisation of RhoGEF2 to proximodistal junctions is observed, but also a decrease in overall levels and the stable fraction of E-cad in this position. This appears to rule out a role for increased E-cad exocytosis on proximodistal junctions. Interestingly, although Sec5 is best characterised as a component of the exocyst, it has also been implicated in endocytosis in the Drosophila oocyte and perhaps this is also true in the wing. It is not clear how planar-polarised E-cad trafficking would contribute to formation of a regular hexagonal array of cells, as removing E-cad from proximodistal distal junctions might be expected to cause shrinkage of these junctions at the expense of anteroposterior junctions. However, during the peak period of junctional rearrangement (from ~18 hours of pupal life), the planar-polarised asymmetric distribution of the core proteins is largely lost, and so it might be that during the crucial stage of morphogenesis the core pathway promotes relatively uniform endocytosis of E-cad (Warrington, 2013).

The observation of a role for the core pathway in modulating E-cad turnover in the epidermis of the embryonic germband is particularly intriguing, as loss of core pathway activity does not result in a defect in embryonic germband extension, even though a planar-polarised distribution of E-cad has been implicated as a key mechanism in promoting cell intercalation in this context. It is speculated that planar polarisation of E-cad might be only one of a number of mechanisms that operate redundantly during the crucial developmental event of germband extension. Among other mechanisms reported are localised actomyosin contraction at vertical junctions, inhibition of Bazooka localisation on vertical junctions by local Rho kinase (Rok) activity and alteration of Arm (β-catenin) dynamics on vertical junctions by localised activity of the Abl kinase. Interestingly, it was found that loss of core pathway activity also abolishes Zipper and Bazooka asymmetry, but not Arm asymmetry (Abl or Rok asymmetry was not examined in this study). Additionally, a planar-polarised distribution of activated Src kinase to horizontal junctions was observed. Although Src kinase is a known modulator of E-cad trafficking in the Drosophila embryo, the significance of its planar-polarised distribution is unclear, as loss of core pathway function did not affect the distribution of Src, but did block the planar-polarised distribution of E-cad (Warrington, 2013).

Another context in which the core pathway might modulate E-cad turnover is during ommatidial rotation in the developing Drosophila eye, in which possible involvement of both RhoA and the kinase Nemo have been reported (Warrington, 2013).

In summary, this study has presented evidence that the core planar polarity pathway acts to locally promote E-cad endocytosis via local recruitment of RhoGEF2 and activation of RhoA activity. This represents a mechanism by which the core pathway can promote planar-polarised cell rearrangements (Warrington, 2013).

Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling

Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. This study reports instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. It was shown that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals (Carvajal-Gonzalez, 2016).

Taken together with observations that Fz/PCP signalling regulates basal body and cilia positioning in vertebrates (Song, 2010; Gray; 2011; Borovina, 2010), the current data on centriole positioning as a Fz/PCP readout in non-ciliated Drosophila wing cells indicate that centriole/MTOC (MT organizing centre)/basal body positioning is an evolutionarily conserved downstream effect of Fz/PCP signalling. Its link with actin polymerization (hair formation in Drosophila wing cells) suggests that actin polymerization effectors also affect cilia positioning, possibly through docking of the basal bodies to the apical membranes. Inturned, Fuzzy and Rho GTPases regulate apical actin assembly necessary for the docking of basal bodies to the apical membrane (Park, 2006; Pan, 2007) and this apical actin membrane accumulation is lost in Dvl1-3-depleted cells (Carvajal-Gonzalez, 2016).

In left-right asymmetry establishment of the Drosophila hindgut, which is not a Fz/PCP-dependent process, asymmetric centriole positioning is observed. During this so-called planar cell shape chirality process, which affects gut-looping and thus embryonic left/right asymmetry, centriole positioning is however still dependent upon actin polymerization downstream of Rho GTPases (Rac and Rho), via MyoD and DE-cadherin control. As Rho GTPases (Rac, Cdc42 and Rho) are downstream effectors of Fz-Dsh/PCP complexes, and their mutants cause PCP-like phenotypes including multiple cellular hairs or loss of hairs in wing cells. It is thus tempting to infer that both processes, planar cell shape chirality and Fz/PCP, regulate centriole positioning through a common Rho GTPase-mediated actin polymerization pathway, initiated by an upstream cellular communication system, although this assumption will require experimental confirmation. In the mouse, Fz/PCP signalling regulates cilia movement/positioning in cochlear sensory cells via Rho GTPase-mediated processes, suggesting a similar mechanism in a representative mammalian PCP model system. In conclusion, the positioning of centrioles appears to be a key and an evolutionary conserved downstream readout of Fz/PCP signalling, ranging from flies to mammals in both ciliated and non-ciliated cells (Carvajal-Gonzalez, 2016).

frizzled: Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

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