InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

The Drosophila 'tissue polarity' genes control the orientation of bristles and hairs in the adult cuticle. Mutant flies have the polarity of cells altered in the plane of the epithelium without gross changes in the overall shape of imaginal structures or the distribution of differentiated cell types within them. Other tissue polarity defects include mirror-image duplications of the tarsal joints, rotations of bristle sockets in the leg, and changes in ommatidial polarity. The tissue polarity genes have been divided into three groups (Wong. 1993). The type 1 genes [dishevelled (dsh), frizzled (fz), and prickle (pk)], affect the whole body surface and are therefore believed to directly establish tissue polarity (Shulman, 1998). In contrast, the type 2 [inturned (in) and fuzzy (fy) and type 3 [multiple wing hairs (mwh)] genes affect distinct subsets of body areas and are thought to interpret the polarity established by the type 1 genes (Gubb, 1999).

prickle and spiny legs (sple) were thought to be two separate, although closely linked genes. Mutations in sple roughen the eye and disrupt ommatidial polarity. pk affects tissue polarity but has no mutant eye phenotype. However, a pk-sple double mutant gives rise to a strongly roughened eye, suggesting that sple and pk act redundantly in eye development. Sections of sple and pk-sple mutant eyes show a disturbed ommatidial polarity with each ommatidium having the normal arrangement of photoreceptor cells. Together with fz, these polarity mutants can be categorized into two classes based on the adult eye phenotypes. One class, which consists of fz, pk-sple and dsh, exhibits all three aspects of polarity phenotype seen in fz adult eyes. Among them, pk-sple had the strongest eye phenotype. The other class, which consists of sple, exhibits only one aspect of the polarity phenotype seen in fz eyes. Although sple ommatidia are still arranged in antiparallel arrays and the equator is still detected, some of the ommatidia are oriented such that their R7 cells are pointing away, rather than towards the equator. Anti-Elav staining of sple eye discs reveals that the disorientation is due to incorrect direction of rotation as in fz mutants. The single mutant alleles pk^pk and pk^sple give the most extreme phenotypes, but in reciprocal regions of the body; pk^pk in the wing and notum and pk^sple in the legs, abdomen, and eyes. Complementation between these classes of allele indicates two subtly different functions at the pk locus (Zheng, 1995 and Gubb, 1999).

Thus loss-of-function lesions in alternatively spliced transcripts of prickle result in two genetically distinct phenotypes, Pk and Spiny-legs. The pk and sple transcripts encode proteins that contain three LIM motifs and a novel conserved domain that has been called PET (Prickle Espinas Testin). Surprisingly, deletion of the entire gene gives a phenotype that is much weaker than that of either the pk or sple single mutants. This unusual result can be explained by the pk and sple gene products acting in concert. The single-mutant phenotypes result from misactivation, rather than simply blocking, of a pathway of polarity formation. It is proposed that the correct balance of the Pk and Sple variants is required for normal planar polarity signaling in the Drosophila imaginal discs (Gubb, 1999).

To investigate the functional relationship between the Pk and Sple protein variants, mutant combinations that alter the ratio of the pk and sple transcripts were generated. The strongest phenotypes result from lack of either one of these two transcripts in homozygous mutants. When the single mutant alleles are combined with pk^pk-sple alleles or deficiencies, the resulting phenotypes are weaker than the corresponding homozygous single mutant phenotypes. Thus, flies that carry only a single functional copy of the sple transcript or the pk transcript, show an intermediate phenotype more similar to pk^pk-sple13 than the single mutant. These results imply that the presence of one transcript without the other (as in pk^pk or pk^sple single mutants) creates an extreme phenotype that is corrected when the dose of the remaining transcript is reduced. The implication is that both the relative and absolute levels of the Pk and Sple proteins are important for the function of putative Pk-Sple homomeric or heteromeric protein complexes (Gubb, 1999).

The importance of the levels of Pk and Sple expression and the balance between them has been investigated further with overexpression constructs. When driven by the expression of the uniform drivers gal4-da (daughterless) or gal4-C765, P[UAS:pk⁺] an extreme tarsal duplication phenotype results, including a duplicated socket structure in the proximal T1 segment. Overexpression of UAS:sple⁺ with these drivers gives a Pk phenotype in the wing triple row bristles; the tarsi, however, remain completely wild type. These results confirm that overexpression of pk gives a phenotype analogous to lack of the sple transcript, whereas overexpression of sple gives a phenotype similar to lack of pk. The da-UAS:pk⁺ and da-UAS:sple⁺ wing phenotypes show an unexpected feature that is not seen in mutant alleles. Instead of uniform polarity patterns, swirls are seen in the wing hair orientation with different patterns from wing to wing. In addition to phenotypes resembling loss of function, misexpression of pk in engrailed (en)-UAS:pk⁺ flies blocks the migration of wing hairs from the distal vertex of wing cells to the central position. This antagonism between the pk and sple transcripts implies either that both proteins compete for a target that is present in limiting amounts, or that they form protein complexes with distinct activities. In pk^pk and pk^sple mutants excess, homodimers would misactivate polarity signaling (Gubb, 1999).

Deletion of the entire prickle gene gives a phenotype over the whole body surface that is similar to dsh and fz. To test whether these mutants affect the same signaling pathway, double mutant combinations were made. Double mutants of pk^pk and dsh in the triple row give a Dsh phenotype, suggesting that dsh is epistatic to pk^pk. In other words, dsh function is blocked; pk function is irrelevant. These data suggest that many of the functions of pk require dsh. The situation within the wing blade is less clear, as the double mutants give an intermediate phenotype. The dsh; pk^sple double mutant retains a dsh phenotype in the triple row, but the wing hair pattern is altered, despite the fact that pk^sple alleles have no wing phenotype. In the leg, the dsh tarsal phenotype is not modified by pk^pk. There is a synergistic interaction between dsh and pk^sple, with the double mutant giving a more extreme mirror-image transformation of the T1 segment than either of the single mutants. This extreme transformation is also seen with UAS:pk⁺ overexpression and in fz (Gubb, 1999).

Clones of fz show a directional nonautonomy that has been interpreted to mean that fz mediates the intercellular communication of a polarity signal. On the other hand, dsh is strictly cell autonomous in clones, implying that it is involved in signal reception. To investigate whether pk participates in both reception and transmission of a polarity signal, pk^pk clones in the wing were studied. Large clones in the wing express the mutant polarity typical of that region of the wing in homozygous flies. There is an occasional nonautonomous disruption of polarity in wild-type cells adjacent to the proximal or lateral margins of a pk^pk clone. In these cases, a short range perturbation aligns wild-type cells with the mutant polarity pattern. There is no clear pattern to the position of such clones, but small peninsulas of pawn (pwn⁺) tissue surrounded by pk^pk pwn tissue tend to adopt the mutant polarity pattern. Smaller clones, induced later than 72-96 hr, do not alter the polarity of adjacent pwn⁺ tissue. In contrast to the autonomous behavior of pk, clones of fz tricornered (trc) cause long-range domineering nonautonomy both distal (Vinson, 1987) and lateral to the clone. Wild-type hairs are oriented toward the clone as though it is acting as a polarity 'sink'. Proximal cells are also directed toward the fz trc clone, but as this is the normal orientation for wing cells no polarity changes would be expected (Gubb, 1999).

There are a number of conserved features within the LIM domains of the pk family proteins. In the first LIM domain, there is a proline residue between the third (H) and fourth (C) zinc binding residues. This internal proline residue might introduce a kink within the zinc binding site. Similar LIMP domains with an internal P at this site are found in the human SLIM 2, SLIM 3, PINCH, Zyxin, and Paxillin proteins and LIM domain kinase 2 (LIK2) from chicken, mouse, rat, and human. In addition, the length of the consecutive LIM domains is constant in the pk family, with LIM1 containing 57 amino acids; LIM2 containing 52 aa, and LIM3 containing 56 aa. The implication is that the conservation of the triple LIM domain itself is important, rather than the individual LIM domains. In the case of the triple LIM domain protein Zyxin, the individual LIM domains may bind different target proteins and act as a template for the assembly of a number of structural components (Beckerle, 1997 and Gubb, 1999). A similar scaffold function for the Pk protein would be consistent with the cell autonomy of pk in clones and the expression of pk transcripts in cells that are changing shape. The blocking of the normal migration of the actin-rich prehair structure to the center of wing cells by overexpression of pk also implies a role in cytoskeletal remodeling. The lack of embryonic phenotype, despite the dynamic expression pattern, implies that pk function is redundant during embryonic development. A putative embryonic pk function could, in principle, be maternally supplied, but pk mutant strains of all three classes are fully fertile and homozygous pk embryos from homozygous mothers remain wild type. Similarly, gal4-da; P[UAS:pk⁺], and gal4-da; P[UAS:sple⁺] flies are completely viable and show no embryonic phenotype, despite the embryonic expression of the gal4-da driver (Gubb, 1999).

Perhaps the most surprising feature of the tissue polarity mutants is the precise polarity patterns seen in the wing hairs (Gubb, 1982). Rather than reflecting fine-grained positional information, however, the precision of the final pattern might be dependent on a tessellation mechanism. The orientation of the first cell would determine the alignment of subsequent cells, like sticking tiles on a bathroom wall. In pk^pk mutants, the alignment of wing hairs deviates progressively with occasional abrupt changes. In the adult mutant wing, hair polarity alters gradually with sudden topological discontinuities that resemble the stacking flaws in liquid crystals. In Df(2R)pk-30 wings, regions where the wing hairs are uniformly oriented retain a predominantly hexagonal array. Regions surrounding topological discontinuities, such as the anterior whorl, show irregular cell shapes frequently associated with duplicated wing hairs (Gubb, 1999).

Strong support for a tessellation mechanism is given by the overexpression phenotype of Pk and Sple when driven by a ubiquitous promotor. Although polarity patterns are variable, hair orientation alters smoothly from cell to cell across the wing surface, indicating that the polarity of cytoskeletal structures is aligned within large fields of cells. It is as if cell packing had nucleated randomly and then spread to neighboring cells until meeting an adjacent domain. The short-range perturbation in polarity that is occasionally seen proximal and lateral to a pk^pk clone is consistent with mechanical adjustment of cells to fit against their immediate neighbors, unlike the long-range domineering nonautonomy lateral and distal to fz clones. With both classes of clones, a tessellation mechanism might impose a threshold. Below this threshold, disruptions in polarity signaling would fail to affect the orientation of neighboring cells (Gubb, 1999).

The tarsal phenotype of dsh is very similar to pk^pksple13 causing duplications of the T3 and T4, segments. A more extreme phenotype including complete duplications of T2 to T4 together with a well-developed ectopic T1 joint are seen in dsh; pk^sple, overexpression UAS:pk⁺ and fz mutant strains, implying that a similar polarity signaling pathway is affected. It has been suggested that pk and fz are upstream in a signaling pathway leading to dsh (Wong, 1993 and Shulman, 1998). There are several problems with a simple linear pathway, however, and the relationship between pk and fz is unclear. (1) Neither fz nor pk is clearly epistatic to the other; rather, the double mutant (pk^pk; fz) phenotype is intermediate between pk^pk and fz (Gubb, 1982 and Wong, 1993). (2) Whereas fz clones cause a long-range nonautonomous disruption in surrounding tissue, pk^pk clones resemble dsh clones in being almost completely cell autonomous. (Both pk^sple1 and pk^pk-sple13 clones are cell autonomous in the eye. In contrast, the pk^pk triple row bristle phenotype (that results from Sple expression in the absence of Pk) is suppressed in dsh; pk^pk wings and the dsh polarity pattern is modified in dsh; pk^sple wings (despite pk^sple on its own not having a wing phenotype). (3) Complete lack of pk transcripts produces a phenotype very similar to dsh. Taken together, these results indicate that Pk is not downstream of Fz but may represent an alternative input into Dsh-mediated planar polarity signaling. If Dsh is acting as a scaffolding or adapter protein then it would be required in stoichiometric ratios to its target proteins, and overexpression would prevent assembly of functional complexes. It may be that the Pk protein isoforms are components of this protein complex that are expressed in cells remodeling their cytoskeletal architecture (Gubb, 1999).

Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity

Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, this study found that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification (Olofsson, 2014).

A model is proposed for coupling Ft/Ds/Fj to the core module. Gradients of Fj and Ds, by promoting asymmetric distribution of Ft/Ds heterodimers, align a parallel network of apical MTs. Vesicles containing Dsh are transcytosed towards MT plus-ends. In the presence of Pk, MT plus-ends are biased towards the high end of the Fj gradient and the low end of the Ds gradient, whereas in the presence of Sple, the MT plus-ends are biased towards areas with low levels of Fj and high levels of Ds expression. Predominance of Pk or Sple, therefore, determines how tissues differentially interpret, or rectify, the Ft/Ds/Fj signal to the core module. It is hypothesized that this signal serves to both orient the breaking of initial symmetry and to provide continual directional bias throughout polarization. Additional validation of this model would require the measurement of Eb1::GFP comet directions while controlling Pk-Sple isoform expression in wings bearing ectopic Ds and Fj gradients, an experiment that is beyond the technical capabilities with currently available reagents. However, further evidence in support of this model is found in the observation that, in Pk-predominant wings, MT polarity and hair polarity point from regions with high toward low Ds expression both in wild-type wings and in wings with ectopic reversed Ds gradients (Olofsson, 2014).

It is noted that the distal plus-end bias of MTs is seen in much of the wild-type wing, but this bias decreases to equal proximal-distal plus-end distribution near to the most distal region of the wing. Thus, the mechanism described in this study might not affect the entirety of the wing; in contrast, plus-end bias was observed across the entire A-abd compartment (Olofsson, 2014).

A model incorporating early Sple-dependent signaling and late Pk-dependent signaling has been proposed to explain PCP in the wing. The current observations and model are compatible with the data presented in support of that model; Sple expression, although always lower than Pk expression in wild-type wing, declined during pupal wing development, suggesting that, in pk mutants, polarity patterns might be set early in development, when Sple is still expressed and when Ds is present in a stripe through the central part of the wing, giving rise to anteroposterior oriented patterns (Olofsson, 2014).

Pk (and presumably Sple, in Sple dependent compartments) is required for amplification of asymmetry by the core PCP mechanism (Tree, 2002; Amonlirdviman, 2005). These results indicate an additional, core module independent, function for these proteins in regulating the polarity of MTs. Furthermore, although the core function of Pk-Sple is not well defined, part of that function might include promoting the formation and movement along aligned apical microtubules of Fz-, Dsh- and Fmi-containing vesicles (Shimada, 2006). The relative abundance of transcytosing vesicles in Pk versus Sple tissues suggests that if Sple promotes MT-dependent trafficking, it does so less efficiently than Pk (Olofsson, 2014).

These activities are remarkably similar to those that have been recently identified for Pk and Sple in fly axons, where Pk promotes or stabilizes MT minus-end orientation towards the cell body, and Sple promotes the orientation of minus-ends toward the synapse, which has effects on vesicle transport and neuronal activity. A common mechanism of differentially adapting the plus- and minus-ends of MT segments is proposed in both instances. In axons, similar to what was observed in this study, Pk also facilitates more robust cargo movement, whereas movement is less efficient when Sple is the dominantly expressed isoform. Furthermore, MT polarity defects might underlie the apical-basal polarity defects and early lethality of mouse prickle1 mutant embryos. As Ft and Ds are not known to regulate MTs in axons, these observations suggest that Pk and Sple are able to modify MT polarity independently of Ft/Ds. However, in wings, a consequence is only evident if MTs are first aligned by Ft/Ds activity (Olofsson, 2014).

How Pk and Sple modulate the organization of MTs remains unknown, but possibilities include modifying the ability of Ft or Ds to capture or nucleate MTs, or altering plus-end dynamics to inhibit capture. These data also suggest the possibility of a more intimate link between the core PCP proteins and Ft/Ds than has been appreciated previously. Other concurrent signals, such as that proposed for Wnt4 and Wg at the wing margin, cannot be ruled out. However, the observations that (1) MTs correlate with the direction of core PCP polarization over space and time, (2) vesicle transcytosis is disrupted in ft clones in which MTs are randomized, (3) chemical disruption or stabilization of MTs disturbs polarity and (4) Pk and Sple isoform predominance rectifies signal interpretation by the core module in a fashion that follows both the wild-type and ectopic Ds gradients provide additional evidence for the model that a signal from the Ft/Ds/Fj system orients the core PCP system in substantial regions of the wing and abdomen (Olofsson, 2014).

Rapid disruption of Dishevelled activity uncovers an intercellular role in maintenance of Prickle in core planar polarity protein complexes

Planar polarity, the coordinated polarization of cells in the plane of a tissue, is important for normal tissue development and function. Proteins of the core planar polarity pathway become asymmetrically localized at the junctions between cells to form intercellular complexes that coordinate planar polarity between cell neighbors. This study combined tools to rapidly disrupt the activity of the core planar polarity protein Dishevelled, with quantitative measurements of protein dynamics and levels, and mosaic analysis, to investigate Dishevelled function in maintenance of planar polarity. Mechanistic insight is provided into the hierarchical relationship of Dishevelled with other members of the core planar polarity complex. Notably, it was shown that removal of Dishevelled in one cell causes rapid release of Prickle into the cytoplasm in the neighboring cell. This release of Prickle generates a self-propagating wave of planar polarity complex destabilization across the tissue. Thus, Dishevelled actively maintains complex integrity across intercellular junctions (Ressurreicao, 2018).

Defining the roles of individual components in signaling networks can be a significant challenge. This is particularly so when the network is not a simple linear pathway, if components play more than one role in the cell (pleiotropy), and if there is 'adaptation' such that over time, the pathway adjusts to the effects of experimental manipulations. However, in many cases, these difficulties can be bypassed through methods that rapidly alter protein activities (Ressurreicao, 2018).

Consistent with this, it has been recently shown that spatiotemporal activation of gene expression is an effective tool for dissecting feedback interactions during planar polarity patterning in the Drosophila wing (Warrington, 2017). The current work used methods for rapidly disrupting protein function to probe the role of the Dsh protein in planar polarity. The main finding is that Dsh regulates Pk membrane association in core planar polarity complexes, acting cell-non-autonomously to prevent its relocalization to the cytoplasm. Notably, this role for Dsh is specifically revealed when Dsh is rapidly depleted from core protein complexes but not in the simple dsh loss-of-function situation, when instead a largely mobile fraction of Pk is seen associated with cell junctions. It is speculated that a Dsh-dependent signal normally passes between cells via the core protein complexes to maintain Pk recruitment. When this signal is disrupted, Pk rapidly leaves the junctions. However, in the long-term absence of Dsh, Pk can return to cell junctions, where it is speculated to weakly associates with cell membranes by virtue of being prenylated (Ressurreicao, 2018).

What might be the nature of the intercellular signal from Dsh to Pk? It is suggested that it passes via the Fmi homodimers that form between cells, as numerous lines of evidence indicate these are essential for cell-cell signaling in planar polarity. A simple possibility is that Dsh binding to Fz induces a conformational change in the complex that passes via the Fmi homodimers to alter the conformation of bound Stbm, thus creating a Pk binding site. Such molecular signaling events mediated by allostery are common features of ligand-receptor interactions. In support of the model that the Dsh signal is transduced via a change in Fz behavior, it is noted that following Dsh disruption, Fz distribution and stability is altered faster than those of Fmi and Stbm (Ressurreicao, 2018).

A related mechanism is suggested by recent observations that the core proteins incorporate into intercellular complexes non-stoichiometrically and that all components contribute to complex stability. These findings are interpreted as suggesting that core complex stability is dependent on a phase transition mediated by multivalent interactions between the core proteins, with Dsh playing a critical role. The rapid destabilization of Fz after Dsh depletion may be a result of loss of multivalent binding interactions mediated between the different domains of Dsh, as also occurs in Wnt signalosome assembly, over time leading to a reduction in multivalent binding interactions between Fmi and Stbm. This would thus produce a gradual 'loosening' of the complex that would result in release of Pk from its binding interactions with Fmi and Stbm. Some support for this model comes from the observation that super-resolution microscopy immediately after Dsh disruption shows subtle changes in the size and distribution of Fmi in junctional puncta (Ressurreicao, 2018).

A striking observation is that if Dsh fails to maintain Pk recruitment in intercellular complexes, free Pk can destabilize Dsh in the same cell, leading to release of Pk in the neighboring cell and a wave of core planar polarity complex destabilization. This observation both supports previous work showing that physiological levels of Pk can effectively destabilize Fz-Dsh complexes at cell junctions (Warrington, 2017) and highlights the importance of sequestering Pk into 'proximal' complexes, to prevent unregulated activity of Pk within the cell. It is proposed that Fmi mediates essential intercellular signals from Fz-Dsh in 'distal' core complexes that actively maintain Pk in 'proximal' core protein complexes. In turn, this promotes the effective segregation of distal and proximal complexes to opposite cell ends, driven in part by destabilizing feedback interactions between Pk and Dsh in the same cell (Ressurreicao, 2018).

Reciprocal action of Casein Kinase Iepsilon on core planar polarity proteins regulates clustering and asymmetric localisation

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. This study shows that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled - which normally localises to opposite cell edges to Strabismus - reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iepsilon. It is concluded that Casein Kinase Iepsilon phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges (Strutt, 2019).

Phosphorylation is a widespread means of controlling protein activity, regulating protein-protein interactions, protein stability and conformation. The activity of most signalling pathways is regulated by phosphorylation of pathway components. This includes the 'core' planar polarity pathway: however, compared to other signalling pathways, the molecular mechanisms are poorly understood (Strutt, 2019).

The core planar polarity proteins (hereafter, the 'core proteins') regulate the production of polarised structures or polarised cell behaviours in the plane of a tissue. This includes polarised production of cilia and of stereocilia bundles in the inner ear, and the coordinated polarisation of tissue movements necessary for convergence and extension of the body axis. In Drosophila, the core pathway controls the production of polarised hairs and bristles on many adult tissues, for example the trichomes that emerge from the distal edge of each cell in the adult wing (Strutt, 2019).

The core pathway specifies polarised structures via the asymmetric localisation of pathway components. In the Drosophila pupal wing, the seven-pass transmembrane protein Frizzled (Fz), and the cytoplasmic proteins Dishevelled (Dsh) and Diego (Dgo) localise to distal cell ends, where the trichome will emerge. The four-pass transmembrane protein Strabismus (Stbm, also known as Van Gogh [Vang]) and Prickle (Pk) localise to proximal cell ends, and the atypical cadherin Flamingo (Fmi, also known as Starry Night [Stan]) localises to both proximal and distal cell ends (see Planar polarity and the cloud model of core protein localisation). Fmi mediates homophilic adhesion that is important for coupling polarity between cells (Strutt, 2019).

The overall direction of polarisation is determined by tissue-specific global cues. Polarity is then thought to be refined and amplified by feedback interactions between the core proteins. Mathematical modelling has suggested that feedback may involve destabilisation of complexes of opposite orientation and/or stabilisation of complexes in the same orientation. This can lead to sorting of complexes such that they all align in the same direction (Strutt, 2019).

With regard to possible stabilising mechanisms, core protein asymmetry is associated with clustering of proteins into punctate membrane subdomains and reduced core protein turnover. Based on a detailed study of core protein organisation in puncta, it was recently proposed that core proteins form a non-stoichiometric 'cloud' around a Fmi-Fz nucleus. Feedback interactions lead to sorting of complexes, and multiple protein-protein interactions are thought to promote a phase transition into higher order 'signalosome-like' structures, where arrays of complexes of the same orientation are stabilised. Interestingly, Stbm stoichiometry was found to be much higher than that of the other core proteins. The reasons for this are unclear, but could relate to a role for Stbm in promoting higher order structures. Furthermore, Pk may stabilise Stbm by promoting complex clustering (Strutt, 2019).

Mechanisms of destabilisation may include competitive binding between core proteins. More specifically, Pk (a 'proximal' complex component) is known to destabilise Fz and/or Dsh ('distal' components) in the same cell. In addition, Pk has been suggested to destabilise complexes containing Stbm and Fmi. However, knowledge of additional molecular mechanisms by which core proteins might become destabilised or clustered together is very poor, and post-translational modifications such as phosphorylation are likely to be a key element (Strutt, 2019).

Indeed, core protein phosphorylation is essential for feedback amplification of asymmetry. In particular, reduced activity of Casein Kinase Iε (CKIε, also known as Discs Overgrown [Dco] or Doubletime [Dbt] in flies) causes planar polarity defects and a reduction in core protein asymmetry. Interestingly, CKIε has been implicated in phosphorylation of both Stbm and Dsh. CKIε was first found to bind to and phosphorylate the vertebrate Dsh homologue (Dvl) in canonical Wnt signalling. In planar polarity in flies, Dsh phosphorylation correlates with its recruitment to cellular junctions by Fz, where it is incorporated into stable complexes, and decreased Dsh phosphorylation is seen in &dco; mutants (Strutt, 2019).

The exact phosphorylation sites for CKIε in Dsh/Dvl are not well defined, but a mutation of a serine/threonine-rich region upstream of the PDZ domain affects Dvl recruitment to membranes in Xenopus. Moreover, mutation of one of these residues (S236 in fly Dsh) blocks phosphorylation of Dsh by Dco in vitro. However, a transgene in which these residues were mutated largely rescued the planar polarity defects of dsh mutants in the adult fly wing (Strutt, 2019).

More recently, CKIε has been implicated in phosphorylating Stbm and its vertebrate homologue Vangl2. In particular, Wnt gradients were proposed to lead to a gradient of Vangl2 phosphorylation and asymmetry in the vertebrate limb. CKIε promotes Stbm/Vangl2 phosphorylation in cell culture. Two clusters of conserved serine and threonine residues were identified as CKIε phosphorylation sites. Mutation of some or all of these residues leads to a loss of Stbm/Vangl2 phosphorylation in cell culture, and defects in planar polarisation (Strutt, 2019).

The fact that CKIε has been implicated in phosphorylating both Stbm/Vangl2 and Dsh/Dvl in cell culture leads to the question of whether both proteins are bona fide targets in vivo. For instance, both Fz and Dsh/Dvl have been proposed to promote Stbm/Vangl2 phosphorylation by CKIε. Thus, it is possible that only Stbm/Vangl2 are direct targets of CKIε and that Stbm/Vangl2 phosphorylation has a secondary effect on Fz-Dsh/Dvl behaviour. Moreover, mechanistic insight into how these phosphorylation events affect core protein sorting and asymmetry is lacking (Strutt, 2019).

This study demonstrates that CKIε has independent and reciprocal actions on Dsh and Stbm during planar polarity signalling in Drosophila. This study used phosphorylation site mutations in Stbm to show that lack of Stbm phosphorylation leads to its clustering in 'mixed' puncta that contain complexes in both orientations. CKIε-dependent phosphorylation increases Stbm turnover at junctions, and thus promotes complex sorting, while phosphorylation of Dsh decreases its turnover. Pk negatively regulates Stbm phosphorylation and increases Stbm stability. These results support a direct role for Dco in phosphorylating both Stbm and Dsh in vivo in planar polarity signalling (Strutt, 2019).

This paper describes a dual role for CKIε/Dco kinase in regulating planar polarity in the fly pupal wing. In the first case, Dco promotes phosphorylation of Stbm. Stbm phosphorylation acts as a switch, changing Stbm from a stable immobile form that can enter junctional complexes, to an unstable mobile form that can redistribute within cells. Inhibiting Stbm phosphorylation causes an increase in Stbm stability at junctions that prevents sorting of complexes: thus complexes are 'locked' in an unsorted state. In contrast, hyperphosphorylation of Stbm destabilises Stbm, allowing it to leave junctions, hence permitting complex sorting. A second role for Dco is to mediate Dsh phosphorylation, which increases Dsh localisation at junctions. Significantly, the effects of Dco on Dsh are independent of Stbm and vice versa (Strutt, 2019).

In the 'cloud model', it is envisaged that multiple binding interactions drive a phase transition from a loosely packed, disordered association of core proteins in non-puncta, towards a highly cross-linked array of complexes within puncta that are all aligned in the same orientation. Stbm is well-placed to be a key component driving such a clustering mechanism, as not only can it multimerise with itself, but it also has a high stoichiometry within junctions. Also consistent with a role for Stbm in complex clustering is the observation that Stbm phosphorylation site mutants act as dominant negatives, recruiting wild-type Stbm into non-polarised puncta. Phosphorylation may inhibit a clustering mechanism, due to an increase in negative charge (Strutt, 2019).

Interestingly, excess clustering of unphosphorylated Stbm in unsorted complexes is also expected to lead to destabilising feedback interactions with the other core components. When Stbm is unphosphorylated, the increase in Stbm stability is sufficient for Stbm to 'win' over Fmi and Fz. Thus, there is an overall increase in Stbm stability in phosphomutant Stbm puncta, that is accompanied by decreased stability of Fmi and Fz (Strutt, 2019).

Pk both promotes Stbm stability and reduces its phosphorylation. A role for Pk in increasing Stbm stability is not surprising, as overexpression of Pk is known to cause excess clustering of the core proteins. A number of mechanisms can be envisioned by which Pk could affect Stbm phosphorylation. A previous study provided evidence that Pk has two roles: firstly, it acts via Dsh to destabilise Fz in the same cell (see Model for how Pk and phosphorylation of Stbm regulate complex sorting and clustering); secondly, it acts via Stbm to stabilise Fz in adjacent cells. In the first case, Pk would promote sorting of complexes, and one possibility is that Stbm is inaccessible to the kinase in sorted complexes, and thus Pk is indirectly reducing Stbm phosphorylation by promoting sorting. Arguing against this, loss of fz or dsh also abolishes core protein asymmetry, but no hyperphosphorylation is seen. The boundary FRAP experiments instead support Pk acting directly in the same cell to stabilise Stbm. A mechanism is therefore proposed whereby direct binding of Pk to Stbm protects Stbm from phosphorylation (Strutt, 2019).

Interestingly, Stbm has a significantly higher stoichiometry within junctions than Pk. One possibility is that Stbm forms multimers, and that association of Pk with these multimers causes a conformational change that reduces accessibility to kinase-binding sites. Alternatively, Pk might recruit a phosphatase (albeit no candidates for such a phosphatase are known). The reduced negative charge might then allow Stbm to form higher order structures, which promotes clustering of the entire core protein complex into puncta (Strutt, 2019).

Puncta formation in both wild-type and phosphomutants is also dependent on Dsh. Dsh is another a good candidate for promoting clustering as it too can multimerise, and thus puncta formation may be dependent on clustering on both sides of the complex. Moreover, direct interactions between Stbm and Dsh may promote clustering of unsorted complexes in the absence of phosphorylation (Strutt, 2019).

Feedback models for core protein asymmetry suggest that particular components of the core pathway signal to other components to either stabilise or destabilise them. An attractive model would be that Fz or Dsh recruits a kinase which phosphorylates Stbm and destabilises complexes of the opposite orientation. Consistent with this, a proportion of Dco localises to apicolateral junctions in pupal wings. However, no change was seen in Stbm phosphorylation in fz or dsh mutants, nor are Fz and Dsh required for the hyperphosphorylation of Stbm seen in pk mutants. Therefore, it is concluded that Stbm phosphorylation is more likely to be constitutive. Such constitutive phosphorylation would be sufficient to keep Stbm mobile and allow complex sorting; and Pk would then counterbalance this and promote complex stability. The balance between Stbm phosphorylation/complex mobility and Pk binding (leading to reduced Stbm phosphorylation) would resolve over time towards a more stable state as complexes segregate to opposite cell edges (Strutt, 2019).

It is noted that in normal development, Stbm downregulates Pk levels. This suggests Pk levels are finely tuned, in order to prevent unrestrained clustering (as seen when Pk is overexpressed) (Strutt, 2019).

Evidence is also provided that Dco regulates Dsh phosphorylation and junctional levels independently of Stbm. These findings are consistent with previous observations that Dsh phosphorylation correlates with its recruitment by Fz into junctional complexes. The mechanism by which Dsh phosphorylation acts in planar polarity remains to be elucidated, but the data show that &dco; overexpression phenotypes are suppressed by reduced dsh gene dosage, and that Dsh phosphomutants have reduced core protein asymmetry in pupal wings. Furthermore, a small but significant decrease in Dsh stability at junctions is observed in Dsh phosphomutants. Overall, these data are consistent with a model in which phosphorylation of Dsh promotes its stable association at junctions (Strutt, 2019).

In summary, it is proposed that Dco regulates the asymmetric localisation of the core proteins by reciprocal actions on Stbm and Dsh. Dco regulates Stbm phosphorylation and turnover and causes it to leave junctions, while phosphorylation of Dsh by Dco promotes its junctional association (Strutt, 2019).

Prickle isoforms determine handedness of helical morphogenesis

Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. This paper describes a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. The Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, it was shown that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. Evidence is provided that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pk(pk) or Pk(sple) predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes (Cho, 2020).

Work in Drosophila indicates that at least two molecular modules contribute to PCP signaling. The core module acts both to amplify molecular asymmetry, and to coordinate polarization between neighboring cells, producing a local alignment of polarity. Proteins in the core module, including the serpentine protein Frizzled (Fz), the seven-pass atypical cadherin Flamingo (Fmi; a.k.a. Starry night), the 4-pass protein Van Gogh (Vang; a.k.a. Strabismus), and the cytosolic/peripheral membrane proteins Dishevelled (Dsh), Diego (Dgo), and the PET/Lim domain protein Prickle (Pk) adopt asymmetric subcellular localizations that predict the morphological polarity pattern such as hairs in the fly wing. These proteins communicate at cell boundaries, recruiting one group to the distal side of cells, and the other to the proximal side, through the function of an incompletely understood feedback mechanism, thereby aligning the polarity of adjacent cells. A global module serves to provide directional information to the core module by converting tissue level expression gradients to asymmetric subcellular Fat (Ft) / Dachsous (Ds) heterodimer localization. The atypical cadherins Ft and Ds form heterodimers which may orient in either of two directions at cell-cell junctions. The Golgi resident protein Four-jointed (Fj) acts on both Ft and Ds as an ectokinase to make Ft a stronger ligand, and Ds a weaker ligand, for the other. Graded Fj and Ds expression therefore result in the conversion of transcriptional gradients to subcellular gradients, producing a larger fraction of Ft-Ds heterodimers in one orientation relative to the other. Other less well defined sources of global directional information appear to act in partially overlapping, tissue dependent ways (Wu et al., 2013; Sharp and Axelrod, 2016) (Cho, 2020).

Various Drosophila tissues depend primarily on either of two isoforms of Pk, Prickle^prickle (Pk^pk) and Prickle^spiny-legs (Pk^sple). These isoforms have been proposed to determine the direction in which core PCP signaling responds to directional information provided by the Ft/Ds/Fj system. Pk^sple binds directly to Ds, orienting Pk^sple-dependent core signaling with respect to the Ds and Fj gradients, while Pk^pk-dependent core signaling has been proposed to couple less directly through a mechanism in which the Ft/Ds/Fj module directs the polarity of an apical microtubule cytoskeleton on which vesicles containing core proteins Fz, Dsh and Fmi undergo directionally biased trafficking. Others, however, have argued that Pk^pk-dependent core signaling is instead uncoupled from the Ft/Ds/Fj signal (Cho, 2020).

The most intensively studied morphogenetic responses to PCP signaling in Drosophila occur in epithelia such as wing and abdomen, in which cellular projections called trichomes (hairs) grow in a polarized fashion from the apical surface, and in ommatidia of the eye, in which photoreceptor clusters achieve chirality via directional cell fate signaling. Chaete (bristles), which serve as sensory organs, are also polarized by PCP signaling. Bristles comprise the 4-5 progeny of a sensory mother cell (SMC), one of which, the shaft, extends a process above the epithelium such that it tilts in a defined direction with respect to the tissue. In mechanosensory microchaete on the notum, one daughter of the SMC divides to produce the shaft and a socket cell that surrounds the shaft where it emerges from the epithelial surface; the other SOP daughter divides to produce a glial cell, a sheath cell and a neuron. Studies of microchaete polarity have shown that the initial division of the SMC is polarized by PCP in the epithelium from which it derives, such that the two daughters are born in defined positions with respect to each other. However, subsequent events that ultimately determine the direction of shaft polarity have not been described (Cho, 2020).

This study bristle chose polarity on the anterior margin of the wing (AWM) for study. A row of stout mechanosensory bristles and a row of curved chemosensory bristles are on the dorsal surface, and a mixed row of mechano and chemosensory bristles is on the ventral side. All of these bristles tilt toward the distal end of the wing in wildtype. In pk^pk mutant wings, and in wings overexpressing Pk^sple, a large fraction of the AWM bristles point proximally rather than distally; the pk^pk phenotype is suppressed by mutation of dsh, implicating the core PCP signaling mechanism in this process. However, the morphogenetic process resulting in polarity and the genetics of its apparent control by PCP signaling have not been explored. Here, focusing on the dorsal mechanosensory bristles, this report describes analysis of the underlying morphogenesis leading to AWM bristle polarization and shows that polarization results from a corkscrew-like helical morphogenetic process involving the shaft and socket cells. Furthermore, the results reveal how interplay between Pk^pk and Pk^sple control the handedness of the helical growth, and how the Ft/Ds/Fj system directs it in opposite orientations depending on whether the core PCP mechanism operates in a Pk^pk- or Pk^sple-dependent mode (Cho, 2020).

Producing structures of defined chirality requires directional information on three Cartesian axes. The results of this study indicate that in determining bristle chirality, PCP provides directional information along the proximal-distal axis. The apical-basal axis is defined by the epithelium, while the dorsal-ventral axis is likely defined by the dorsal-ventral compartment boundary (Cho, 2020).

This study has shown that the polarity of wing margin bristles (proximal or distal tilt) is determined by controlling the handedness of helical growth. The entwined shaft and socket cells undergo an apical clockwise or counterclockwise rotation that results in a left-handed or right-handed helical structure, placing the shaft to the proximal (wildtype) or distal (pk^pk mutant) side of the socket cell. The direction of rotation depends on PCP signaling among and between margin and socket cells. Helical cellular structures of defined handedness, such as the bristles resulting from properly directed rotation, have been noted in bacterial and plant species, but few examples have been described in animals (Cho, 2020).

The entwined twisting of the shaft and socket is a coordinated morphogenetic event, and the apparent stereotyped junctional rearrangement of additional margin cells suggests that at least some other cells are involved as well. It is not known in which cell or cells mechanics are regulated to drive this morphogenesis. One possibility is that an internal cytoskeletal mechanism induces the helical growth of the socket and/or shaft cells. Another possibility is that the side of the socket cell crescent marked by Pk at 24 hr is anchored, while the other side grows to wrap around the shaft, inducing junctional rearrangements and propelling the rotation of the apical portion of the shaft relative to the socket cell. Apical rotation could then cause twisting of the more basal portions of the socket cell, and could in turn direct the shaft to the corresponding side of the socket cell (Cho, 2020).

The precise location at which the PCP signal is required to determine rotation direction is unclear. Because asymmetric core complexes were observed at margin-socket cell junctions, but very little at shaft-margin or shaft-socket junctions, it is hypothesized that interaction between the socket and surrounding margin cells is the essential determinant of rotation. PCP proteins at these junctions could control junctional dynamics, as is known to occur in other systems. This will require further investigation (Cho, 2020).

Characterization of prickle isoform-specific pk^pk1 and pk ^sple1 mutations in Drosophila melanogaster

Paired-end next generation sequencing (NGS) was used to characterize the classic isoform-specific pk^pk1 and pk^sple1 mutations of the prickle gene in Drosophila melanogaster. Evidence is provided that these previously reported null mutations are caused by either a tirant transposon insertion into the 5' UTR of pk^pk1 or a premature stop codon in the second exon of pk^sple1. Additional likely benign missense mutations were identified in both mutant isoforms (Lilienthal, 2022).

The prickle gene in Drosophila melanogaster has been studied for decades for its involvement in planar cell polarity (PCP), and more recently, epilepsy. prickle expresses three isoforms, prickle-prickle (pk^pk11 ; pk-RA, FBtr0089042), prickle-M (pk_M ; pk-RB, FBtr0089043) , and prickle-spiney-legs (pk^sple ; pk-RC, FBtr0089044). While pk_M expression is initiated during embryogenesis, expression ceases prior to the end of pupation. On the other hand, both pk^pk11 and pk^sple are only expressed post-embryonically. All isoforms arise from alternative start sites and differ based on 5' coding or non-coding exons. Mutations that affect pk^pk11 or pk^sple isoforms have been shown to alter development of the fruit fly body plan including the orientation of eye ommatidia as well as positioning of epidermal hairs and bristles of the wing and body. In addition, pk isoforms are required in the nervous system to ensure proper neuronal wiring and function, while mutations specifically affecting pk_M have not been identified. Although some mutations affecting Drosophila prickle isoforms have been approximately defined ( pk^pk-sple13) or mapped ( pk^pk30), the original pk^sple1 and pk^pk1 mutations have heretofore remained uncharacterized (Lilienthal, 2022).

Paired-end Illumina next generation sequencing was used to characterize and map pk^pk1 and pk^sple1 mutations. On average, for the pk^pk1 (and pk^sple1) mutants, 96.5% (and 96.8%, respectively) of the genome had at least 1X read coverage, 92.3% (and 93%, respectively) had at least 10X read coverage, and 84.3% (and 85.8%, respectively) had at least 30X read coverage. For the genomic region encompassing the prickle locus, both mutants had at least 30X read coverage. No mutations affecting predicted splice-sites were found in either mutant. Two nonsynonymous mutations, C902A and T2690C, were identified in pk^pk1, neither of which fall within the evolutionarily conserved protein-protein-interaction PET and LIM domains. Additionally, alignment of paired-end reads to the dm6 D. melanogaster genome revealed a region 13 bases downstream of the transcription start site of prickle transcript variant A that only showed alignment of one out of the two reads of numerous paired-end sequences. Analysis of the unmapped read of these pairs identified sequences from a tirant transposon which was confirmed with PCR followed by Sanger sequencing. This transposon insertion is thus likely responsible for the loss-of-function phenotype in the pk^pk1 mutant, particularly given the numerous start and stop codons at the 5' end of the tirant sequence which would be predicted to be utilized in favor of the correct distal downstream pk^pk11 start codon. In pk^sple1 homozygous mutants, nonsense (C1593T) and missense (A1601C) mutations were identified in the coding sequence, both of which were located at the 3' end of the pk^sple-specific second exon of prickle transcript variant C and upstream of the PET and LIM domain exons. These data suggest that the premature stop codon is likely responsible for loss-of-function of pk^sple in the pk^sple1 mutant (Lilienthal, 2022).

In order to assess whether the nonsynonymous mutations found in the pk^pk1 or pk^sple1 sequences could significantly affect prickle gene function, the conservation of amino acids in these regions was examined. In pk^pk1, while both mutations fall in conserved regions (as predicted by PhyloP in the UCSC Genome Browser), they are likely tolerated and non-deleterious. C902A of pk^pk1 replaces proline with threonine, an amino acid that is similar in structure to the serine found at that position in a close outgroup to the Drosophilidae family (Aedes aegypti, the mosquito), although this region of the protein shows relatively poor conservation between D. melanogaster and A. aegypti. T2690C also is likely non-deleterious, as Drosophila melanogaster is the only member of family Drosophilidae shown to contain a serine at this location while all others carry the amino acid that is inserted due to the missense mutation (proline). Further evidence is found in D. melanogaster sequences submitted to GenBank that indicate a proline at this position. In pk^sple1, the nonsynonymous mutation occurs immediately downstream from the premature stop site and replaces a glutamine with proline, the amino acid found at the analogous position in another member of the Drosophilidae , D. ananassae. Additionally, this region of pk^sple is poorly conserved. Collectively, these results argue that the missense mutations found in pk^pk1 and pk^sple1 are less likely to be deleterious (Lilienthal, 2022).

A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). In this study, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3) was dissected. Pk and Dsh/Dvl were shown to bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment (Humphries, 2023).

This study identified through mass spectrometry analyses with mouse skin epidermis samples phosphorylation on mouse Vangl2 Y308 residue (equivalent to Y374 in Drosophila Vang). This tyrosine lies within with the overlapping binding regions of Pk and Dsh in Vang/Vangl, and, importantly, its charge/phosphorylation status regulates selective binding between Pk and Dsh, with phosphorylation tipping the balance towards Pk binding. It was demonstrated in vivo that binding of Vang to both cytoplasmic core PCP factors is physiologically important (which is the first in vivo evidence for a Vang-Dsh binding requirement). This study provides novel insight into the critical importance of Vang tyrosine phosphorylation and reveals mechanistic features of how regulation of the binding of antagonistic PCP factors to Vang/Vangl during the process of PCP complex segregation and polarity establishment is achieved (Humphries, 2023).

While previous work defined a broad region within the C-tail of Vang to interact with both Pk and Dsh, the mechanistic regulation and physiological significance of these interactions remained unresolved. Importantly, the defined region is conserved between Drosophila Vang and mammalian Vangl1/2 genes. These data reveal that a small conserved stretch of amino acids within this broader region is both necessary (as shown in the whole Vang protein) and sufficient (as deduced from the in vitro peptide assays) to interact with both cytoplasmic core PCP factors. This region is well conserved between all Vang family members and centered on the tyrosine, which can be phosphorylated, as our mass spec data reveal. Mutational studies define that Pk binding is mediated by tyrosine phosphorylation and associated negative charge, while Dsh requires the aromatic ring found in tyrosine (and also phenylalanine) for its binding to Vang. It is worth noting that this Vang region, shared by both Pk and Dsh for binding, is specific for these two factors, as other Vang associated cytoplasmic PCP proteins, for example, Dgo and Scrib, are not affected by mutations within this domain (Humphries, 2023).

The importance of the Vang-Pk complex has been well documented in vivo and is also the core of one of the two stable PCP 'core complexes' that result from PCP factor interactions and signaling. In contrast, an interaction between Vang/Vangl and Dsh/Dvl family members has only been documented biochemically. The dissection of binding requirements allowed us to generate single point mutations in Vang that separate binding to only one of the cytoplasmic factors, either Pk or Dsh. The associated in vivo rescue experiments provided the possibility for physiological testing of a functional requirement of the individual interactions between Vang and Dsh or Pk. While all three point mutations display partial rescue, their function is reduced and thus the respective amino acids are physiologically required. Interfering with binding of Vang to both factors (VangY374A) shows the weakest rescue, with the mutant displaying defects that are more similar to the Vang- null phenotype than the other point mutants. Nevertheless, as the point mutations affecting individual Vang-Pk or Vang-Dsh interactions also displayed only partial rescue of the Vang-/- defects, these data indicate that interactions with either cytoplasmic PCP factor, Pk and Dsh, are critical for in vivo Vang function in PCP core complex localization and hence PCP establishment. The phenotypic defects with the Vang-V376A mutant seen in adult wings suggest that it might behave as a mild neomorph, although no dominant effect was observed when heterozygous over Vang-WT. Importantly, this is the first physiological in vivo evidence demonstrating that Vang has a requirement to interact with Dsh during PCP complex segregation. Of all point mutants tested, Vang-Y374F, showed the strongest partial rescue (appeared closest to wild-type). This is consistent with the notion that Pk does not strictly require binding to Vang for membrane association, and that formation of a Vang-Pk complex is much more complcated than a single interaction between the two proteins. Of note, each single point mutant affects the formation of the stable polarized core PCP complexes, as evident in the protein localization studies in 28h APF pupal wings, suggesting that interfering with any interaction among the core PCP factors causes a significant disruption to the PCP interaction cascade and network, needed for normal asymmetric complex polarizations (Humphries, 2023).

It is intriguing to think about how phosphorylation, and lack thereof, affects the formation of stable core PCP complexes. The data indicate that binding of Dsh/Dvl to Vang/Vangl is physiological, and yet in standard co-staining studies Vang and Dsh do not co-localize. How does Dsh binding to an unphosphorylated Y374 region affect core PCP complex formation (Humphries, 2023)?

There are a few potential scenarios, and importantly Vang is not a major membrane recruiter of Pk, if at all and thus other factors likely contribute to this. First, a Vang-Pk association, which is assumed to be stable in wing cells in the proximal junctional membrane region, should likely not form in other areas of the cell membrane. As such a Vang-Dsh transient/intermediary interaction might serve a function to prevent Vang-Pk binding. If the kinase in question is asymmetrically localized or active, for example in the proximal area, then-and only then-a switch from Vang-Dsh to a Vang-Pk interaction would occur. If the kinase in question is not asymmetrically active or localized, Dsh binding to this Vang region might be required to prevent the kinase to act on Vang in cell membrane domains, where formation of a Vang-Pk complex should not form, for example the distal vertex of a wing cell. In such a mechanistic scenario Dsh would keep Vang/Vangl 'flexible' to find the right cellular context, where/when the presence of the kinase would initiate the switch to a Vang-P at Y374 (Y308 in mVangl2) and thus support an interaction with Pk and its local effectors. While these are intriguing mechanistic models, they remain speculative (Humphries, 2023).

In general, the function of Vang/Vangl proteins in PCP establishment remains unresolved. While Vang family proteins are critical for the process and they can physically interact with all other core PCP factors, their contribution to the stability of the intercellular junctional complexes remains unclear, which seem to mainly require Fz-Fmi::Fmi interactions, although Vang/Vangl proteins are part of these asymmetric complexes and bind to Fz intercellularly. Moreover, the non-stoichiometric manner in which the stable PCP complexes form, with for example one single Pk molecule per 6 Vang molecules, suggests complicated mechanistic scenarios that do not rely on one-to-one protein interactions. Importantly, the formation and maintenance of stable PCP complexes requires also Dgo (Diversin in vertebrates) and extends beyond interactions among the core factors, including Scribble and CK1ε and many regulatory interactions are still to be discovered. Complex in vivo experiments will be necessary to better understand the mechanistic sequence of events (Humphries, 2023).

It is currently unclear which tyrosine kinase(s) act on Vang to mediate its phosphorylation on the Y374 residue (Y308 in mVangl2) and this is one of the regulatory interactions to be still discovered. Sequence motif searches in the Vang Y374 flaking region suggest that Src family kinases could be involved, with no other kinase family having a higher probability (by sequence alignment searches). It is however technically difficult to prove that Src kinases indeed act on this Vang residue in a physiological context, and unfortunately in vitro kinase assays have proven uninformative, as most tyrosine kinases tested could phosphorylate Vang in such assays on multiple residues. Redundancy of Src kinases is an issue in in vivo studies in both the systems (mouse skin and Drosophila wing epithelia), as there are several Src family kinases in both Drosophila and mice, for example. Moreover, in addition to cell survival requirements, many cellular functions are associated with Src family kinases. For example, in Drosophila the two main Src family members are either viable with no overt developmental phenotypes in imaginal discs (Src64, redundant with Src42) or are largely cell lethal (Src42) when analyzed in vivo. They have also been linked to a vast variety of cellular functions, ranging from cytoskeletal regulation and cell adhesion, to synaptic plasticity, proliferation, cell death, and others. Src kinases remain nonetheless likely candidate(s), as (i) GOF phenotypes were observed consistent with a PCP function and (ii) genetic interactions with these Src GOF defects suggest that Vang is required in these contexts. However, again, a loss-of-function scenario to really demonstrate a Src function in PCP establishment remains elusive and should be the focus of future studies (Humphries, 2023).

REGULATION

Dachsous-dependent asymmetric localization of Spiny-legs determines planar cell polarity orientation in Drosophila>

In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. This study shows that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. This model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing (Ayukawa, 2014).

The orientation of Fz localization relative to the Ds/Fj gradients (the Ds/Ft asymmetries) in the Drosophila wing is opposite to Fz orientation in the eye. This observation has been a puzzle in the PCP field and a barrier to understanding how the Ds-Ft system and PCP core protein asymmetries are connected. The current experiments and computational simulations have demonstrated that it is the Pk:Sple ratio that governs the relationship between the Ds/Ft asymmetries and core protein localization in the Drosophila eye and wing. Importantly, this model is supported by a loss-of-function experiment in the eye from a previous study. The pksple mutant, which shows specific loss of the Sple isoform, exhibits a polarity reversal in the orientation of the eye ommatidia. The pkpk mutant does not exhibit a complete reversal of PCP in the wing, perhaps because the remaining endogenous amount of Sple is small and/or the timing of expression of endogenous sple is altered. These data reinforce the conclusion that skewing the Pk:Sple ratio alters PCP establishment in the wing and eye (Ayukawa, 2014).

It is hypothesized that tissues in which Sple complexes (Sple-Pk and Sple-Sple) are predominant will tend to have one polarity, whereas tissues containing mainly Pk complexes will show the opposite polarity. However, the possibility that uncomplexed Pk and Sple molecules may influence PCP determination even if Pk-Pk, Sple-Pk, and Sple-Sple complexes localize asymmetrically in each cell cannot be excluded. Alternatively, multimeric protein complexes containing multiple Pk and/or Sple molecules may be responsible for establishing PCP (Ayukawa, 2014).

This study has found that, in tissues where Sple was relatively abundant, Sple (or the Sple complex) was recruited at the cell edge exhibiting the highest Ds level. Furthermore, biochemical and genetic experiments suggested a model in which Sple-Ds cooperation polarizes Sple (or Sple complexes) at the cell edge exhibiting the highest Ds level. It was also demonstrated that the atypical myosin Dachs is heavily involved in the process of Sple polarization in the wing. This observation is intriguing because dachs loss of function does not show a PCP defect as strongly as that of ds or ft loss of function in Drosophila tissues and Dachs does not appear to be as important to PCP in the eye as in the wing. There may be a redundant unknown mechanism responsible for Sple asymmetry (Ayukawa, 2014).

Intriguingly, in the wing of the pkpk mutant, loss of lowfat (lft), one of the members of the Ds-Ft group, affects wing hair polarity in a manner similar to loss of ds or ft. This is despite the fact that, in contrast to the ds or ft mutant, the lft mutant does not show any PCP defect in Drosophila tissues including the wing and eye. These observations are consistent with result showing that Dachs is involved in Sple asymmetry. These results have profound implications regarding the relationship between Pk isoforms and the Ds-Ft system. In addition, this study revealed that Pk physically and genetically interacts with Dachs, even though the subcellular localizations of these two proteins are opposite. There are several possibilities to explain the physiological relevance of the Pk-Dachs interaction. For example, Pk and Sple-Dachs complexes may have mutually antagonistic functions at the opposite cell edges, which is similar to the relationship between Pk (which is localized at the proximal cell border) and Dsh and Dgo (which are localized distally). To understand the molecular mechanism governing global PCP patterning, it will be important to elucidate (1) whether and/or how Dachs is involved in Sple-Ds cooperation/interaction and (2) how Pk becomes engaged in Dachs function and vice versa (Ayukawa, 2014).

Although these experiments do not directly reveal the molecular mechanism by which polarized Sple complexes regulate the asymmetry of the core proteins, a mathematical model was developed based on this study that supports the proposed mechanism governing the core protein asymmetry. The model includes a possible reaction where Sple stabilizes the membrane localization of Stbm on the cell edge with the highest Ds level. An alternative possibility is that, in addition to the above mechanism, Sple directly promotes the formation of the Fz asymmetry via reversing the direction of Fz transport, by changing the orientation of the microtubule array. Future work will include elucidating the molecular mechanism by which the Pk:Sple ratio regulates the core protein asymmetry, as well as determining how the Pk:Sple ratio is differentially regulated in various tissues (Ayukawa, 2014).

The balance of Prickle/Spiny-Legs isoforms controls the amount of coupling between core and Fat PCP Systems

The conserved Fat and Core planar cell polarity (PCP) pathways work together to specify tissue-wide orientation of hairs and ridges in the Drosophila wing. Their components form intracellularly polarized complexes at adherens junctions that couple the polarity of adjacent cells and form global patterns. How Fat and Core PCP systems interact is not understood. Some studies suggest that Fat PCP directly orients patterns formed by Core PCP components. Others implicate oriented tissue remodeling in specifying Core PCP patterns. This study used genetics, quantitative image analysis, and physical modeling to study Fat and Core PCP interactions during wing development. Their patterns were shown to change during morphogenesis, undergoing phases of coupling and uncoupling that are regulated by antagonistic Core PCP protein isoforms Prickle and Spiny-legs. Evolving patterns of Core PCP are hysteretic: the early Core PCP pattern is modified by tissue flows and then by coupling to Fat PCP, producing sequential patterns that guide hairs and then ridges. These data quantitatively account for altered hair and ridge polarity patterns in PCP mutants. Premature coupling between Fat and Core PCP explains altered polarity patterns in pk mutants. In other Core PCP mutants, hair polarity patterns are guided directly by Fat PCP. When both systems fail, hairs still align locally and obey signals associated with veins. It is concluded that temporally regulated coupling between the Fat and Core PCP systems enables a single tissue to develop sequential polarity patterns that orient distinct morphological structures (Merkel, 2014).

Planar polarized features of wing development are influenced by two different molecular systems, termed Core PCP and Fat PCP. Both consist of proteins that form asymmetric complexes at cell junctions that couple the polarity of adjacent cells and develop tissue-wide polarity patterns. Perturbing each pathway produces distinct alterations in hair and ridge orientation. Fat PCP also influences growth orientation in the larval wing. How global patterns of Fat and Core PCP emerge and the functional relationship between them is not completely understood (Merkel, 2014).

One-half of the Core PCP complex is composed of the seven-pass transmembrane cadherin Flamingo (Fmi), the seven-pass transmembrane protein Frizzled (Fz), and the peripherally associated proteins Dishevelled and Diego. These interact across adherens junctions with a complementary complex in the adjacent cell consisting of Fmi, the transmembrane protein Strabismus (Stbm), and a peripherally associated protein derived from the prickle locus. Two protein isoforms produced by this locus, Prickle (Pk) and Spiny-legs (Sple), differ in their N terminus and are required tissue specifically. Core PCP complexes with opposite polarities segregate to different sides of the cell, generating intracellular polarity that is coupled between neighbors. Global patterns of Core PCP emerge during larval growth and change dynamically during wing morphogenesis. At the time hairs form, Core PCP complexes are uniformly aligned along the PD axis of the wing with Fz-containing domains facing distally. Loss of Core PCP components causes reproducible changes in the hair pattern: hair polarity throughout the wing has a strong anterior-posterior (AP) component and tends to point either toward or away from the third wing vein (L3), depending on the specific mutation (Merkel, 2014).

Positive and negative interactions between Core PCP proteins within and between cells appear to self-organize polarity and align it between small groups of cells. Global Core PCP patterns are thought to rely on cues that bias the direction of the feedback interactions. The Fat system has been proposed to be one such cue, and genetic evidence for its involvement is strongest for tissues and structures whose polarity depends on Sple. However, Fat and Core PCP can also operate independently to orient hairs and bristles. In the wing, distal hair orientation depends on Pk and not on Sple. Nonetheless, loss of Fat PCP perturbs hair polarity in the proximal wing blade. How it does so is controversial (Merkel, 2014).

The Fat PCP system is based on the atypical cadherins Fat (Ft) and Dachsous (Ds), which bind heterophilically across cell contacts, and the Golgi-kinase Four-jointed (Fj), which modulates their affinities for each other. Intracellular asymmetry of Ft:Ds heterodimers is established in response to opposing tissue-wide expression gradients of ds and fj such that intracellular Ds polarity points down the Ds gradient. Fat:Ds heterodimer polarity causes intracellular polarization of the atypical myosin Dachs. In the larval wing disc, Fat PCP vectors, which are defined as pointing toward the side of the cell that accumulates Ds and Dachs, are aligned with growth orientation, pointing in a roughly radial pattern toward the center of the wing pouch. This pattern is similar to that of Core PCP vectors (with Ds and Fz orienting the same direction). However, Fat PCP vectors (deduced from transcriptional gradients of Fj and Ds) point opposite to Core PCP in the eye and have the opposite relationship to hair and bristle polarity in the anterior abdomen. Despite the similarity of their patterns in larval wing discs, Fat PCP influences the disc Core pattern only near the presumptive hinge; elsewhere, it is guided by signals from the AP and dorsoventral (DV) compartment boundaries (Merkel, 2014).

After pupariation, Core PCP reorganizes to form a fan-shaped pattern. Later, during pupal morphogenesis, it reorients distally, forming the pattern that guides wing hair outgrowth. Whether Fat PCP orientation changes similarly during pupal development has never been examined, and the functional relationship between the two pathways at this stage is unclear. Distal realignment of Core PCP depends on oriented epithelial remodeling that reshapes the wing blade at this time. Gain and loss of cell contacts that are biased in the PD axis, along with PD cell elongation, suffice to shift the Core PCP axis in simulations. However, epithelial remodeling also coincides with PD alignment of microtubules, which is perturbed in Fat pathway mutants and has been proposed to bias transport of Fz-containing vesicles (Merkel, 2014).

This study sought to clarify the functional relationship between the Fat PCP and Core PCP systems in the wing. Their global patterns of polarity were quantified and compared throughout development, how these systems influence each other's polarity was examined, ande their respective functions in hair and ridge orientation were probed (Merkel, 2014).

The relationship between the Core and Fat PCP systems has been a vexed issue. Some argue that Fat PCP provides global cues to direct the pattern of Core PCP, which then orients structures such as hairs and bristles. Others have suggested that these systems contribute independently to morphological polarization. This study has resolved this problem by showing that the balance of Pk and Sple activities regulates coupling between Core and Fat PCP. Pk allows uncoupling, whereas Sple links the two systems with opposite vector orientation (i.e., Fz and Ds are on opposite cell boundaries). In the wing, Pk is needed to uncouple Core and Fat PCP during tissue flows. This allows Core PCP to reorient distally and guide hair formation, while the Fat system stays oriented along the AP axis. When Pk is lost and Sple dominates, the systems are coupled during flow and Core PCP is misoriented. Later, Sple is required to couple the two systems and generate the cuticular ridge pattern. It has been proposed based on genetic observations that Sple allows the Core PCP system to respond to cues from Fat PCP; these findings provide concrete molecular support for this idea. They further suggest that when Core and Fat PCP are coupled, either partner can determine the orientation of their joint pattern: Core PCP guides Fat PCP to form the cuticular ridge pattern (Merkel, 2014).

Although Fat and Core PCP vectors can uncouple when Pk is present, they are aligned in the wing disc, despite the presence of Pk. Here, they point in the same direction -- unlike when Sple dominates. This may suggest that Core and Fat PCP have a weak tendency to align when Pk is present, but that this can be overcome later by tissue flows. This may explain why Fat PCP is needed for some aspects of the larval Core PCP pattern near the hinge. Alternatively, Fat PCP may influence Core PCP through effects on growth orientation (Merkel, 2014).

The findings highlight the hysteresis of Core PCP patterns, i.e., they depend not only on current input (such as tissue flows or changed balance between Pk and Sple) but also on the patterns that precede them. Early Sple overexpression is an illuminating example. Sple overexpression prior to tissue flows reverses the orientation of the early Core PCP pattern; in the absence of further Sple expression, this reversal is preserved as the Core PCP axis realigns with the PD axis in pupal wings, eventually reversing hair orientation. Thus, pupal reorienting signals act mainly on the axis, rather than the vector, of Core PCP. Axial information may be provided by oriented cell divisions and rearrangements. It may also be present in PD cell elongation and alignment of microtubules with the PD axis. However, an axial signal is inconsistent with vectorial input from signaling gradients, or with a requirement for polarized (as opposed to axially aligned) microtubules at this stage. The observed hysteresis of Core PCP patterns suggests that the disturbed orientation of Core PCP (and hairs) in ft mutant pupal wings could derive from the altered pattern in larval discs, as polarity defects propagate during pupal tissue flows. The data are inconsistent with a direct role for Fat PCP in orienting Core PCP distally at pupal stages, because their patterns do not agree at this time (Merkel, 2014).

Understanding hysteresis in PCP patterns also helps resolve the puzzle as to how they specify distinct orientations of hairs and ridges. Based on phenotypes caused by timed Sple overexpression, it was proposed that the ridge pattern posterior to L3 is specified by an early (pre-18 hAPF) Sple- and Fat-dependent signal oriented along the AP axis, while the pattern anterior to L3 depends on a later Pk-dependent signal oriented in the PD axis. It was unclear what cellular features posterior to L3 might persist through subsequent Core PCP reorganization to guide cuticle deposition many hours later (40 hAPF). The current findings confirm the general idea that Sple allows Fat PCP to influence Core PCP and ridge orientation. But this study showed that this occurs just before cuticle secretion, as loss of Pk allows Sple to couple Fat and Core PCP and generate the ridge pattern. Earlier Sple overexpression (like loss of Pk) affects ridges because the PD-oriented Core PCP pattern does not develop properly. This affects the subsequent evolution of the ridge pattern (Merkel, 2014).

The reproducible hair polarity patterns characteristic of different Core PCP mutants have always been hard to explain. A physical model is presented that accounts for all the hair polarity patterns in wild-type wings, and in wings overexpressing Sple at different times. These patterns can be quantitatively understood by taking into account just three physical principles: (1) an axial PD-oriented signal (tissue shear) that acts on differently oriented initial patterns, (2) the Sple-dependent coupling of Core PCP to Fat PCP that is stronger than the axial signal, and (3) the tendency of Core PCP domains to align with each other. Thus, while Sple-dependent coupling of Core to Fat PCP contributes importantly to the hair polarity patterns seen in Sple-overexpressing wings (and Pk mutant wings), it acts in the context of these other factors and the resulting patterns cannot be understood quantitatively without including them (Merkel, 2014).

While hair polarity in pk wings is still guided by Core PCP, this system loses polarity in fz and stbm mutants. Hair patterns in these mutants are oriented directly by Fat PCP. Thus, both PCP systems can orient hairs (consistent with results in the abdomen, but when these two systems disagree, Core PCP dominates. Strikingly, even in the absence of both PCP systems hairs align with each other locally and form global patterns exhibiting new features never observed in either type of mutant alone. Overall, these findings suggest that tissue polarity relies on multiple self-contained mechanisms that can be flexibly linked to each other (Merkel, 2014).

Protein Interactions

Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling

Planar cell polarity signaling in Drosophila requires the receptor Frizzled and the cytoplasmic proteins Dishevelled and Prickle. From initial, symmetric subcellular distributions in pupal wing cells, Frizzled and Dishevelled become highly enriched at the distal portion of the cell cortex. A Prickle-dependent intercellular feedback loop is described that generates asymmetric Frizzled and Dishevelled localization. In the absence of Prickle, Frizzled and Dishevelled remain symmetrically distributed. Prickle localizes to the proximal side of pupal wing cells and binds the Dishevelled DEP domain, inhibiting Dishevelled membrane localization and antagonizing Frizzled accumulation. This activity is linked to Frizzled activity on the adjacent cell surface. Prickle therefore functions in a feedback loop that amplifies differences between Frizzled levels on adjacent cell surfaces (Tree, 2002).

Using a glutathione-S-transferase (GST) pull-down assay, it was found that full-length, in vitro translated Pk^Sple (one of the three alternatively spliced forms of Prickle) binds to a GST-Dsh fusion protein. To determine the domain dependency of this interaction, domains of Pk were tested for binding to individual Dsh domains. A Pk construct containing just the conserved PET and LIM domains that are common to all Pk isoforms (PkPETLIM), but not either domain individually, is sufficient for binding to full-length Dsh. Furthermore, of the three defined Dsh domains, DIX, PDZ, and DEP, it was found that PkPETLIM binds a construct containing the DEP domain but not other Dsh domains. The binding between Pk and Dsh was confirmed, and the domain specificity was verified, using a yeast two-hybrid assay. PkPETLIM fused to a DNA binding domain interacts specifically with the Dsh DEP domain but not with the other domains of Dsh. It is concluded that Pk, via its conserved PET and LIM domains, binds the Dsh DEP domain (Tree, 2002).

Since the Dsh DEP domain is required for membrane localization, it was hypothesized that Pk antagonizes Fz activity by interfering with Dsh membrane localization. To test this, it was necessary to find conditions in which Pk activity is uncoupled from Fz/Dsh activity in the adjacent cell. Fz-dependent Dsh localization was therefore reconstituted in cultured cells. In U20S cells, transfected GFP-tagged Dsh (Dsh::GFP) can be seen in punctate patches in the cytoplasm. On cotransfection with Fz, Dsh::GFP is translocated to the cell membrane, as was seen in a similar frog animal cap assay. To test whether the physical interaction between Pk and Dsh affects Dsh membrane localization, Fz, Dsh::GFP, and Pk were cotransfected. Dsh is localized in the cytoplasm in 90% of the cotransfected cells, resembling cells transfected with Dsh::GFP alone. In contrast, Pk lacking the PET domain is severely impaired in its ability to block Dsh membrane localization. Pk, therefore, interferes with Dsh membrane association in this heterologous system and by extension may interfere with Dsh membrane localization at the proximal boundary of pupal wing cells. Since Dsh is required to generate asymmetric localization of Fz, it is suggested that blocking Dsh membrane localization also inhibits the accumulation of Fz at the proximal boundary (Tree, 2002).

Fz activity on the distal side of the cell is linked to the accumulation of Pk on the proximal side of the adjacent cell and that accumulation of Pk suppresses Fz/Dsh localization at the proximal cell cortex. In this way, Fz and Pk appear to function in a feedback loop rather than a linear signaling pathway. The ordering of these members of the PCP pathway was analyzed through epistasis analysis. The loss-of-function phenotypes of pk^pk-sple, dsh, and fz are very similar, suggesting that they activate the same signaling mechanism. However, these similarities make classical genetic epistasis tests difficult to interpret. In previous work, Fz overexpression phenotypes have been used to show that Fz signaling requires Dsh. These results were interpreted as implying that the PCP genes form a linear pathway with Dsh acting downstream of Fz. Consistent with this is the finding that Dsh localization requires Fz protein. In contrast, other observations do not fit this model. For example, localization of Fz requires Dsh, a presumed 'downstream' element of the pathway. Fz localization also requires the other presumed downstream elements Pk, the 7 cadherin Flamingo (Fmi), and novel TM protein Van gogh/Strabismis (Vang/Stbm) (Tree, 2002).

To clarify these results, several further epistasis tests were performed. Dsh was found to be required to generate a Fz overexpression phenotype and Fz is required to produce a Dsh overexpression phenotype. In addition, Pk is required for the Dsh and Fz overexpression phenotypes. These results argue against a simple linear pathway but are consistent with Fz, Dsh, and Pk participating in a feedback loop (Tree, 2002).

Evidence is provided that Fmi acts with Pk on the proximal side of the cell. Like Pk, overexpression of fmi using ptc-Gal4 causes hairs to point toward the midline of the wing. fmi overexpression clones show distal-domineering nonautonomy, phenocopying fz loss-of-function and pk overexpression clones. Thus, it is possible that Fmi could be acting similarly to Pk in feedback amplification of PCP signaling. Fmi is likely to be present at both sides of the cell, but it may act with Pk at the proximal side to suppress Fz signaling. However, Fmi must have an additional function since, within and surrounding fmi clones, very little Pk and Dsh localize to cell boundaries. In addition to functioning with Pk to suppress Fz/Dsh activity, it is suggested that Fmi could have a role in stabilizing the Fz complex at the membrane, without which no signaling occurs (Tree, 2002).

Whether differences in Pk activity between adjacent cells affect the localization of Dsh and Fz was examined. pk was overexpressed in the posterior of the wing (using UAS-pk, engrailed-Gal4), and Dsh and Fz localization were assessed near the edge of the pk overexpression domain. Dsh and Fz were relocalized to the antero-posterior cell boundaries, indicating that accumulation of Dsh and Fz occurs at interfaces between cells expressing high and low levels of Pk. Furthermore, within the posterior domain, Dsh and Fz are seen to accumulate to higher levels than in the anterior, consistent with a role for Pk in promoting PCP signaling. Two conclusions are drawn from these observations: (1) Dsh and Fz localization occur preferentially at boundaries between cells where one cell expresses high levels and the other expresses lower levels of Pk; (2) providing high levels of Pk amplifies Fz signaling, as measured by the increased accumulation of both Fz and Dsh at cell peripheries. In the wild-type, therefore, Pk at the proximal side of the cell drives Fz and Dsh accumulation at the distal side of the adjacent cell. Conversely, providing high levels of Fz induces higher levels of Pk accumulation (Tree, 2002).

Since Pk accumulates at boundaries between cells expressing different levels of Fz, and Dsh accumulates at boundaries between cells expressing different levels of Pk, it is suggested that Fz and Dsh on one side and Pk on the opposite side of an intercellular boundary form a self-organizing complex. Indeed, evidence is found for this in the posterior, pk-overexpressing domain of the same wings. In the posterior, Dsh and Fz accumulate in discrete patches around the cell peripheries. Simultaneous staining for Pk, Dsh, and Fmi, or Fz and Fmi, reveals that all four proteins colocalize to these patches. Thus, Pk, Dsh, Fmi, and Fz form self-organizing complexes bridging adjacent cells (Tree, 2002).

Various models have been proposed for the distribution of PCP information within Drosophila epithelia. Regardless of which model is correct, it is likely that this cue induces only a slight asymmetry within each cell. In the Drosophila wing, this initial small asymmetry may result in slightly higher levels of Fz accumulation or activation on the distal than on the proximal side of cells. However, this initial level of asymmetry is insufficient to polarize the cytoskeletal architecture of the cell and is undetectable as assessed by Dsh localization. Fz and Dsh localization subsequently become highly polarized through the action of a feedback amplification loop. Although the mechanism for such a feedback loop has not been demonstrated, it has previously been speculated that Fz activity on distal cell surfaces might promote expression of an inhibitory Wnt molecule that would suppress Fz activity on the adjacent cell surface. This study demonstrates suppression of Fz activity on adjacent cell surfaces but finds that it is mediated through asymmetric localization of Pk, which antagonizes Fz/Dsh accumulation by blocking cortical Dsh localization (Tree, 2002).

A model is proposed for a Pk-dependent feedback mechanism that functions across each P-D cell interface to amplify the difference between the initial levels of Fz/Dsh activity. Pk and Dsh are initially distributed nearly symmetrically, but in largely nonoverlapping distributions around the cells' apices. As a result of an initial, slight asymmetry in one or more components, Pk suppresses Fz accumulation on the proximal side of the cell by antagonizing Dsh cortical localization. Dsh association with the cortex is required for asymmetric Fz accumulation. Reduced proximal Fz accumulation then decreases Pk activity on the adjacent, distal side of the neighboring cell, allowing even greater Dsh cortical localization and Fz accumulation. Conversely, Pk localization is induced by Fz localization on the neighboring cell. The result is the highly asymmetric distribution of Fz, Dsh, and Pk observed at 30 hr APF. This mechanism is bidirectional, constituting a negative feedback loop that is predicted to be unstable; once any asymmetry is initiated, high levels of Fz/Dsh are promoted on one side of the boundary and low levels on the other. Fmi is proposed to play two roles. The localization of all components to the apical cell cortex depends on Fmi, which is thought to be on both sides of the boundary. Since Fmi overexpression mimics Pk overexpression, it is proposed that Fmi is activated selectively on the proximal side of the cell, where it works with Pk to suppress Fz/Dsh activity. In essence, the function of this regulatory loop depends on the balance of forces regulating Dsh membrane association on either side of the boundary. Fz recruits Dsh to the cell cortex, and Pk blocks this recruitment. Once an initial asymmetry is induced, these forces become different on either side of the boundary, and the feedback mechanism amplifies the differences (Tree, 2002).

Because fmi overexpression produces polarity phenotypes very similar to those seen with pk overexpression, it is likely that Fmi activity is also polarized across cell boundaries and participates in blocking Fz/Dsh accumulation on the distal side of the interface. However, Fmi has been proposed to exist on both sides of the boundary, and Fmi is required for the efficient localization of Fz, Dsh, and Pk to boundaries. Fmi, therefore, appears to have an additional, complex-stabilizing function. Diego (Dgo), an ankyrin repeat protein, is required for PCP signaling and localizes to P-D cell boundaries, though it has not been possible to resolve on which side of the boundary Diego is found. Dgo may function as a scaffold for assembly of PCP-signaling components (Tree, 2002).

The core PCP protein Van Gogh (Vang, also known as Strabismus [Stbm], a transmembrane protein, is likely to be involved in the feedback amplification mechanism. In a vang/stbm mutant background, Fz is localized symmetrically to the membrane in the same manner as in a pk null background, and vang/stbm has been proposed to function downstream of pk. Interestingly, in Xenopus and zebrafish, Stbm binds Dsh and seems to toggle its activity between the canonical Wnt and a PCP-like signaling pathway. Stbm antagonizes canonical Wnt signaling and activates a PCP-like pathway that regulates convergent extension and induces JNK signaling. It is proposed that both fly and vertebrate Vang/Stbm may function together with Pk, facilitating PCP signaling by antagonizing Dsh activity (Tree, 2002).

The feedback amplification mechanism described here provides a clue to understanding the long-standing problem of domineering nonautonomy. Loss-of-function clones of fz, vang/stbm, and to a lesser extent pk induce polarity phenotypes in neighboring wild-type tissue. The feedback loop model predicts that loss of Fz will disrupt the localization of these components in the neighboring distal cells, causing their polarity to reverse. Furthermore, if the immediate clone neighbors have reversed polarity, this could then cause the reversal to propagate over a distance. This phenomenon is observed near the lateral borders of a fz clone, where Pk accumulation is seen to run parallel to the clone, even at a distance from the mutant cells. Thus, the reversal in Pk localization may underlie, in part, the domineering nonauontomy observed within wild-type tissue distal to fz clones (Tree, 2002).

Once asymmetrically localized, the Fz/Dsh complex must direct reorganization of the cytoskeleton by both determining the location for prehair initiation and by limiting the number of prehairs to one. Rho and Rho-associated kinase directly regulate myosins to limit the number of prehairs. The recently described Formin homology protein, Daam1, links Dsh to Rho during vertebrate PCP-like signaling, and a Drosophila homolog may function similarly. However, further studies will be required to understand how localized Fz and Dsh orient prehair initiation (Tree, 2002).

Strabismus is asymmetrically localized and binds to Prickle and Dishevelled during Drosophila planar polarity patterning

Planar polarity decisions in the wing of Drosophila involve the assembly of asymmetric protein complexes containing the conserved receptor Frizzled. This study analyses the role of the Van Gogh/strabismus gene in the formation of these complexes and in determination of cell polarization. The Strabismus protein becomes asymmetrically localized to the proximal edge of cells. In the absence of strabismus activity, the planar polarity proteins Dishevelled and Prickle are mislocalized in the cell. Strabismus binds directly to Dishevelled and Prickle and is able to recruit them to membranes. Furthermore, the putative PDZ-binding motif at the C terminus of Strabismus is not required for its function. A two-step model is proposed for assembly of Frizzled-containing asymmetric protein complexes at cell boundaries. First, Strabismus acts together with Frizzled and the atypical cadherin Flamingo to mediate apicolateral recruitment of planar polarity proteins including Dishevelled and Prickle. In the second phase, Dishevelled and Prickle are required for these proteins to become asymmetrically distributed on the proximodistal axis (Bastock, 2003).

The subcellular localiaation of Stbm protein was investigated during wing morphogenesis using both a Stbm-YFP (Stbm yellow fluorescent protein) expressing transgene and using specific antibodies raised against Stbm. During the third instar stage, Stbm-YFP in the wing pouch localizes unevenly around apicolateral cell boundaries. Based on its molecular homology as a multi-pass transmembrane protein, it is assumed that Stbm is present in the outer cell membrane. At 18 hours of pupal life, a similar pattern is seen, Stbm-YFP still being distributed patchily in an apicolateral ring. By 24 hours, there is preferential distribution of Stbm-YFP to proximodistal cell boundaries; this distribution is clearly present at 28 hours and persists until at least 32 hours, which corresponds to the time of trichome initiation. The pattern seen with Stbm antibodies confirms that Stbm-YFP is a faithful reporter of Stbm protein distribution (Bastock, 2003).

The timecourse and distribution of Stbm broadly fits that described for other planar polarity proteins such as Fmi, Fz, Dsh and Pk-Sple. Consistent with this, good colocalization is found between Stbm-YFP and other polarity proteins. The localization of Stbm-YFP to the adherens junction zone was confirmed by costaining for Armadillo distribution. Conversely, Stbm-YFP shows no overlap with the distribution of Discs-Large, which is localized in the septate junction region. Mosaic analysis revealed that Stbm-YFP becomes preferentially distributed to the proximal edges of cells with no appreciable accumulation at distal edges (Bastock, 2003).

The three putative multipass transmembrane proteins Fmi, Fz and Stbm all play important roles in the first step of localizing planar polarity proteins to the apicolateral adherens junction zone. It is thought that Fmi acts at the top of the hierarchy in this process, since, in its absence, negligible amounts of any planar polarity proteins become apicolaterally localized. Stbm is also key, because, in its absence, both Fz and Fmi recruitment are reduced. Additionally, Stbm is also required for Dsh apicolateral recruitment and for efficient localization of Pk to membranes. Fz is not significantly required for apicolateral recruitment of Fmi, but is partly needed for apicolateral localization of Stbm and is absolutely required for apicolateral localization of Dsh. Hence, in the absence of Fmi, Fz or Stbm, one or more planar polarity proteins do not become apicolaterally localized and the process of asymmetric localization on the proximodistal axis does not occur (Bastock, 2003).

An important question is which of these factors are directly binding together, in the process of apicolateral recruitment. So far no direct protein interactions have been reported for Fmi, although it is tempting to speculate that Fmi might bind directly to Fz and Stbm in the process of apicolateral recruitment. However, Fz is able to recruit Dsh to membranes in a heterologous cell type, suggesting that these factors directly interact. In addition, vertebrate Stbm and Dsh homologs have been shown to directly interact. Direct interactions are shown between Drosophila Stbm and Dsh, and Stbm and Pk. This suggests a model in which Dsh and Pk both become apicolaterally localized as a result of direct interactions with Fz and Stbm. Notably, in the absence of Stbm, Pk accumulates in the cytoplasm, suggesting that its interaction with Stbm is important for regulating its level in the cell in addition to its subcellular localization (Bastock, 2003).

At the stage when the planar polarity proteins are apicolaterally localized, but prior to the stage when they are asymmetrically localized on the proximodistal axis of the wing, it is possible that they are present in either 'symmetric' or 'asymmetric' complexes assembled across cell-cell boundaries. If the complexes were symmetric, then Fmi, Fz, Stbm, Pk and Dsh would all be present in a complex together on the same side of the cell-cell boundary. Such symmetric complexes would then subsequently evolve into asymmetric complexes, with Fz/Dsh at distal cell edges and Stbm/Pk at proximal cell edges and Fmi on both sides. Alternatively, the initial apicolateral complexes formed could be asymmetric, with Fz/Dsh always on the opposite side of the cell-cell boundary from Stbm/Pk. These asymmetric complexes would initially be randomly oriented relative to the axes of the wing, but would gradually become aligned to the proximodistal axis. The possibility is favored that planar polarity protein complexes are initially symmetric, since Stbm directly interacts with Dsh and these molecules colocalize during earlier stages of wing development. However, it has been reported that Pk and Dsh-GFP do not precisely colocalize in early pupal wings: this observation supports the early presence of asymmetric complexes (Bastock, 2003).

After the apicolateral recruitment of planar polarity proteins, over a number of hours their localization alters such that they become asymmetrically distributed on the proximodistal axis of the wing. Although Dsh and Pk play negligible roles in the apicolateral recruitment of proteins, both are required for this subsequent proximodistal redistribution. Since overexpression of both factors leads to the accumulation of polarity proteins at apicolateral cell boundaries, it is suggested that they both function to promote the assembly and/or stabilization of protein complexes. Removal of the function of the planar polarity gene dgo also blocks asymmetric proximodistal localization but not apicolateral localization of other polarity proteins. Furthermore, overexpression of Dgo causes an accumulation of other polarity proteins at cell boundaries similar to that seen when Dsh and Pk are overexpressed. Therefore, it is proposed that Dsh and Pk act together with Dgo in the assembly of asymmetric complexes (Bastock, 2003).

Recently, it has been proposed that the function of Pk in asymmetric complex assembly is to antagonize Dsh localization to membranes. This model is mechanistically attractive, in providing an explanation for the formation of asymmetric complexes in which Dsh and Pk are found on opposite sides of cell-cell boundaries. However, it is found that in the presence of Stbm, Dsh and Pk will colocalize at the same membranes. Furthermore, it was not possible to show an effect of overexpressing Pk on the association of Fz and Dsh at membranes. In addition, high level Pk expression in vivo does not cause Dsh to lose its membrane localization but instead appears to increase levels of Dsh at the membrane. Resolution of these issues will require a more detailed understanding of the composition and properties of the protein complexes involved (Bastock, 2003).

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle and strabismus are thought to antagonize Fz signaling. They act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. Stbm and Pk interact physically; Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus the data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling (Jenny, 2003).

pk function is required in the R4 precursor, as opposed to fz PCP signaling in R3, for control of polarity establishment. Stbm, a transmembrane protein also required in R4, interacts genetically and physically with Pk. This interaction is important for the recruitment of Pk to the plasma membrane. In Xenopus animal-cap explants, Stbm and Pk relocalize each other to subdomains of the membrane. A model is proposed of how Pk/Stbm might regulate Fz/Dsh signaling activity (Jenny, 2003).

The in vitro molecular interaction between Pk and Stbm and their mutual relocalization in Xenopus animal caps suggest that they form multiprotein complexes. Several pieces of evidence indicate that the physical interaction is physiologically important: (1) correct membrane localization of Pk depends on stbm function because in stbm mutant tissue Pk staining is diffuse and absent (or strongly reduced) at the membrane; (2) Pk and Stbm interact genetically by mutually enhancing each other's GOF and LOF phenotypes in the eye; (3) pk is necessary for PCP signaling in the R4 precursor, the same cell in which stbm is required; (4) expression of the interacting domains of Pk or Stbm interferes with polarity establishment. In particular, a subfragment of 131 amino acids of the C-terminus of Pk, required for the molecular interaction, is sufficient to affect polarity (Jenny, 2003).

Both Pk and Stbm act as if they antagonize Fz signaling. (1) In zebrafish, Stbm overexpression can prevent Wnt11 from rescuing a wnt11 mutation. (2) In the Drosophila wing, overexpression of Pk leads to wing hairs pointing towards the source of the overexpressed protein, behaving like a fz LOF clone (whereas overexpression of Fz leads to hairs pointing away from the Fz source). stbm LOF clones show the opposite behavior to fz LOF clones: wing hairs point away from the mutant patch, consistent with the mutant tissue having a higher Fz-activity (Jenny, 2003).

In the Drosophila eye, evidence that pk acts antagonistically to fz comes from the fact that the Notch-signaling-responsive R4-specific reporter mdelta0.5-lacZ is expressed for a prolonged period in both R3/R4 precursors in a pk^sple1 mutant. This is explained if Fz activity in the R4 precursor is increased, resulting in higher levels of Dl there. This in turn leads to N activation and concomitant mdelta0.5-lacZ reporter expression in both cells of the R3/R4 pair. Conversely, in fz and dsh mutant eye discs (where Fz signaling is absent or reduced and thus Dl should not be upregulated) N-signaling activity and mdelta-lacZ expression is initially reduced in both cells. Fz activity is also antagonized by stbm in the eye. Mosaic analysis of stbm shows that it has the capability to instruct a cell to become R4 as long as the other cell of the R3/R4 pair is mutant for stbm. Therefore, in such an all-or-nothing situation, Stbm in the R3 precursor can override a positive signal of Fz, resulting in a cell fate switch to R4 fate (Jenny, 2003).

In a wild-type situation with all PCP components present in both cells, it is crucial that Stbm activity is higher in R4 than in R3 to ensure proper Fz-signaling regulation. Therefore it is an intriguing possibility that a Pk/Stbm complex in the R4 precursor ensures such higher Stbm activity, and the associated higher Fz repression there is important for a proper R3/R4 cell fate decision (Jenny, 2003).

How does the Stbm/Pk complex regulate Fz-signaling activity? During PCP establishment in the wing, Fz, Dsh, Dgo, Fmi and Pk are initially localized uniformly around the apical circumference of wing cells. During and after PCP signaling, these proteins relocalize and become differentially enriched: Pk concentrates on the proximal side of the cell, whereas Fz and Dsh become enriched distally. Fmi becomes enriched at both sides (Jenny, 2003).

In the eye, the situation is analogous. During PCP establishment, signaling components at the R3/R4 cell border are relocalized from a uniform to a more restricted pattern. Stbm-YFP is localized on the R4 but not on the R3 side, and Fz-GFP ends up on the R3 but not the R4 side. The analogy between the R4/R3 and proximal/distal cell borders is corroborated by the genetic requirements in R3 and R4: the distally localized factors Dsh and Fz are required in R3, while proximally localized Pk is required in R4. Fmi is localized on both poles of each wing cell and also required in both cells of the R3 and R4 pair. The function of fmi has been linked to both proposed complexes, the 'Fz/Dsh side' (fmi is required for apical localization of both Fz and Dsh) and the Stbm/Pk complex. In addition to the genetic interactions between fmi and stbm or pk, a reduced membrane staining of Pk in fmi^- clones in wing cells has been shown (Jenny, 2003).

How do these changes in localization occur? Localization studies in Drosophila and Xenopus suggest that Pk and Stbm influence each other's localization and form clusters in subdomains of the cell membrane. Interestingly, such Stbm/Pk complexes also affect Fz-dependent Dsh membrane localization. Thus it is an intriguing possibility that the patches observed in animal cap cells upon coinjection of Stbm with Pk represent the result of a similar, though unpolarized, symmetry-breaking step during PCP signaling (Jenny, 2003).

The PET/LIM domain of Pk can interact with the DEP-domain/C-terminus of Dsh. This interaction has been suggested to prevent Dsh membrane recruitment. Also, the C-terminus of Stbm can interact with Dsh as long as the PDZ domain is present. Since the data suggest that Pk regulates the activity and localization of Stbm, this regulation might promote or stabilize the interactions of Dsh with Stbm and/or Pk, thereby helping to pull Dsh away from a Fz-signaling complex. The Stbm/Pk complexes could then cause active release of Dsh from the membrane or target it for degradation, resulting in low levels of Dsh (and by inference Fz) at places where Pk and Stbm are enriched. Furthermore, in the R3 cell (or distally in the wing) an unknown factor might act to prevent either the formation of the Stbm/Pk complex or its effect on Dsh (Jenny, 2003).

In conclusion, Pk and Stbm form a functional complex during PCP signaling in Drosophila and during convergent extension in Xenopus. Interestingly, in zebrafish, in addition to its function in convergent extension, Stbm is also required for the caudal migration of hindbrain motor neurons. This function of Stbm is independent of Dsh and the PCP genes tested so far. It will be interesting to determine whether Stbm and Pk function together in this context as well (Jenny, 2003).

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

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

Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. Evidence is provided that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. The initial membrane association of Dgo depends on Fz, and Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi (Das, 2004).

A crucial region for PCP signaling in the eye is in rows 2-5 in the 3rd instar larval disc behind the morphogenetic furrow (MF). Four lines of evidence support this assumption: (1) cells that take part in PCP signaling (R3/R4) are specified as photoreceptor subtypes in this region; (2) Frizzled-Notch signaling-dependent transcription in the R4 cell is initiated in this region, as detected by the mdelta0.5 reporter for the E(spl)mdelta gene; (3) the sev-enhancer, which is active in R3/R4 cells in this region, can drive a PCP gene in order to fully rescue the respective mutant phenotype; and (4) in the region ahead of the MF to the first row behind it, the PCP proteins are uniformly apically localized in all cells, before they begin at row 2 to display the characteristic PCP protein localization pattern (Das, 2004).

Following their initial symmetric apical localization, the PCP factors become asymmetrically enriched across the respective cell boundaries in the proximodistal axis in the wing or the dorsoventral axis in the eye. Although several models have been proposed as to how these complexes might be formed and maintained, the mechanism behind the early aspect of PCP establishment remains largely unclear. The data suggest a complex mechanism that involves redundancy among several PCP genes (Das, 2004).

Based on the analysis of single mutant clones in the eye, only Fz and Fmi affect PCP gene localization in a general non-redundant manner (and Stbm affects Pk localization). The single and double mutant clone data indicate the following (Das, 2004).

1. Fz is required for membrane localization of Dgo and this step precedes any apparent PCP signaling requirement. Fz also affects the apical localization of Dsh but not of Fmi, Pk, or Stbm significantly.
   
2. Dgo alone does not affect the apical localization of other PCP genes, but instead it shares this function redundantly with Stbm and Pk.
   
3. Pk alone does not affect the apical localization of other PCP proteins significantly, but does so in conjunction with Dgo and Stbm.
   
4. Fmi is responsible for the apical localization of Fz.
   

In addition to these initial requirements for apical localization and maintenance, the subsequent asymmetric resolution of the respective PCP proteins to the R4 cell is affected and often delayed in mutant backgrounds (Das, 2004).

How is the initial apical localization of all these factors maintained? As outlined above, none of the single mutant PCP genes, except fz and fmi, has a significant effect on the whole complex. However, in double mutant clones for either dgo and pk, or dgo and stbm, localization of the PCP proteins is severely affected. Most strikingly, the apical localization of Fmi and Fz is affected in these double mutant combinations. In addition, the localization of Stbm and Dsh are also affected. This could be either a direct effect of Dgo and Pk or could be mediated through their effect on Fmi [as in fmi^- tissue, Stbm and Dsh as well as Fz are reduced apically]. These data suggest that the cytoplasmic PCP proteins, which are initially recruited to the membrane by Fz (i.e. Dgo and Dsh) and Stbm (i.e., Pk), form a protein complex that is required to maintain Fmi apically. This interpretation is supported by the observation that Dgo physically interacts with Stbm and Pk, and thus possibly stabilizes the initial complex. Thus, these studies reveal that Dgo, Stbm and Pk are required to maintain apical Fmi localization, possibly through the physical interactions among themselves and possibly other PCP factors, during the early stages preceding PCP signaling (i.e., anterior to MF in eye). In turn, apical Fmi promotes the maintenance of an initial PCP complex at adjacent cell membranes to facilitate their signaling specific interactions (Das, 2004).

It is possible to speculate on further implications of these data. During later stages of PCP signaling, the localization of the PCP factors is resolved into two types of complexes on adjacent cell membranes. The differential localization of either Fz/Dgo or the Stbm/Pk complex in the neighboring cells (R3 versus R4) suggests that asymmetric localization of PCP factors is maintained across the border of the R3 and R4 cells in the eye and across proximodistal cell borders in the wing. In the eye, the PCP proteins analyzed in this manner indeed localize to specific sides of the R3/R4 cell border. Similarly, proximodistal localization in the wing correlates with the respective R3/R4-specific localization. For example, the localization of Fz and Diego in the distal side of a wing cell correlates with the localization on the R3 side of the R3/R4 border; conversely, Stbm localization to the proximal side of a wing cell correlates with its localization on the R4 side of the R3/R4 border. The localization to either the R3 or R4 side also corresponds to the genetic requirements in either cell, as established in mosaic analyses. Thus, since Dgo, which is initially recruited by Fz, localizes to R3 and the pk/stbm complex localizes to R4, it is likely that at later stages during PCP signaling (posterior to MF) Fmi localization is maintained and stabilized through feedback loops on both sides of the R3/R4 boundary (Das, 2004).

A prediction from such a scenario is that Fz/Dgo are performing this function in R3 and the Stbm/Pk complex in R4. Since Fmi is known to function as a homophilic cell-adhesion molecule, the removal of the feedback loop on one side could be overcome through the homophilic recruitment of Fmi from the other side. Only when both feedback loops are weakened on either side, can Fmi localization become affected. This is supported by the different effects of the respective double mutants posterior to the MF; those that affect both sides of the R3/R4 boundary, e.g., dgo and stbm (R3side/R4side) or dgo and pk (R3side/R4side) can cause Fmi delocalization, whereas double mutants affecting only one cell, e.g., pk and stbm (both R4side), have no significant effect (Das, 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, Fz^autonomous 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 fz^autonomous 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 fz^autonomous 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drosophila Rab23 is involved in the regulation of the number and planar polarization of the adult cuticular hairs

The planar coordination of cellular polarization is an important, yet not well-understood aspect of animal development. In a screen for genes regulating planar cell polarization in Drosophila, Rab23, encoding a putative vesicular trafficking protein, was identified. Mutations in the Drosophila Rab23 ortholog result in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. Rab23 is required for hexagonal packing of the wing cells. Rab23 is able to associate with the proximally accumulated Prickle protein, although Rab23 itself does not seem to display a polarized subcellular distribution in wing cells, and it appears to play a relatively subtle role in cortical polarization of the polarity proteins. The absence of Rab23 leads to increased actin accumulation in the subapical region of the pupal wing cells that fail to restrict prehair initiation to a single site. Rab23 acts as a dominant enhancer of the weak multiple hair phenotype exhibited by the core polarity mutations, whereas the Rab23 homozygous mutant phenotype is sensitive to the gene dose of the planar polarity effector genes. Together, these data suggest that Rab23 contributes to the mechanism that inhibits hair formation at positions outside of the distal vertex by activating the planar polarity effector system (Pataki, 2010).

The formation of properly differentiated organs often requires the planar coordination of cell polarization within tissues, a feature referred to as planar cell polarity (PCP) or tissue polarity. Although planar polarity is evident in many vertebrate tissues (such as fish scales, bird feathers, and cochlear epithelium) and it has recently been shown that PCP regulation is highly conserved throughout the animal kingdom, such polarization patterns are best studied in Drosophila melanogaster. PCP in flies is manifest in the mirror-image arrangement of ommatidia in the eye, in the adult cuticle, which is decorated with parallel arrays of hairs and sensory bristles, and in the wing, which is covered by distally pointing hairs (or trichomes). Wing hairs form during the pupal life when each cell produces a single microvillus-like prehair stiffened by actin and microtubules. In wild-type wing cells prehairs form at the distal vertex of the cells and extend distally as they grow (Pataki, 2010).

Mutations in PCP genes result in abnormal wing hair polarity patterns and wing hair number. On the basis of their cellular phenotypes (i.e., prehair initiation site and number of hairs per cell), initial studies placed PCP genes into three groups: the first group (often called the core group) includes frizzled (fz), dishevelled (dsh), starry night (stan) (also known as flamingo), Van Gogh (Vang) (also known as strabismus), prickle (pk), and diego (dgo); the second group consists of inturned (in), fuzzy (fy), and fritz (frtz) (referred to as planar polarity effectors or In group); whereas the third group includes multiple wing hairs (mwh). Double mutant analysis demonstrated that these phenotypic groups also represent epistatic groups, and it was proposed that the PCP genes may act in a regulatory hierarchy, where the core group is on the top, whereas the In group and mwh are downstream components. Subsequent work identified several other PCP genes as well. Some of these have been placed into the Fat/Dachsous group, while another group consists of cytoskeletal regulators, including Rho1 and Drok. Genetic analysis of these two groups has led to models in which the Fat/Dachsous group acts upstream of the core proteins, while Rho1 and Drok act downstream of Fz. Although the existence of a single, linear PCP regulatory pathway is debated, it is clear that in the wing, PCP genes regulate (1) the number of prehairs, (2) the place of prehair formation, and (3) wing hair orientation (Pataki, 2010).

While the molecular mechanism that restricts prehair formation to the distal vertex of the wing cells is elusive, it has been well established that the core PCP proteins adopt an asymmetrical subcellular localization when prehairs form, which appears to be critical for proper trichome placement. In addition, it has recently been found that the In group of proteins and Mwh also display an asymmetrical pattern with accumulation at the proximal zone. These studies concluded that the core PCP proteins are symmetrically distributed until 24 hr after prepupa formation (APF), when they become differentially enriched until prehair formation begins at 30-32 hr APF. This transient asymmetric localization ends by 36 hr APF. It has recently been shown that Fz and Stan containing vesicles are transported preferentially toward the distal cell cortex in the period of 24-30 hr APF, and hence, polarized vesicular trafficking might be an important determinant of PCP protein asymmetry. Other recent studies, however, challenged the view that PCP protein polarization is limited to 24-32 hr APF. Instead, it has been suggested that at least a partial proximal-distal polarization is already evident at the end of larval life and during the prepupal stages (6 hr APF). Polarity is then largely lost at the beginning of the pupal period, but becomes evident again in several hours until hair formation begins. Thus, molecular asymmetries are clearly revealed during wing hair formation, yet the molecular mechanisms that contribute to the establishment of these asymmetrical patterns are not well understood (Pataki, 2010).

In a large-scale mosaic type of mutagenesis screen, Drosophila Rab23, encoding a vesicle trafficking protein, was identified as a PCP gene involved in the regulation of trichome orientation and number in various adult cuticular structures, including the wing, abdomen, and leg. This study shows that Rab23 plays a modest role in cortical polarization of the core PCP proteins in the wing and that Rab23 associates with at least one core protein, Pk. Additionally, it was found that Rab23 contributes to the mechanism that restricts actin accumulation and thus, prehair initiation to a single site within each wing cell (Pataki, 2010).

Rab23 appears to regulate two main aspects of trichome development, hair orientation and hair number. In pupal wing cells, the absence of Rab23 leads to increased actin accumulation in the subapical region and the formation of multiple hairs. In addition, Rab23 mutations impair hexagonal packing of the wing cells, and to a lesser degree, affect cortical polarization of the PCP proteins. Although, Rab23 does not appear to exhibit a polarized distribution in wing cells, it was found that Rab23 associates with Pk, which normally accumulates in the proximal cortical domain (Pataki, 2010).

Careful comparison of the Rab23 mutant phenotype with that of the other PCP mutations reveals that the phenotypic effect of Rab23 differs from all of the known PCP genes. Most notably, Rab23 has a specific requirement in the development of one particular type of subcellular structure (i.e., the cuticular hair) in every body region examined. However, it does not appear to play any role in the planar orientation of multicellular units such as ommatidia in the eye or the sensory bristles of the adult epidermis. In contrast to this, other PCP genes typically exhibit a tissue specific, but not structure specific, requirement, or, such as mutations of the core group, affect the polarization of every tissue and structure, regardless of whether they are hairs, bristles, or unit eyes. Focusing on the wing, loss of Rab23 results in weak trichome orientation defects and a relatively strong multiple hair phenotype (mostly double hairs). This is clearly different from the core PCP phenotypes (strong hair orientation defects and few multiple hairs), or the phenotypes of the In group and mwh (strong orientation defects and multiple hairs in almost every cell). As compared to Rho1 and Drok, Rab23 displays a similar adult wing hair phenotype in mutant clones with respect to multiple hairs, while the orientation defects are less clear in Rho1 and Drok mutants than in Rab23. Moreover, a significant difference exists at the molecular level, because, unlike Rab23, Rho1 and Drok do not play a role in cortical polarization of the core PCP proteins. Given that Rab23 alleles genetically behave as strong LOF or null alleles, Rab23 identifies a unique class of PCP genes dedicated to the regulation of trichome planar polarization (Pataki, 2010).

Although some recent data suggested that the establishment of properly polarized cortical domains is not an absolute requirement for correct trichome polarity in the wing, asymmetric accumulation of the PCP proteins is thought to serve as a critical cue for cell polarization. Thus, the Rab23-induced weak alterations in wing hair polarity are best explained by the similarly modest effect on PCP protein asymmetries. Because Rab23 is able to associate with Pk, it follows that Rab23 is likely to play a role in the proximal accumulation of Pk. Given that the Rab family of proteins is known to control membrane trafficking, the results provide further support for models suggesting that polarized membrane transport is an important mechanism for the asymmetric accumulation of the PCP proteins. Although Rab23 showed a specific interaction with Pk, technical limitations might have prevented the detection of interactions with other core PCP proteins, and hence it is possible that the mechanism whereby Rab23 contributes to cortical polarization is not limited to Pk regulation. One additional candidate is the transmembrane protein Vang that partly colocalizes with Rab23 in S2 cells and has been shown to bind Pk. Thus, through binding to Pk, Rab23 might affect Vang localization or signaling capacity. Irrespective of whether Rab23 directly affects the localization of only one or more PCP proteins, in the wing Rab23 has a relatively modest effect on protein localization, and, as a consequence, on hair orientation, indicating that Rab23 has a minor or largely redundant role in this tissue. Interestingly, however, Rab23 induces much stronger trichome orientation defects on the abdominal cuticle. Although it is not proven formally, genetic analysis suggests that asymmetric PCP protein accumulation (or at least polarized activation) is likely to occur in the abdominal histoblast cells as well. Hence, with respect to protein polarization Rab23 may act in a tissue-specific manner playing a largely dispensable role in the wing, but having a critical role in the abdominal epidermis (Pataki, 2010).

Correct trichome placement at a single distally located site is clearly a crucial step in planar polarization of the wing cells. Current models suggest that prehair initiation is controlled by an inhibitory cue localized proximally in a Vang-dependent manner, and by a Fz-dependent cue that positively regulates hair formation at the distal vertex. Whereas it is not clear how the distal cues work, with regard to the proximal cues it is known that Vang and Pk colocalize with the effector proteins In, Fy and Frtz that control the localization and activity of Mwh, which is thought to regulate prehair initiation directly by interfering with actin bundling in the subapical region of cells. This study found that Rab23 severely impairs trichome placement in the wing leading to the formation of multiple hairs, which indicates a role in the repression of ectopic hair initiation. Where does Rab23 fit into the regulatory hierarchy of trichome placement? Double mutant analysis suggests that Rab23 is upstream of the In group and mwh, and acts at the same level as the core PCP genes. The synergistic genetic interaction between Rab23 and the core PCP mutations indicates that they function in parallel pathways during the restriction of prehair initiation. Remarkably, the pk^pk; Rab23 double mutants exhibit an almost identical phenotype to mutations of the In group, suggesting that, unless the existence of an In independent restriction system is assumed, Pk and Rab23 together are both necessary and sufficient to fully activate the In complex. In pk single mutants the proximal accumulation of In is severely impaired, yet multiple hairs rarely develop, indicating that proper In localization plays only a minor role in the restriction mechanism. Conversely, in Rab23 single mutants In localization is weakly affected, but multiple hairs often form, suggesting that the major function of Rab23 is related to In activation. Thus, it appears that the proximally restricted activation of In on the one hand is ensured by Pk, that mainly plays a role in proper In localization, and on the other hand by Rab23, that seems to be required for In activation. At present, the molecular function of the In system is unknown, and it is therefore also unclear how Rab23 might contribute to the activation of the In complex. Nevertheless, because Rab23 has a weaker multiple hair phenotype than in, but the pk^pk; Rab23 double mutant is nearly as strong as in, it is conceivable that In activation is not exclusively Rab23 dependent but, beyond a role in protein localization, Pk has a partial requirement as well (Pataki, 2010).

The regulation of cellular packing is an interesting, yet only lately appreciated aspect of wing development. It has been reported that the wing epithelium is irregularly packed throughout larval and prepupal stages, but shortly before hair formation it becomes a quasihexagonal array of cells. Hexagonal repacking depends on the activity of the core PCP proteins. However, defects in packing geometry do not appear to directly perturb hair polarity in core PCP mutant wing cells. The possible exception to this rule is pk that exhibits very strong hair orientation defects and induces the strongest packing defects within the core PCP group. Additionally, another study revealed that irregularities in cell geometry are associated with polarity defects in the case of fat mutant clones. Thus, cell geometry is not the direct determinant of cell polarity, but in some instances cell packing seems to impact on PCP signaling and hair orientation. This study has shown that in the wing Rab23 is predominantly involved in the regulation of wing hair number, and it is also required for hexagonal packing of the wing epithelium. Do these packing defects correlate with the severity of the multiple hair phenotype? The data argue against this idea for the case of Rab23, and also for the cases of other strong multiple hair mutants, such as in, frtz, and mwh. Therefore, cell shape has no direct effect on the regulation of the number of prehair initiation sites, and Rab23 appears to regulate hexagonal packing and hair number independently (Pataki, 2010).

As Rab23 and Pk are both required for cellular packing, and Rab23 associates with Pk, it is possible that they cooperate during the regulation of packing. This is in agreement with the observation that pk^pk; Rab23 double mutant wings do not show stronger packing defects than a pk^pk single mutant. However, other interpretations are also possible, hence further investigations will be required to understand how Rab23 and Pk regulates cellular packing and to clarify the impact of packing geometry on PCP establishment in the wing (Pataki, 2010).

Unlike the vertebrate orthologs, Drosophila Rab23 is not an essential gene and does not appear to regulate Hedgehog signaling. Given that Rab GTPases are thought to regulate vesicular transport and that mouse Rab23 localizes to endosomes, it was expected that Rab23 regulates the trafficking of vesicle-associated Hedgehog signaling components. However, in the mammalian systems no clear link between endocytosis, Rab23, and the subcellular localization of Hedgehog signaling elements has been identified. The finding that Rab23 associates with Pk suggests that Rab23 might be directly involved in the regulation of Pk trafficking, and therefore Pk could be the first known direct target of Rab23. Interestingly, there is a significant overlap reported in the embryonic expression domains of the vertebrate Pk and Rab23 genes in the region of the dorsal neural ectoderm, the somites, and the limb buds. Moreover, it is also known that blocking of Rab23 or Pk function in vertebrate embryos can both lead to a spina bifida phenotype. These observations raise the possibility that, unlike the Rab23 involvement in Hedgehog signaling, the Rab23-Pk regulatory connection is evolutionarily conserved (Pataki, 2010).

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. This study found that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. Pk was found to participate in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. these results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling (Cho, 2015).

This study has shown that Cul1 complex-mediated ubiquitinylation of Pk is required for correct function of the core PCP signaling module, thereby ensuring proper alignment of hairs on the Drosophila wing. Ubiquitinylation by the Cul1 complex targets Pk for proteasome-dependent degradation, and in its absence, excess Pk accumulates, resulting in disruption of core PCP function. In several previous reports, ubiquitinylation has been recognized to regulate PCP signaling. In a mouse model, Smurf E3 ligases were shown to regulate PCP signaling by modulating Pk levels. However, mutation of Drosophila smurf failed to show PCP defects. In Drosophila, Cul3 E3 ligase-BTB protein-mediated regulation of Dsh ubiquitinylation modulates PCP signaling, as does the de-ubiqutinylating enzyme Faf, possibly acting on or upstream of Fmi, or more recently proposed to act on Pk. Loss of either activity shows subtle effects on final PCP outcomes in Drosophila. In no case is there a demonstrated mechanism for how these events impact the characteristic asymmetric subcellular localization of PCP proteins that underlies cell polarization (Cho, 2015).

Slimb was found to be the F-box protein that mediates Pk and Cul1 complex association in vivo. It appears likely that the motif that mediates interaction between Pk and Slimb resides in the C-terminal half of the protein, as do the Vang interaction domain and the farnesylation (CaaX) motif. Of note, the amount of Slimb protein in the cell was also dependent on Pk. In previous cell culture studies, F-box proteins themselves were targeted for ubiquitinylation by their own Cul complexes when not bound by other substrates, and this appears to be the case here, as Slimb levels are increased in cul1 knock-down clones. Furthermore, this result supports the idea that Pk is the major target of the Cul1 complex during pupal wing development (Cho, 2015).

If the Cul1-SkpA-Slimb complex targets Pk for degradation, why do Slimb and Pk accumulate together on the proximal side of wildtype cells? Pk is known to bind to Vang, and to localize with it in the proximal complex. Slimb adapts the Cul1 complex to Pk and is seen to colocalize with Vang on the proximal side, as well as with overexpressed Pk. However, this suggests that the Pk in this location is resistant to Cul1 complex-dependent degradation. Pk levels have long been known to be limited by a Vang-dependent activity. Recently, it has been shown that farnesylation of Pk is required for Pk to interact with Vang and promote its degradation, and that levels of Pk also depend on SkpA, leading to the suggestion that farnesylation-dependent Pk-Vang interaction results in SkpA-dependent Pk degradation. This study provides evidence suggesting that the Cul1-SkpA-Slimb E3 complex directly targets Pk for destruction, but in contrast, the finding that Pk with deleted CaaX domain accumulates to elevated levels in cul1 knock-down cells indicates that Cul1/SkpA/Slimb-dependent degradation is independent of farnesylation. Furthermore, the finding that Pk promotes internalization of Fmi-Vang-Pk during mutual exclusion of oppositely oriented core PCP complexes leads to a model, that is consistent with the shared observation that Pk associated with stable intercellular complexes ([Dsh-Fz-Fmi]-[Fmi-Vang-Pk]) is protected from degradation (Cho, 2015).

In theory, generation of cell polarity requires the combination of a local self-enhancement of a cell polarity factor and a long range inhibition of the same factor. In isolated cells, likely the evolutionarily more ancient mechanism, intracellular local self-enhancement can arise through cooperativity among P proteins. Intracellular long range inhibition is most easily accomplished by limiting amounts of a component of the P complex, such that aggregation of P complexes in one location decreases the probability of aggregation elsewhere by depletion of that component (Cho, 2015).

Cell polarization within a multicellular system introduces additional possible intercellular mechanisms for both the local self-enhancement and the long range inhibition (see Cell polarity establishment and the involvement of Pk-mediated endocytosis). If two polarity complexes, P and Q, exist, and can interact at junctions between adjacent cell boundaries, then both the local and long range effects can be mediated through these intercellular interactions. If P complexes recruit Q complexes to opposing sides of junctions, and if mutual antagonism between P and Q occurs, then long range inhibition can occur by P recruiting Q to the neighbor, where P is then excluded. Similarly, exclusion of P decreases Q in that region of the original cell, enabling the accretion of more P (in effect, cooperativity) (Cho, 2015).

The peripheral membrane associated core PCP proteins Pk, Dsh and Dgo appear to mediate these polarization events, but how they do so is not known. They are not required for assembly of asymmetric [Fz-Fmi]-[Fmi-Vang] complexes, but were known to share the ability to induce clustering, and are all required for the feedback amplification that results in the asymmetric subcellular localization of PCP signaling complexes. While their action somehow promotes the assortment of proximal and distal core proteins to opposite sides of the cell, how they carry out this function, and in particular whether this is through intracellular or intercellular mechanisms, is unclear (Cho, 2015).

To understand how excess Pk resulting from mutation of the Cul1 E3 complex disrupts PCP, Pk's role in establishment of core asymmetry was studied further. pk mutation causes symmetric distribution of other core proteins without substantially diminishing or enhancing their junctional recruitment. On the other hand, Pk overexpression causes both accumulation of higher levels of all proximal and distal core proteins and induces their clustering at apical membrane domains, generating discrete puncta. A Pk induced clustering of similarly oriented core complexes could explain both the aggregated punctate appearance and the increased levels of accumulated proteins if one assumes a steady state relationship between free asymmetric complexes and unassembled components as asymmetric complexes are sequestered into puncta (Cho, 2015).

Pk over-expression study shows that Fz is not required for making Fmi clusters, but Vang is. This suggests an intracellular mechanism in which Pk interacts with Vang at the apical membrane to induce clustering. However, since Vang over-expression does not cause accumulation of other core proteins, a specific function for Pk beyond stabilization of Vang must be considered to explain the accumulation of other core proteins. Furthermore, the depletion of Fmi from the membrane achieved by the very high levels of Pk upon simultaneous Pk overexpression and Cul1 depletion argues for a function for Pk beyond clustering (Cho, 2015).

Pk might stimulate amplification simply by promoting clustering, with long range inhibition mediated by other mechanisms, or perhaps by limiting amounts of Pk. However, the data suggest an alternative interpretation, as Pk-dependent mutual exclusion of oppositely oriented complexes is observed, forcing local accumulation of distal proteins induced the Pk-dependent removal of proximal proteins within the same cell. Exclusion is associated with Pk mediated internalization of Pk-Vang-Fmi complexes, suggesting that this exclusion involves endocytosis. The requirement for Vang in this internalization is consistent with a previous study showing that Vang contributes to Fmi internalization. It is therefore proposed that Pk is involved in an intercellular long range inhibition to promote feedback amplification (Cho, 2015).

Like clustering, the Pk-induced routing of Fmi into intracellular vesicles was dependent on Vang, and Pk, Vang and Fmi colocalize in vesicles both apically and more basally, indicating that Fmi-Vang complex trafficking is regulated by associated Pk. However, unlike clustering, it is also dependent on Fz. This suggests a model for feedback inhibition in which oppositely oriented asymmetric complexes interact within clusters, leading to endocytosis and removal of Pk-Vang-Fmi. Competitive interaction between the proximal protein Pk and the distal protein Dgo for Dsh binding is known to occur, suggesting that these interactions might result in either of two alternative outcomes, one of which would be disruption of the proximal complex, and the other disruption of the distal complex. It is proposed that if the distal complex 'wins,' thus remaining stable, the proximal Pk-Vang-Fmi complex becomes internalized in a Pk-dependent step. Once there is a predominance of complex in a given orientation, Vang will be enriched on one side of the intercellular boundary with relatively little Fz present. Since Pk and Slimb associate with Vang, they too will be enriched, but the absence of competitive interactions from the Fz complex allows them to remain within clusters, accounting for the accumulation of Pk and Slimb on the proximal side of wildtype wing cells. According to this model, Pk and Slimb are observed primarily at sites where they are inactive and therefore not internalized (Cho, 2015).

Modest levels of Pk overexpression both enhance accumulation of PCP protein complexes at the membrane and disrupt the normal orientation of polarization. This may be explained by enhanced feedback amplification that overwhelms the ability to interpret directional inputs. In contrast, the depletion of Fmi from the membrane observed with the very high levels of Pk induced by simultaneous Pk overexpression and Cul1 depletion suggests that sufficient Pk can induce internalization even without the competitive interactions from the Fz complex that normally stimulate internalization (Cho, 2015).

The mechanism for Pk-dependent clustering is not known. As previously proposed, clustering may result from a scaffolding effect; the possibility of decreased endocytosis accounting for clustering was previously discounted. Whatever the mechanism, clustering by Pk must occur independent of Fz. Furthermore, Pk must enable the multimeric aggregation of complexes containing [Vang-Fmi]-[Fmi] or [Vang-Fmi]-[Fmi-Fz]. Induction of multimeric clustering would also provide a context for the dose-dependent competition that determines internalization of either the proximal or distal complex. Additional work will be required to determine how Pk facilitates clustering (Cho, 2015).

Since Cul1 depletion increases the amount of Pk, and excess Pk produces clustering and amplification, how Cul1 might produce the observed phenotype is now considered. The simplest possibility is that in the Cul1 mutant, excess Pk produces excess clustering and amplification that overwhelms the directionality in the system. However, because Pk is associated with Slimb and yet stable in the polarized state, and because Pk degradation is dependent on Vang, the possibility is also entertained that Cul1-dependent degradation is somehow functionally coupled to Pk-mediated internalization. Additional studies will be required to distinguish these possibilities (Cho, 2015).

In summary, a model is proposed in which Pk-dependent internalization of proximal complexes provides an intercellular long range inhibition that contributes to amplification of core protein asymmetric localization. At the same time, Pk provides a local cooperative effect by inducing clustering and accumulation of proximal complexes. The mechanism for clustering are not known, but a simple model is that Pk mediates closely related internalization events (Cho, 2015).

It is noted that a similar intercellular long range inhibition was initially discussed long ago, except that [Vang-Pk] was proposed to disrupt [Fz-Dsh]. This interpretation was based largely on inference. The current study provides evidence that [Fz-Dsh] disrupts [Vang-Pk] (by promoting internalization). On theoretical grounds, either one would be sufficient to cause polarization, but the possibility cannot be excluded that both may occur. Indeed, vesicles containing Fz, Dsh and Fmi have been shown to be transcytosed in a microtubule-dependent fashion with a directional bias, and these vesicles appear to derive from apical junctions, where they may arise by exclusion (Cho, 2015).

Although knock-down of smurf in flies reveals no function in PCP; the mechanism described in this study is similar to that inferred for Smurf in mouse PCP. Mice mutant for both Smurf1 and Smurf2 show PCP defects and lose asymmetric localization of core PCP proteins. Furthermore, biochemical evidence was provided that Smurfs, in the presence of the Dsh homolog Dvl2 (and Par6) mediated ubiquitinylation of mouse Pk1. From this, a model was proposed that proximal complexes containing Pk1, and presumably Vang and Celsr (Fmi), are disrupted upon proximity to distal complexes containing Fzd and Dvl2. This model is similar to the model of mutual exclusion, except that the mode of disruption was not directly addressed. While this study proposes disruption by internalization, perhaps coupled to degradation, the mouse stud was only able to address degradation. Furthermore, it is not known if, in mouse, Pk1 mediates clustering, perhaps by a related mechanism, as as is described in flies (Cho, 2015).

The de-ubiquitinase USPX9 was recently identified as a regulator of Pk in the context of Pk's role in epilepsy in human, mouse, zebrafish and flies. Similarly, the orthologous Drosophila de-ubiquitinase Faf modulates the pk^sple dependent seizure phenotype in flies. These observations suggest that while the ubiquitinylating and de-ubiquitinylating activities of Smurf and USPX9 control the ubiquitinylation state of vertebrate Pk's, Cul1 and Faf may serve the analogous function to regulate ubiquitinylation of Drosophila Pk (Cho, 2015).

DEVELOPMENTAL BIOLOGY

The expression patterns of the pk transcripts were investigated on developmental Northern blots and by tissue in situs using probes to the common exons and the unique 5' exons. Both the temporal and spatial patterns of expression of the three transcripts were indistinguishable, with the exception that the pkM transcript was only detected during the embryonic stages. In 28- to 34-hr pupal wings, pk and sple transcripts are expressed uniformly in intervein cells but leave the presumptive vein regions unstained. At the same stage in the pupal legs, pk and sple transcripts are expressed in a similar pattern, uniform in most cells, but excluded from the segmental boundaries. In third larval instar imaginal discs, a low level of pk transcripts can be detected in restricted domains that correlate with the places where pk is required. In the eye disc, maximal expression is detected in a stripe of cells behind the morphogenetic furrow (in the region where ommatidial organization and polarity is being specified). In wing discs, pk transcripts are expressed at higher levels along the dorsoventral (D/V) compartment boundary, where the bristles of the wing margin will form. In the embryo, pk probes show a dynamic expression pattern in cells engaged in morphogenetic movements, such as invaginating mid-line cells, in the cephalic fold, and at parasegmental boundaries (Gubb, 1999).

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).

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).

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, epithelial morphogenesis has been identified as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. This study used quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients (Sagner, 2012).

To study the emergence of polarity in the wing disc, the subcellular distribution of the PCP proteins Flamingo (Fmi) and Prickle (Pk) were quantified. Planar cell polarity (PCP) nematics were calculated based on Fmi staining and PCP vectors based on the perimeter intensity of EGFP::Pk clones. At 72 hr after egg laying (hAEL), the wing pouch has just been specified and is small. EGFP::Pk localizes to punctate structures at the cell cortex that are asymmetrically distributed in some cells, but PCP vectors exhibit no long-range alignment. By 96 hAEL, PCP vector magnitude increases and a global pattern emerges. Later, PCP vector magnitude increases further and the same global polarity pattern is clearly apparent. It is oriented with respect to three signaling centers: the dorsal-ventral (DV) boundary (where Wingless [Wg] and Notch signaling occur), the anterior-posterior (AP) compartment boundary (where Hedgehog [Hh] and Decapentaplegic [Dpp] signaling occur), and with respect to the hinge fold (where levels of the atypical Cadherin Dachsous [Ds] change sharply) (Sagner, 2012).

PCP vectors in the wing pouch near the hinge fold point away from it toward the center of the pouch. Within the Wg expression domain at the DV boundary, PCP vectors parallel the DV boundary and point toward the AP boundary. Just outside this domain, PCP nematics and vectors turn sharply to point toward the DV boundary in central regions of the wing pouch. However, where the DV boundary intersects the hinge-pouch interface, they remain parallel to the DV boundary over larger distances such that PCP vectors orient away from the hinge around the entire perimeter of the wing pouch (Sagner, 2012).

The AP boundary is associated with sharp reorientations of PCP. First, PCP vectors that parallel the DV boundary point toward the AP boundary in both anterior and posterior compartments. Second, although PCP vectors in the central wing pouch are generally orthogonal to the DV boundary, they deflect toward the AP boundary where Hh signaling is most active (as defined by upregulation of the Hh receptor Patched [Ptc]). On either side of this region, PCP vectors turn sharply to realign parallel to the AP boundary. Third, PCP vectors in the hinge point away from the AP boundary and align parallel to the hinge fold (Sagner, 2012).

The atypical Cadherins Fat (Ft) and Ds limit disc growth and orient growth perpendicular to the hinge. Their loss perturbs the PCP pattern in pupal wings and alters hair polarity. To investigate whether they influence the larval pattern, PCP was was quantified in ft and ds mutant discs. The PCP pattern is similar to wild-type (WT) in the central wing pouch but altered in proximal regions close to the hinge fold. Polarity vectors deviate from their normal orientation (away from the hinge fold) in many regions of the proximal wing pouch. This is especially clear near the intersection of the DV boundary with the hinge - here, PCP vectors orient toward the DV boundary rather than away from the hinge. Furthermore, near the AP boundary, vectors form a reproducible point defect, with vectors pointing away from the defect center (Sagner, 2012).

After pupariation, morphogenesis reshapes the wing disc, apposing its dorsal and ventral surfaces such that the DV boundary defines the margin of the wing blade. During reshaping the PCP pattern evolves, but specific local features are retained through pupal development. Consistent with this, hair polarity in ds adult wings proximal wing near the anterior wing margin orient toward the margin rather than away from the hinge. Near the AP boundary, hairs form swirling patterns. Thus, Ft and Ds are required during larval growth to ensure that PCP vectors in the proximal wing orient away from the hinge (Sagner, 2012).

Notch and Wg signaling at the DV boundary organize growth and patterning in the developing wing. These pathways maintain each other via a positive feedback loop; Notch induces transcription of Wg at the DV interface, and Wg signaling upregulates expression of the Notch ligands Delta (Dl) and Serrate (Ser) adjacent to the Wg expression domain, further activating Notch signaling at the DV boundary. To study how the DV boundary organizer affects PCP, Ser was ectopically expressed along the AP boundary with ptc-Gal4 (ptc > Ser). In the ventral compartment, Ser induces two adjacent stripes of Wg expression, which then upregulate Dl expression in flanking regions (dorsally, Fringe prevents Notch activation by Ser. The posterior Wg and Dl stripes are distinct, but the anterior stripes are broader due to the graded activity of ptc-Gal4. In these discs, the ventral compartment overgrows along the AP boundary, parallel to the ectopic 'organizers'. PCP nematics and vectors near the posterior Wg/Dl stripes are organized similarly to those flanking the normal DV boundary, running parallel to the stripe and turning sharply outside this region to orient toward the ectopic organizer). PCP nematics anterior to the ectopic Ser stripe run parallel to it over larger distances before turning sharply, consistent with the broader Wg/Dl expression in this region. In resulting adult wings, hairs orient toward the ectopic wing margin that forms along the AP boundary. Ectopically expressing Wg along the AP boundary also generates an ectopic organizer that reorients growth and PCP (Sagner, 2012).

To ask how loss of the DV boundary organizer affected PCP, a temperature-sensitive allele of wg was used that blocks Wg secretion (wgTS), or wings were populated with wg null mutant clones. Loss of Wg signaling severs the feedback loop with Notch such that both decay. PCP nematics were quantified in wgTS discs shifted to the restrictive temperature shortly after the second to third-instar transition (earlier, Wg is required to specify the wing pouch). wgTS discs have smaller wing pouches than WT and are missing a large fraction of the central region of the pouch where polarity orients perpendicular to the DV boundary. Polarity still orients away from the hinge, thus the PCP pattern in wgTS discs appears more radial (i.e., oriented toward the center of the wing pouch). Analogously, adult wings populated by wg null clones are missing those regions of the distal wing blade where hairs normally point perpendicular to the wing margin. The remaining proximal tissue is normally polarized except at its distal edges. Here, polarity deflects from the proximal-distal axis to parallel the edge of the wing. Normally, hair polarity in the wing blade parallels the margin only in proximal regions, where Ft/Ds influences polarity. Thus, the DV organizer is needed to orient PCP in distal regions perpendicular to the margin. Ft/Ds is required for a complementary subset of the PCP pattern in the proximal wing. Their influences largely reinforce each other (i.e., away from the hinge and toward the DV boundary or wing margin) except where the hinge and wing margin intersect. Here, loss of one signaling system expands the influence of the other. Wg is distributed in a graded fashion and is a ligand for Frizzled (Fz). Thus, it could bias the PCP pattern directly, e.g., by asymmetrically inhibiting interactions between Fz, Strabismus (Stbm), and Fmi or causing Fz internalization. If so, uniform Wg overexpression should prevent intracellular polarization or reduce cortical localization of PCP proteins. To investigate this, Wg was overexpressed uniformly (C765 > wg::HA). Uniform Wg expression elongates the wing pouch parallel to the AP boundary. It broadens the pattern of Dl expression, such that sharp Dl stripes at the DV boundary are lost, but Dl expression remains excluded from the Hh signaling domain anterior to the AP boundary. Fmi and EGFP::Pk polarize robustly in these discs; thus, the Wg gradient does not act directly on PCP proteins to induce or orient polarity. However, the pattern of PCP vectors and nematics is altered. PCP points away from the hinge (rather than perpendicular to the DV boundary) over larger distances compared to WT and then turns sharply to face theDV boundary in the middle of the wing pouch. Because specific alterations in the PCP pattern are induced by uniform Wg overexpression, Wg protein distribution does not directly specify the new PCP pattern (Sagner, 2012).

To identify signals that influence the PCP pattern near the AP boundary, the effects of uniform high-level expression of Dpp and Hh, two morphogens that form graded distributions near the AP boundary, were examined. Uniform Dpp expression does not influence the magnitude of PCP or the range over which PCP deflects toward the AP boundary. Interestingly, uniform Hh expression dramatically increases the range over which PCP deflects toward the AP boundary, suggesting that Hh is important for this aspect of the pattern. However it clearly indicates that PCP vectors are not oriented directly by the graded distribution of Hh or by the graded activity of Hh signaling, because both are uniformly high in the anterior compartment of Hh overexpressing discs. Whether the apposition of cells with very different levels of Hh signaling might produce sharp bends in the PCP pattern was therefore considered. In WT discs, Hh signaling levels change at two interfaces: one along the AP boundary and one along a parallel line outside the region of highest Hh signaling where Ptc is upregulated. PCP vectors orient parallel to the AP boundary in the cells posterior to it, deflect toward the boundary anteriorly, and then reorient sharply outside of this region to align parallel to the AP boundary. Discs uniformly overexpressing Hh have only one signaling discontinuity (at the AP boundary), because Hh signaling is high throughout the anterior compartment. This could explain why PCP in these discs remains deflected toward the AP boundary over longer distances (Sagner, 2012).

To test this, clones mutant for the Hh receptor Ptc, which constitutively activate signaling in the absence of ligand, were generated. Quantifying PCP nematics in these discs reveals reproducible patterns of polarity reorientation at interfaces between WT and ptc^- tissue. In WT tissue adjacent to ptc^- clones, PCP aligns parallel to the clone interface. Due to the typical clone shape, this orientation is often consistent with the normal PCP pattern. However, PCP also aligns parallel to ptc^- clones in regions where this is not so. Thus, ptc^- clones exert a dominant effect on adjacent WT tissue. In contrast, on the mutant side of the clone interface, polarity tends to orient perpendicular to the interface. Thus, apposition of high and low levels of Hh signaling causes a sharp bend in the PCP pattern. Corresponding polarity reorientation by ptc^- clones is also seen in adult wing. Thus, Hh signaling has two effects in WT discs: within the Hh signaling domain, it deflects PCP toward the AP boundary, and just outside the Hh signaling domain, it orients PCP parallel to the AP boundary. In this region, the tendency for polarity to align parallel to Hh signaling interfaces is consistent with the orientation of polarity toward the DV boundary and away from the hinge. Thus, these three polarity cues reinforce each other throughout much of the wing pouch, making the global PCP pattern robust (Sagner, 2012).

Simulations have highlighted the difficulty of establishing long-range polarity alignment in a large field of cells from an initially disordered arrangement. The pattern typically becomes trapped in local energy minima, forming swirling defects. Introducing a small bias in each cell removes such defects - this has been an attractive argument for the involvement of large - scale gradients in orienting PCP. The graded distribution of Ds along the proximal-distal axis (orthogonal to the hinge-pouch interface) suggested a plausible candidate for such a signal. Strikingly, the Ds expression gradient gives rise to intracellular polarization of both Ft and Ds, and the recruitment of the atypical myosin Dachs to the distal side of each cell. Nevertheless, most of the PCP defects in ft mutants can be rescued by uniform overexpression of a truncated Ft version that cannot interact with Ds, and PCP defects in ds mutants can be rescued by uniform overexpression of Ds. Moreover, blocking overgrowth through removal of dachs also suppresses PCP phenotypes in both mutants. The remaining disturbances in PCP in each of these backgrounds are restricted to very proximal regions, both in adult wings and the wing disc. Thus, the graded distribution of Ds does not provide a direct cue to orient PCP over long distances; rather, it appears to be important only locally near the hinge. Furthermore, this study shows that the two other key signaling pathways that contribute to the global PCP pattern in the disc do not act directly through long-range gradients. How do these signals specify the PCP pattern, if not through gradients (Sagner, 2012)?

Simulations in the vertex model have suggested that long-range polarity can be established in the absence of global biasing cues if PCP is allowed to develop during growth. PCP easily aligns in a small system, and globally aligned polarity can then be maintained as the system grows. Such a model obviates the necessity of long-range biasing cues like gradients, at least to maintain long-range alignment of PCP domains. The finding that a global PCP pattern develops early during growth of the wing makes this idea plausible. It may be that a combination of local signals at the different organizer regions specifies the vector orientation of PCP when the disc is still small, and that the pattern is maintained during growth. This may explain why loss-of-function studies have failed to identify the signaling pathways at the AP and DV boundaries as important organizers of the PCP pattern (Sagner, 2012).

In addition to local signals, the orientation of growth may provide additional cues that help shape the PCP pattern. Simulating the interplay between PCP and growth in the vertex model showed that oriented cell divisions and cell rearrangements orient PCP either parallel or perpendicular to the axis of tissue elongation, depending on parameters. Interestingly, each of the signaling pathways that influence PCP in the disc also influences the disc growth pattern. Wg/Notch signaling at the DV boundary drives growth parallel to the DV boundary, consistent with the pattern of clone elongation at the DV boundary. Growth near the AP boundary, where Hh signaling is most active, is oriented parallel to the AP boundary. This behavior probably reflects oriented cell rearrangements rather than oriented cell divisions. Finally, Ft and Ds orient growth away from the hinge. Suppressing overgrowth in ft or ds mutant wings by altering downstream components of the Hippo pathway rescues normal PCP except in the most proximal regions of the wing. Thus, altered growth orientation may contribute to the PCP defects seen in ft and ds mutants (Sagner, 2012).

Growth orientation reflects the orientation of both cell divisions and neighbor exchanges, and these can each exert different effects on the axis of PCP. Understanding the influence of local growth patterns on PCP will require a quantitative description of the patterns of cell divisions and rearrangements in the disc. More refined simulations incorporating local differences in the orientation of cell divisions and rearrangements will allow exploration of how planar polarity patterns can be guided by different growth patterns (Sagner, 2012).

EFFECTS OF MUTATION

To undertake a thorough analysis of the pk locus a variety of genetic strategies were used to isolate new alleles. These alleles can be divided into three phenotypic classes: Pk, Pk-Sple, and Sple. None of these classes show any embryonic phenotype (even when homozygous mutant embryos develop from homozygous mutant mothers). Consistent with this lack of either a zygotic or maternal requirement, deletions of the pk gene are fully viable and fertile. The defects of double mutant pk^pk-sple alleles are the same as those seen with overlapping deletions that remove the entire gene and eliminate all pk functions. Paradoxically, these pk^pk-sple alleles do not produce the most severe phenotypes. Instead the single mutant alleles pk^pk and pk^sple give more extreme phenotypes, but in reciprocal regions of the body; pk^pk in the wing and notum and pk^sple in the legs, abdomen, and eyes. Complementation between these classes of allele indicates two subtly different functions at the pk locus (Gubb, 1999).

Complete lack of Pk function, in pk^pk-sple alleles, gives a weak polarity phenotype in the wing, notum, abdomen, eye, and leg. pk^pk alleles cause an extreme polarity phenotype in the wing and notum; pk^sple alleles affect eye, abdomen, and leg. The pk^pk wing phenotype shows a characteristic reversal in the triple-row bristles along the anterior margin; a whorl in the wing hairs near the tip of vein 2, and abrupt discontinuities in hair polarity, e.g., pk^pk1. The weak Pk^Pk-sple phenotype shows a slight effect on triple-row bristle orientation and gives gently curved hair polarity vectors, e.g., pk^pk-sple13. sple alleles are completely wild type, e.g., pk^sple1. The eye phenotype is wild-type in pk^pk1, showing a line of mirror symmetry along the equator. On both sides of the equator the R3 photoreceptor cell is aligned toward the pole. In addition to being rotated through 180° ommatidia show reversed chirality around the equator, so that both a rotation and a reflection in the plane of the epithelium is required to superimpose the ommatidial patterns. pk^sple1 eyes contain a mixture of ommatidia with reversed polarity and chirality in both hemispheres of the eye. These ommatidia remain aligned along the polar axis, but with their R3 photoreceptors directed toward the equator rather than the pole giving rise to D/V mirror-image reversals of the normal rhabdomere pattern. In addition, all the pk^sple alleles exhibit ~1% anteroposterior (A/P) reversed ommatidia. pk^pk-sple13 eyes contain a mixture of chiral forms of ommatidia. Some ommatidia fail to rotate properly, and the resulting imperfections in the hexagonal stacking give a slightly rough eye phenotype. Some ommatidia are aligned at 60° to the equator and some show A/P reversals, with the R3 rhabdomere anterior to R4. The tarsi of pk^pk1 are wild type; In pk^pk-sple13, the T3 and T4 segments carry medial duplications of the proximal and distal joint structures, with the middle of each segment deleted. This results in alternating reversed-proximal and reversed-distal tarsal joint structures with half the length of a normal segment. In pk^sple1 the tarsal duplications affect T2, T3, and T4 segments, with an occasional incipient ectopic joint in the distal T1. The distal T5 segment remains unaffected in all mutant alleles (Gubb, 1999).

The duplicated wing hair phenotype typical of most type 1 tissue polarity mutants (Gubb, 1982; Wong, 1993) also affects pk^pk alleles, but only 2%-3% of cells show doubled hairs. Where the polarity vectors are changing sharply, however, cells frequently show doubled hairs. After the last cell division in the pupal wing, the cytoskeleton is reorganized. Cells become hexagonal, and prehairs grow out from the distal vertex of each cell (for review, see Eaton, 1997). It is not possible, however, to fill an irregular shape such as the wing blade with a perfect hexagonal array of cells, and occasional defects, such as a distorted four-pentagon array, are seen. The relationship between hair orientation and the cell shape, implied by the localization of prehair initiation sites, is confirmed by the doubled hairs near stacking flaws and the lack of regular hexagonal packing in the vicinity of the anterior whorl (Gubb, 1999).

Mutations in the Van Gogh gene, shown to be allelic to strabismus, result in the altered polarity of adult Drosophila cuticular structures. The two original Vang alleles were recovered because of a dominant phenotype -- a swirl in the wing hair pattern in the C' region of the wing (this is the region that lies between the third and fourth veins proximal to the proximal cross vein). On the wing, Van Gogh mutations cause an altered polarity pattern that is typical of mutations that inactivate the frizzled signaling/signal transduction pathway. Flies homozygous for Van Gogh alleles show a tissue polarity bristle phenotype on the wing, thorax, leg and abdomen. On the abdomen, bristles point almost orthogonally to the midline instead of posteriorly. The tarsus joints are often duplicated as is typical for tissue polarity mutants. The phenotype differs from those seen previously in other polarity mutants, since the number of wing cells forming more than one hair is intermediate between that seen previously for typical frizzled-like or inturned-like mutations. Consistent with Van Gogh being involved in the function of the frizzled signaling/signal transduction pathway, Van Gogh mutations show strong interactions with mutations in frizzled and prickle. pk is a slightly haploinsufficient gene. A deficiency for pk (and some pk point mutants) shows a weak, partially penetrant dominant tissue polarity phenotype. This effect is enhanced by several Vang alleles. Phenotypic data suggest that genetics of Vang and its interactions with pk are complex. Several, but not all, Vang alleles act as enhancers of the pk haploinsufficient tissue polarity phenotype, and the pkTBJ21 antimorphic phenotype. Mutations in pk can also act as suppressors of Vang dominant phenotypes. Mitotic clones of Van Gogh display domineering cell nonautonomy. In contrast to frizzled clones, Van Gogh clones alter the polarity of cells proximal (and in part anterior and posterior) but not distal to the clone. In further contrast to frizzled clones, Van Gogh clones cause neighboring wild-type hairs to point away from rather than toward the clone. This anti-frizzled type of domineering nonautonomy and the strong genetic interactions seen between frizzled and Van Gogh suggest the possibility that Van Gogh is required for the noncell autonomous function of frizzled. As a test of this possibility, frizzled clones were induced in a Van Gogh mutant background and Van Gogh clones were induced in a frizzled mutant background. In both cases the domineering nonautonomy is suppressed consistent with Van Gogh being essential for frizzled signaling (Taylor, 1998).

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. The mechanisms by which this pattern formation occurs have been investigated and the results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. To determine if nemo expression is regulated by fz, fz mutant flies were generated that carry an enhancer trap in the nemo gene. Expression of beta-galactosidase from the enhancer trap resembles the expression pattern of nemo transcripts. The expression of beta-galactosidase is greatly reduced in fz eye imaginal discs, especially in the morphogenetic furrow. It is proposed that frizzled mediates a bidirectional signal emanating from the equator (Zheng, 1995).

To gain further insight into the mechanism of mirror-image symmetry formation, other polarity mutants were examined. Mutations in the sple and dsh genes are seen weakly to roughen the eye and disrupt ommatidial polarity. A third gene, pk, affects tissue polarity but has no mutant eye phenotype (Gubb, 1993). However, a pk-sple double mutant gives rise to a strongly roughened eye, suggesting that sple and pk act redundantly in eye development. Sections of sple, pk-sple and dsh mutant eyes show a disturbed ommatidial polarity with each ommatidium having the normal arrangement of photoreceptor cells. Together with fz, these polarity mutants can be categorized into two classes based on the adult eye phenotypes. One class, which consists of fz, pk-sple and dsh, exhibits all three aspects of polarity phenotype seen in fz adult eyes. Among them, pk-sple had the strongest eye phenotype. The other class, which consists of sple, exhibits only one aspect of the polarity phenotype seen in fz eyes. Although sple ommatidia are still arranged in antiparallel arrays and the equator is still detected, some of the ommatidia are oriented such that their R7 cells are pointing away, rather than toward the equator. Anti-Elav staining of sple eye discs reveals that the disorientation is due to incorrect direction of rotation as in fz mutants (Zheng, 1995).

To determine which cells require the sple gene for ommatidial polarity, mosaic analysis was performed by producing clones of sple- cells. There was no evidence of the non-autonomous or biasing effects that are observed in fz- clones. Since the sple mutant phenotype is partially penetrant and photoreceptor identities could be assigned in mutant ommatidia, only mosaic ommatidia that had rotated incorrectly were examined. Far fewer mosaic ommatidia exhibit a mutant phenotype than genotypically mutant ommatidia. At the borders of seven clones, fourteen mosaic ommatidia with mutant polarity were scored. In all cases, the presumptive R4 cell was sple- and, in almost all cases, the presumptive R3 and R5 cells were sple-. Thus, removal of sple from R3, R4 and R5 cells can lead to incorrect rotational direction. If any one of these cells is sple+, the ommatidium almost always rotates normally. Therefore, sple function in either the R3, R4 or R5 cell appears to be sufficient to drive an ommatidium in the right direction (Zheng, 1995).

In each facet of the Drosophila compound eye, a cluster of photoreceptor cells assumes an asymmetric trapezoidal pattern. These clusters have opposite orientations above and below an equator, showing global dorsoventral mirror symmetry. However, in the mutant spiny legs, the polarization of each cluster appears to be random, so that no equator is evident. The apparent lack of an equator suggests that spiny legs+ may be involved in the establishment of global dorsoventral identity that might be essential for proper polarization of the photoreceptor clusters. Alternatively, a global dorsoventral pattern could be present, but spiny legs+ may be required for local polarization of individual clusters. Using an enhancer trap strain in which white+ gene expression is restricted to the dorsal field, it has been shown that white+ expression in spiny legs correctly respects dorsoventral position even in facets with inappropriate polarizations; the dorsoventral boundary is indeed present, whereas the mechanism for polarization is perturbed. It is suggested that the boundary is established before the action of spiny legs+ by an independent mechanism (Choi, 1996).

Presented here is a cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster. Within this interval, 27 complementation groups have been identified by extensive F2 screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described (Heitzler, 1993).

In wild-type flies, the body surface is covered with cuticular structures that reflect the polarity of the underlying epithelial cells. In most regions, cuticular bristles and hairs are aligned approximately along the long axis of the structures that they cover. A set of mutants causing discrete changes in the orientations of bristles and hairs was described by Gubb and Garcia-Bellido (1982). Mutations of different genes affect different regions of the body, but different mutant alleles at each locus are very similar to each other and have the same regional specificity. Most of these polarity mutants; dishevelled (dsh), frazzled (frz), fritz (frtz), frizzled (fz), fuzzy (fy), inturned (in), multiple wing hairs (mwh) and prickle (pk) affect the wing disc. In addition, the abdomen is affected by dsh, frtz, fz, fy, in and spiny-legs (sple); the legs by dsh, frz, frtz, fz, fy, in and sple; while three of the mutants give a rough eye phenotype: dsh, frz and fz. Taken together, these mutants affect all regions of the body. Two of the mutants, dsh and fz, affect the whole body surface although a class of fz alleles have been recovered that do not affect the eye. The pk and sple mutants are unusual in that they affect reciprocal regions of the body and represent discrete classes of lesion in a complex genetic locus. Double mutant pk-sple alleles affect the whole body surface and give a rough eye phenotype (Heitzler, 1993 and Gubb, 1993). In general, the topography of the regions affected by these mutants remains wild-type as does the distribution of specialized cell types (Gubb, 1982). In this sense, cellular polarity is affected independent of other elements of the pattern. In addition to altering the orientation of hairs, the polarity mutations cause additional hairs to be secreted by wing cells. This phenotype appears to be common to all the mutations that affect the wing blade although the effect is very slight with pk1 (Gubb, 1982) and fz1, with only occasional wing cells giving duplicated hairs. Some combinations of pk mutations and some fz alleles, however, give a significant frequency of cells with doubled hairs. At the other end of the range, mwh expresses several hairs in every wing cell. The duplicated hair phenotype is associated with abnormal organization of the F-actin bundles that are present at the normal site of hair initiation. This suggests a link between polarity and cytoskeletal architecture as in yeast cells (Gubb, 1993 and references).

As a general rule, the hair and bristle polarity of these mutants is cell autonomous in clones. Mosaics of mutant tissue within a wild-type background express the pattern typical of that region in a homozygous wing. Both pk and fz clones, however, can cause a disruption in the polarity of neighboring cells. In the case of fz, this disruption may be quite extensive, but is restricted to cells distal to the fz clone. In addition to altering the orientation and number of wing hairs, the imaginal polarity mutants cause fine-scale rotations and mirror-image reversals. In the tibia, the bristles, together with their associated socket cells and bracts, are rotated as a unit. Similar rotations are shown between the ommatidial units of the compound eye in dsh, fz, frz and pk-sple. In these mutants the overall shape of the eye remains normal, but the surface becomes roughened as the corneal lenses fail to pack into an hexagonal array. The internal structure of the ommatidia shows mirror-image reflections as well as rotations. In sple1 flies, ommatidia show reflections, but no rotations and the surface of the eye remains smooth (Gubb, 1993 and references).

The different classes of mutants at the pk-sple locus give a phenotypic series. In pk eyes, the ommatidial organization is wild-type; sple mutations show mirror-image reflections while pk-sple13 eyes show both rotations and reflections. This last phenotype is particularly informative in that even when the ommatidial units are rotated, their spacing remains relatively uniform. In this sense, the hexagonal array is retained, although the orientation and handedness of adjacent units is no longer co-ordinated. The idea that the ommatidial units in the eye and the bristle sense organs behave as discrete fields fits the known development of these structures, which has been well studied. In contrast, very little is known about the process of segmentation in the adult leg of Drosophila. Classical experiments in the cockroach, however, show that individual tarsi behave as discrete developmental units. The tarsal segments of the legs of dsh, pk-sple, sple, in, fz, and frz flies show mirror-image duplications. The tarsal pattern transformation is similar in all the mutants and corresponds to a mirror-image duplication of both the proximal and distal regions of each segment with loss of medial pattern elements. In addition, frtz tarsi show a weak transformation consisting of occasional ectopic joints. The tarsi, therefore, are regions in which these polarity mutants alter both topographical shape and the distribution pattern of specialized cell types, in addition to the polarity of individual cells. These mirror-image duplications, together with the small size of the tarsi, suggest that the tarsal segments represent discrete fields, within which the fate of a cell is dependent on its immediate neighbor. In the more proximal regions of the legs of dsh, pk-sple, sple, in, fz, frz and frtz mutants, the orientation of bristles and hairs is affected across large regions, but mirror-image duplications are not seen. The different classes of morphogenetic change caused by the imaginal polarity mutants can be rationalized as follows: the primary effect of the mutations is to alter cellular polarity. In regions of the imaginal discs where fields of specialized cell types are specified by interactions between adjacent cells, mirror-image duplications and rotations of the fields can occur. The fields are separated by regions of 'background' cells that are not visibly differentiated with respect to one another. In these regions, the polarity of individual cells is altered, as indicated by the polarity of cuticular hairs. What is not observed are distortions in the shape of imaginal structures, frizzledwhich would be expected if the regions of background cells were rotated. This is a surprising result. The implication is that cellular polarity is altered independent of spatial displacement, as if similar cells have equivalent positional information and do not know their precise position. Taken to an extreme, this view suggests that imaginal disc cells only know their position with respect to adjacent cells. The separation of fields of differentiated cells by regions of background cells allows the integration of patterns to occur across entire imaginal discs without the requirement that cells know their precise position with respect to a global co-ordinate system (Gubb, 1993 and references).

A surprising result in the initial study of the imaginal polarity mutants (Gubb, 1982) was the demonstration of a large class of mutations that are viable, but which cause discrete alterations in the polarity of adult structures. The mutations correspond to complete lack of function, at least by genetic tests, and yet the adult patterns are uniform within a population, but more complex than the wild-type pattern. In addition, most of the genes affect different pathways, by the criterion that double mutants have intermediate phenotypes. The exceptions to this are that mwh and in are epistatic to pk, in that pk;mwh and pk;in flies express the mwh and in phenotypes, respectively. An indication that fy might affect a pathway related to both pk and mwh is that fy;mwh and fy;pk flies give a weak tarsal duplication phenotype showing ectopic tarsal joints, which are not found in the fy, pk or mwh single mutant stocks, although fy affects bristle and hair polarity and mwh causes multiple hairs in the legs. Similarly, dsh and sple mutations together give a more extreme tarsal joint pattern transformation than either mutation alone and the wing polarity of dsh;sple flies is much less extreme than dsh flies, although sple wings have wild-type polarity (Gubb, 1993 and references).

It seemed unlikely that such a large set of genes should be required for the correct orientation of bristles and hairs, particularly as lack of function of these gene products produces not random polarity but discrete alternative patterns. The demonstration of fine-scale mirror-image duplications in the tarsi and ommatidia shows that the imaginal polarity mutants are affecting cellular polarity at a fundamental level. This makes it all the more surprising that the gene functions should be non-essential. One possibility is that these polarity mutants control related products that are functionally redundant. While this may well be true for some of these products, it would predict that some of the double mutant combinations of viable mutations might be lethal. This is not the case for any of the double mutant combinations of dsh, pk, sple, frz, frtz, fz, fy, in and mwh, with the possible exception of frtz;pk and frtz;fz flies, which eclose at less than 1% the expected frequency. The alternative hypothesis is that these gene products are only required during the last few cell divisions in the imaginal discs, so that even severe perturbations in cell polarity do not cause a major developmental crisis (Gubb, 1993 and references).

The pattern of connections between R1-R6 neurons and their targets in the lamina is one of the most extraordinary examples of connection specificity known. An interwoven set of connections precisely maps R cells in different ommatidia that 'see' the same point in space onto the same group of postsynaptic cells, the lamina cartridge. R1-R6 cells that see the same point in space are distributed over six neighboring ommatidia as a consequence of the curvature of the eye and the angular placement of their light-sensing organelles. Conversely, each of the R1-R6 axons from a single ommatidium sees a different point in space and connects to a different set of lamina target neurons arranged in an invariant pattern. Each cartridge is innervated by a complete set of R1-R6 neurons from six different ommatidia (i.e., an R1 from one ommatidium, an R2 from another, and so on). By superimposing multiple inputs from the same point in visual space upon a single synaptic unit, the signal-to-noise ratio of the response to a signal in the visual field is enhanced. This phenomenon is called neural superposition (Clandinin, 2000 and references therein).

The R1-R6 projection pattern develops in two temporally distinct stages. During the third larval stage, R cells extend axons into the brain, where they terminate between two layers of glia, forming the lamina plexus. These glia act as intermediate targets for R1-R6 neurons. R cell axons induce the differentiation and organization of lamina target neurons and glia. At this stage of development, R cell axons from the same ommatidium form a single fascicle. A column of lamina neurons forms above the lamina plexus, in tight association with a single R cell axon fascicle. By the sequential addition of ommatidial bundles and their associated columns of lamina neurons, a precise retinotopic map forms in which fascicles from neighboring ommatidia terminate adjacent to each other. As lamina neurons differentiate, they send axons along the surface of R cell axons through the plexus and fasciculate with R7 and R8 as they project into the medulla. Although lamina neurons are in close association with R cell axons at this early stage, no synaptic contacts are formed (Clandinin, 2000).

In the second phase of development, ~30 hr after reaching the lamina plexus, R cell axons defasciculate from each ommatidial bundle and project across the surface of the lamina to their synaptic partners, making the pattern of connections characteristic of neural superposition. Growth of R cell axons toward their targets occurs approximately simultaneously in all ommatidial bundles and is presaged by an invariant sequence of contacts between R cell growth cones. This reorganization of terminals converts a strictly anatomical retinotopic map that reflects neighbor relationships between ommatidia into a new topographic map that reflects R cell visual response and reconstructs visual space in the first layer of the optic ganglion (Clandinin, 2000 and references therein).

R cell projections from a single ommatidium display two prominent features. (1) Each R cell axon terminates in an invariant position relative to the other axons from the same ommatidial fascicle. (2) The projection is oriented with respect to the dorsoventral midline of the eye (i.e., the equator), with the R3 axon extending toward the equator -- as a result, the projection patterns on opposite sides of the dorsoventral midline of the eye are mirror images. Using mutations that eliminate specific subsets of R cells or alter ommatidial polarity, tests were performed to see whether R cell synaptic specificity requires interactions among neighboring afferent axons or reflects independent navigation of each axon to its target. It has been demonstrated that interactions between specific R cells are required for target selection, and it is proposed that the precise composition of R cell axons within a fascicle plays a critical role in target specificity (Clandinin, 2000).

Neural superposition was first noted 90 years ago and the R1-R6 connection pattern in the lamina was first described using serial reconstruction of electron microscopic images in 1965. This pattern is cited as a classic example of extreme connection specificity. However, mechanistic analysis of this pattern was prevented by the absence of a rapid method for assessing R cell projections. In particular, the complexity of the pattern precludes conventional approaches based on visualizing all R cell axons in the target region, yet the assessment of connection specificity requires visualization of all R cell axons from one ommatidium. A method has been developed to label individual ommatidia with DiI and visualize the projection pattern using confocal microscopy. R1-R6 axons form a single bundle as they project into the brain. They defasciculate, project across the surface of the lamina, and then turn 90° and extend into the lamina cartridge. R cell axons elaborate a complex en passant presynaptic structure with lamina interneurons within the lamina cartridge. The axons of R7 and R8 project through the lamina, into the medulla. The relative positions of lamina targets chosen by each R1-R6 growth cone are invariant between ommatidia. This labeling method facilitates analysis of R1-R6 projections in various genetic backgrounds and creates a unique experimental system in which synaptic partner choices made by identified neurons can be directly assessed (Clandinin, 2000).

Serial electron microscopic reconstruction studies have revealed that, during pupal development, individual R cell axons leave their original bundle and migrate outward, in the precise direction of their final targets. This process was visualized using confocal microscopy. Early in pupal development, each ommatidial bundle forms a compact mass of expanded growth cones in the lamina plexus. This spherical mass then flattens, as distinct filopodial extensions corresponding to individual R cell axons become visible. This pattern of connections forms within a spatially patterned environment containing lamina target neurons and glial cells, as well as R cell axons. Since extension from the bundle is not preceded by extensive filopodial exploration, interactions between axons within ommatidial bundles may specify the initial trajectory of each growth cone. To address whether cell intrinsic mechanisms or interactions between R cell growth cones or both control target specificity, R cell projections were examined in mutant animals lacking specific subsets of R1-R6 cells. R cell axons from single ommatidia were labeled with DiI and visualized by confocal microscopy. In this series of experiments, animals were analyzed in which the eye was genetically mutant and the lamina neurons and glia in the target were wild type. Three mutant backgrounds were examined: (1) phyllopod, in which R1, R6, and R7 are transformed into nonneuronal cone cells; (2) lozenge^sprite, in which R3 and R4 are transformed into R7 cells; and (3) seven-up, in which R1, R3, R4, and R6 are transformed into R7 cells (Clandinin, 2000).

The first step of lamina target innervation is the coordinated defasciculation of R cell axons from bundles comprising axons from the same ommatidium. To determine whether interactions between specific subsets of R1-R6 axons are necessary for this defasciculation, R cell projections were assessed in phyllopod, seven-up, and lozenge^sprite mutants. In all three of the R cell transformation mutants examined, R cell axons migrated outward from the bundle. In particular, 4 R cell fibers in the lamina of 14/15 phyllopod mutant animals (missing R1, R6, and R7) and 20/24 lozenge^sprite mutants (missing R3 and R4) defasciculated from the bundle and projected to local targets. Similarly, in 17/23 seven-up mutants (missing R1, R3, R4, and R6), it was observed that the two remaining R cell axons defasciculated from the ommatidial bundle and innervated separate cartridges. In some cases, additional R cell axons also defasciculated, consistent with the reported incomplete expressivity of cell fate transformations in these mutants. In each case, axons projected to lamina targets in the local environment of the fascicle terminus. It is concluded that each R cell subtype is programmed to initiate a search for targets in a local region of the lamina target, independent of interactions between other R cell subtypes. In the following sections, whether interactions between specific R1-R6 cells regulate target specificity is assessed (Clandinin, 2000).

Two models could explain the mechanisms that determine the precise projection of R3 and R4 axons toward the dorsoventral midline and, by extension, the relative orientations of the other R cell axons. The growth cones of R3 and R4 may respond to an orienting cue in the lamina that promotes extension toward the dorsoventral midline. Alternatively, the orientation of R cell bodies in the retina may determine the orientation of R cell growth cones in the lamina, independent of any environmental cues. To assess the role of ommatidial polarity on projection specificity, projections from misoriented ommatidia were assessed (Clandinin, 2000).

If a lamina cue can promote equatorial extension of the R3 and R4 axons, ommatidia that rotate incorrectly should project their axons normally, toward the equator. Alternatively, if ommatidial orientation determines the direction of axon projection in the lamina, incorrectly oriented ommatidia should project their R3 and R4 axons away from the equator (Clandinin, 2000).

In wild-type animals, ommatidia are mirror image reflected about the dorsoventral equator of the eye. R cell projections are also mirror image symmetric about the equator but are rotated 180° with respect to the retina. That is, while the R3 cell body is oriented toward the pole in each ommatidium, its axon projects toward the equator in the lamina. This rotation is generated by a twist in the axon fascicle that occurs between the retina and the lamina (Clandinin, 2000).

To test the effects of large changes in ommatidial orientation, two mutations, spiny legs (in homozygous animals) and frizzled (in somatic mosaic animals in which a mutant eye projects to a wild-type target), were examined. In these mutants, ommatidia frequently adopt orientations that are 180° rotated; that is, the R3 cell body is frequently oriented toward the equator in the eye. In these two mutant backgrounds, the orientation of projections from ommatidia that were correctly oriented was normal. Therefore, neither gene is required for R cell axons to respond to orienting cues in the target. However, almost 90% of the ommatidia that were ~180° misoriented in the eye made projections that were also 180° misoriented in the lamina. Rare, abnormal projections of single R cell axons in both of these mutant backgrounds were observed, irrespective of ommatidial orientation. Therefore, the orientation of R cell projections along the dorsoventral axis of the lamina is largely determined by the orientation of ommatidia in the retina (Clandinin, 2000).

Three exceptional cases, in which misoriented ommatidia projected axons toward the equator, were observed. Thus, a cue in the lamina may reinforce the ommatidial orientation cue to ensure the correct direction of outgrowth along the dorsoventral axis. To test whether such a cue contributes to directionality of R cell projections, a mutation that causes a more moderate defect in ommatidial orientation was examined. In nemo mutant animals, ommatidia are misoriented up to 45°. If ommatidial orientation directly determines the directionality of R cell projections, they would be misoriented 45° with respect to the equator; the angle between ommatidial orientation and the axon projection pattern would remain 180°. However, while ommatidial orientation was disrupted in nemo, R cell projections were normal with respect to the equator. This observation suggests that in addition to ommatidial polarity, a cue in the lamina can influence R cell projection orientation (Clandinin, 2000).

It is concluded that interactions between R cell afferents play a crucial role in target specification, and it is proposed that the spatial relationships between axons within a fascicle influence synaptic specificity. It is hypothesized that the interactions between R cell subtypes that are required for target specificity are mediated by direct contacts between specific growth cones. R3 and R4 are required for the remaining R cell axons to choose their normal targets. R1 and R6 are required for R2 and R5 projections but are not required for the projections of R3 and R4. These interactions could occur between growth cones from the same or neighboring ommatidial bundles. The characteristic morphological changes of these growth cones as revealed through electron microscopic reconstruction studies are consistent with the notion that precise spatial relationships between specific growth cones within the lamina plexus are required for these critical interactions to occur. This sequence of interactions determines the relative positions of targets chosen by R cell axons from the same ommatidium (Clandinin, 2000).

R cell transformation mutants could disrupt these interactions in two ways. First, transformation of specific R cells could directly disrupt the instructive signals between R cell growth cones within the plexus that determine growth cone trajectories. Alternatively, these mutations could affect the interactions indirectly, by disrupting the spatial relationships between the remaining R cell axons. That is, outgrowth trajectory could be determined passively by the position each growth cone occupies as it leaves the ommatidial fascicle. In this view, these mutant backgrounds alter the composition of axons within each ommatidial bundle and, hence, disrupt the precise packing of axons within the fascicle. The differential requirements for particular R cell subtypes would reflect their specific roles in directing the spatial relationships between growth cones within the fascicle, rather than interactions between specific growth cones in the target region (Clandinin, 2000).

Ommatidial polarity is defined by the relative positions of R cells within an ommatidium. Each R cell occupies an invariant position; R1-R6 cells within each ommatidium create a pattern that is mirror-image symmetric about the dorsoventral midline of the eye. The observation that ommatidial polarity determines projection orientation requires that the spatial relationships between R cell bodies be maintained in ommatidial axon fascicles. Indeed, a striking feature of Drosophila visual system connectivity is the perfect conservation of spatial relationships between R cell axons, both within each bundle and with respect to the dorsoventral axis of the eye. It is hypothesized that the developmental mechanisms that determine where each R cell differentiates in the retina also control where each R cell axon lies within the fascicle and how the fascicle is oriented along the dorsoventral axis (Clandinin, 2000).

Control of projection orientation by ommatidial polarity also requires that the relative positions of R cell axons within a fascicle, as well as the dorsoventral orientation of the fascicle itself, be 'read out' in the lamina. In this view, the relative positions of axons within the fascicle allows the specific interactions between growth cones that control synaptic specificity to 'self-organize' the pattern of targets. Since axons from both correctly oriented and misoriented ommatidia choose targets arranged in a normal pattern, these interactions between growth cones must occur independent of orientation along the dorsoventral axis. In this model, fascicle orientation determines whether the pattern of targets chosen is oriented either dorsally or ventrally but does not determine the relative positions of the targets within the pattern. This approach of 'encoding' the spatial arrangement of sensory neuron cell bodies within an axon fascicle followed by 'reading out' the preserved orientation cues within the target may provide a general mechanism to generate highly precise patterns of connections (Clandinin, 2000).

Projection specificity in the lamina is not solely controlled by interactions between R cell axons. The observation that the R cell projections are correctly oriented in nemo mutant animals provides evidence that a cue(s) in the target can reorient R cell axons. Such a cue need only orient a subset of R cell axons, likely R3 and R4; these axons could then organize the remaining R1-R6 projections. This cue could be a weak signal that directs R3 and R4 axon outgrowth toward the equator. Alternatively, this cue could simply confine the outgrowth of R3 and R4 to the dorsoventral axis, without determining whether outgrowth is either dorsal or ventral. These target-derived cues could correct for small variations in ommatidial orientation (and fascicle orientation) that exist in the pupal eye prior to programmed cell death in the retina (Clandinin, 2000).

Previous studies suggest that connection specificity can be generated by activity-dependent refinement of synaptic contacts. Indeed, interactions between synapses mediated by electrical activity are clearly important for several aspects of neuronal connectivity in the vertebrate visual system, including the maintenance of ocular dominance columns and formation of eye-specific layers in the lateral geniculate nucleus. In the Drosophila retina-lamina projection, the role of neuronal activity is unclear. These connections likely develop independent of visual input since the projections form when R cells display little light-evoked response. Moreover, phototransduction-defective mutants display normal numbers of R cell termini in each cartridge, suggesting that R cell target selection occurs normally in these backgrounds. These connections also form prior to the development of morphologically distinct synaptic contacts between R cells and lamina neurons (Clandinin, 2000).

To test the effects of changes in neuronal activity and synaptic transmission on R cell targeting, known mutations affecting both sodium and potassium channel subunits, as well as synaptotagmin were examined. None of these backgrounds displays defects in R cell synaptic specificity. However, these genetic approaches are confounded by the significant molecular redundancy present in these protein families within the Drosophila genome. Synaptobrevin-mediated vesicle release was disrupted by expressing tetanus toxin specifically in R cells. Synaptobrevin blockade, however, affects expression of cell adhesion molecules in the Drosophila visual system and disrupts axonal morphology. Hence, it remains unclear whether disruption reflects a role for synaptic transmission in R cell target specificity or is an indirect result of effects on other cellular processes. In summary, while these experiments demonstrate that developmental mechanisms that are likely to be independent of neuronal activity are sufficient to generate the precise pattern of retina-lamina connections, a role for neuronal activity cannot be excluded (Clandinin, 2000).

The cellular mechanisms described here provide a conceptual framework for understanding the molecular basis of synaptic specificity. While the DiI method facilitates the analysis of R1-R6 specificity on a scale sufficient to analyze many mutants, it is too laborious to accommodate large-scale screening. Hence, a genetic screen based on visual behavior driven specifically by R1-R6 is required to extend these studies to the molecular level. A wealth of visual behaviors have been described in Drosophila, one of which, the optomoter response, is mediated by these cells. Techniques that generate mosaic flies in which only R cells are made homozygous for randomly induced mutations, while the rest of the fly is heterozygous, have recently been described. Currently, projects are underway, combining this specific behavioral screen with genetic mosaics, in order to screen for genes controlling R1-R6 synaptic specificity (Clandinin, 2000).

The frizzled gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. This hypothesis was tested in two ways. In one set of experiments the domineering non-autonomy of fz and Vang Gogh (Vang) clones was used to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, pk mutations alter the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments, cells in the pupal wing were ablated. A lack of cells that could be propagating a long-range signal does not alter hair polarity. It is suggested that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal (Adler, 2000).

The directional domineering non-autonomy of fz clones in the wing was originally suggested to be due to a failure in the proximal to distal propagation or transmission of a polarity signal. This model predicted a special population of cells that serve as a source (or origin) of the signal. If this model is correct how could mutations in pk and dachsous (ds) result in an altered direction of fz domineering non-autonomy? An obvious possibility is that these mutations could change the fate of some cells so that an ectopic source of signal was produced. This hypothesis is inconsistent with the cell autonomy/non-autonomy of pk clones. Consider the possibility that pk produces a tissue polarity phenotype by causing the formation of an ectopic source of signal at a new location in the wing. If a pk clone is located in such a region, then the clone would be predicted to show domineering non-autonomy. This is infrequent in pk clones, but is not restricted to clones in one or a few regions of the wing. If a pk clone is located elsewhere, it would be expected to have no consequences for polarity. However, it was observed that cells inside of all pk clones show altered hair polarity. The source and directional transmission model also fails to explain the results of temperature shift experiments with a cold-sensitive fz allele. If fz function is required for the propagation of a signal along the proximal distal axis of the wing it is predicted that a temperature shift from the permissive to the restrictive temperature during the middle of the temperature sensitive period would result in a wing with a permissive phenotype proximally and a restrictive phenotype distally. This is not what was seen. Instead an intermediate phenotype in all regions of the wing is found. This result argues that fz functions in all regions of the wing at the same time and is not consistent with fz functioning in the propagation of a signal down the wing (Adler, 2000).

The complementary nature of the domineering nonautonomy of fz and Vang clones is striking. It is true for the anatomical direction of the non-autonomy (i.e. distal vs. proximal); the relationship of the domineering non-autonomy to the clone (i.e. affected wild-type hairs pointing toward or away form the clone), and for the interactions with pk mutations. It is suggested that the domineering non-autonomy of fz clones is a consequence of a failure of the clone cells to send a locally acting polarity signal. The domineering non-autonomy of Vang clones could be due to the Vang clone sending excess signal (models that reverse this arrangement are also possible) (Adler, 2000).

A model for tissue polarity signaling in the wing is presented. Early models to explain planar polarity in the insect epidermis suggested it could be a reflection of the vector of a concentration gradient and this idea has remained popular. It is suggested that a distal/proximal gradient of fz activity is produced in the early prepupal wing (or wing disc). One way this could be achieved is by a gradient of a Wnt (or other type of ligand) resulting in a gradient of ligand bound Fz. Later in development cells would produce a locally acting second signal in amounts proportional to Fz activity. This hypothetical signal is referred to as Z and it is suggested that ligand bound Fz activates more Z production than unbound Fz. In this way a gradient of Fz activity would be translated into a gradient of Z. Cells would respond by initiating prehair morphogenesis on the side of the cell where Z level was lowest. This would result in hair polarity being oriented in the same direction as the vector of the Z concentration gradient. This is consistent with previous results showing that a directed gradient of fz expression results in cells with higher Fz levels producing hairs that point toward cells of lower levels. The absence of fz activity in clone cells would result in no Z being produced by the clone and a local decrease in Z levels that would cause surrounding cells to produce hairs that point toward the clone as is observed. Such a model can effectively incorporate the affects of pk and ds mutations on the direction of fz domineering non-autonomy. Mutations in these genes could alter the relationship between the ligand bound state of fz and Z production. For example, in a new antimorphic dominant pk allele, pk^D wing unbound Fz receptor could act as a super-activator of Z production. This would lead to a reversed gradient of Z and to the reversal of both polarity and the direction of fz domineering non-autonomy. This model can also explain the observation that cells inside of a pk clone display the same polarity as do cells in a similar position in an entirely pk wing, since the alternative polarity caused by pk mutations would be due to abnormal amounts of Z. Such a model can also explain some of the results seen with Vang. The domineering non-autonomy of Vang could be due to Vang cells being constitutive for the production of high levels of Z. This would lead to locally elevated Z levels and cells surrounding Vang clones producing hairs that point away from the clone, as is observed. The model can also explain the ability of pk^D to enhance the extent of fz domineering non-autonomy and suppress the extent of Vang domineering non-autonomy. In the model the level of Z will be higher in all regions of a pk^D wing since now both bound and unbound Fz receptor will be strongly activating the production of Z. Thus, when a clone of cells lacking functional Fz protein is produced, the difference between the amount of Z produced by the clone cells and their neighbors will be increased over that seen in an otherwise wild-type wing. The ability of pk^D to inhibit the extent of domineering non-autonomy of Vang clones can be explained by the reduced difference in the level of Z produced by the clone and neighboring cells (Adler, 2000).

At first glance the model cannot explain the suppression of Vang domineering non-autonomy in a fz mutant background, because the clone should produce high levels of Z in a background where there is little or no Z produced. One possibility is that in the absence of functional Fz no Z can be produced. A second possibility is that fz has multiple functions in wing tissue polarity and that an additional function is what suppresses the domineering non-autonomy of Vang. The model can also explain the relatively weak and poorly penetrant domineering non-autonomy of pk clones. The cells in such clones would produce aberrant amounts of Z, however the difference between the normal and mutant levels would be less than is seen in a fz mutant clone (that produces no Z) or in a Vang mutant clone (that produces constitutive high levels of Z). Thus, it is reasonable that pk (and ds) clones would show weak domineering non-autonomy (Adler, 2000).

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 (pk^pk-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 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, Pk^pk-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).

Tissue polarity in Drosophila is regulated by a number of genes that are thought to function in a complex, many of which interact genetically and/or physically, co-localize, and require other tissue polarity proteins for their localization. The enhancement of the strabismus tissue polarity phenotype by mutations in two other tissue polarity genes, flamingo and prickle, is reported. Flamingo is autonomously required for the establishment of ommatidial polarity. Its localization is dynamic throughout ommatidial development and is dependent on Frizzled and Notch. Flamingo and Strabismus co-localize for several rows posterior to the morphogenetic furrow and subsequently diverge. While neither of these proteins is required for the other's localization, Prickle localization is influenced by Strabismus function. The data suggest that Strabismus, Flamingo and Prickle function together to regulate the establishment of tissue polarity in the Drosophila eye (Rawls, 2003).

In an attempt to define more precisely the role of Stbm in the tissue polarity pathway, genetic interactions were identifed between stbm and two other tissue polarity genes, fmi and pk. Characterization of the fmi-stbm interaction reveals a requirement for Fmi in ommatidial polarity and a dynamic pattern of Fmi localization that depends on Fz and N. An antibody was raised against Stbm, its subcellular localization was characterized, and the localization of Fmi and Stbm was shown to differ in two ways: first, Fmi is enriched in R4, whereas Stbm is not, and second, Fmi, but not Stbm, is endocytosed. Characterization of the pk-stbm interaction shows that pk enhances the stbm phenotype and that Pk localization requires Stbm (Rawls, 2003).

Three alternatively spliced transcripts are encoded by the pk locus: pk^pk, pk^M and pk^pk-sple. Although these three isoforms differ in the 5' region, they all contain the single PET and three LIM domains characteristic of the Pk protein. PET and LIM domains are thought to mediate protein-protein interactions. Isoform-specific mutations in the 5' region of the transcript result in the pk^pk phenotype, affecting only the wing and notum, whereas mutations in the LIM- or PET-encoding domains result in pk^pk-sple alleles, null alleles that affect the eye, legs and abdomen in addition to the wing and notum (Rawls, 2003).

The observation that Pk distribution is altered in a null stbm background suggests that its localization is, at least in part, dependent on Stbm. The possibility that Pk localization is mediated directly by Stbm has not yet been explored, but the PET and LIM domains are candidates for domain-specific interactions with Stbm. Disruption of these domains would result in genetic null alleles, consistent with the pk^pk-sple phenotype described in this study (Rawls, 2003).

Although ommatidial polarity is not affected in individuals carrying the pk^pk1 allele, this allele enhances the stbm eye phentoype. Functional redundancy could account for the ability of pk to enhance the stbm phenotype such that there is no phenotype when pk is knocked out but a reduction in pk gene dose can be detected by Stbm. Furthermore, the balance of Pk isoforms contributes to the establishment of tissue polarity. Perhaps this balance is also required for Stbm function (Rawls, 2003).

In a deficiency screen, pk was identified as a dominant genetic modifier of stbm. The genetic interaction between stbm and pk may have its basis in a physical interaction that enhances or stabilizes these proteins at the R3/R4 boundary. To explore this possibility, Stbm localization was examined in a pk mutant background, and Pk localization in a stbm mutant background. Stbm localization does not appear to be affected in a pk^eq background (a genetic null that fails to complement pk^pk-sple alleles). However, Pk localization is disrupted in a stbm^6cn null background. The distribution of Pk was characterized in wild-type eye imaginal discs; it is indistinguishable from that of Stbm. Pk is significantly reduced overall in the stbm^6cn background. While some protein does accumulate at the boundary between R3 and R4, Pk is not detectable at the R8/R1/R7/R6 boundary. Physical interactions have not been demonstrated between either of these proteins, nor have genetic interactions between fmi and pk been shown. These data are consistent with the possibility that Stbm, Fmi and Pk may all function together in a complex (Rawls, 2003).

In order to differentially affect signal transduction through the N pathway, the assembly and/or activity of proteins that set up polarity must be different in R3 and R4. The model presented below requires that Stbm and Pk be restricted to the R4 cell to properly modulate N signaling. Stbm has been shown to be restricted to R4 at the R3/R4 boundary; the subcellular location of Pk in the eye has not yet been determined (Rawls, 2003).

It is proposed that the direct interaction between N and Dsh blocks N signaling, and that the different subset of proteins bound to Dsh is the basis of the asymmetry of the complex. In the future photoreceptor R3, N binds Dsh (which is part of the Fmi/Diego/Dsh scaffold) thereby inhibiting N activity in R3. In the future R4 cell, where Stbm and perhaps Pk are localized, Fmi, Diego and Dsh also form a complex. However, in this case, the re-organization of the Fmi/Diego/Dsh complex to include Stbm and Pk bound to Dsh may prevent N from binding to Dsh, leading to high levels of N-mediated signaling in R4. Ultimately, these differences in gene activity in the R3 and R4 precursors direct the fate specification of these cells (Rawls, 2003).

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).

A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple^13/26), neighbor number was quantitated over time (Classen, 2005).

pk-sple^13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple^13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk¹ mutant wings are even more irregularly packed than pk-sple^13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk¹ wings. Even four-sided cells accumulate significantly in pk¹ mutant wings. Individual cell contact lengths are also much more variable; while pk-sple^13/26 boundary lengths were 9% more variable than wild-type, those of pk¹ were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).

Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)³, are mild but significant. The null allele fmi^E59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmi^E59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmi^E59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).

The packing geometry was examined of two different frizzled (fz) alleles, fz^R52 and fz^P21. fz^P21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fz^P21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fz^P21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fz^R52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fz^P21, suggesting that little repacking occurs in fz^R52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).

stbm⁶ and dgo³⁸⁰ mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple^13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).

To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmi^E59) or nonautonomous (fz^R52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fz^R52 and fmi^E59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fz^R52 and fmi^E59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fz^R52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmi^E59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).

To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).

In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).

At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).

It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo³⁸⁰, stbm⁶, stbm¹⁵³, stbm^D, stan³, pk-spl¹, or pk¹, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk¹ or shi;dgo³⁸⁰ mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).

Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fz^P21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmi^E59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).

To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).

To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).

The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).

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 pk^pk 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 pk^pk30 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 pk^pk 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 pk^pk 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 pk^pk-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 pk^pk 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 pk^pk mutant anterior wing are lost in a pk^pk-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 pk^pk 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 pk^pk 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 pk^pk mutant wing. According to this model, cell polarization occurs in the Early PCP Phase in a pk^pk 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).

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. This study presents data showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point - the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance (Mrkusich, 2011).

The dorsal cluster sensory axon stalling defect reported in this study in the pk mutant is the first demonstration in Drosophila of a role for a core PCP gene other than flamingo in regulation of neurite growth. This finding raises the question as to whether prickle and flamingo interact in this context. The similarity in the phenotypes of fmi RNAi and pk mutant embryos supports this proposition and further weight is provided by the finding that embryos lacking a single copy of both fmi and pk show the same axon stalling phenotype as pk homozygotes. A pk-fmi interaction is not required for axon advance in all sensory neurons, since lateral cluster axons grow into the CNS normally in pk mutant embryos (Mrkusich, 2011).

One possible molecular mechanism for a Prickle-Flamingo interaction -- direct binding between Prickle protein and the intracellular tail of Flamingo -- is not supported by the current study. A mutant Flamingo protein lacking the C-terminal tail is at least partially effective in rescuing the fmi mutant phenotype. Whether Prickle interacts directly with Flamingo in some other way, or whether an intermediary protein is involved, remains to be established (Mrkusich, 2011).

Do other core PCP genes cooperate with flamingo and prickle in regulating sensory axon growth? Whereas Frizzled has been shown to be necessary for the formation of several axon tracts in the mouse brain, there is no apparent requirement for the Drosophila homologue to promote sensory axon advance. It was previously found that sensory axon growth is normal in frizzled mutant embryos and, in addition, no signs of sensory axon stalling was seen in frizzled RNAi embryos (Mrkusich, 2011).

However, an axon stalling phenotype similar to pk mutants was observed following overexpression of Dishevelled in all sensory neurons. This finding mirrors findings in mammalian cell culture systems: overexpression of murine Prickle1 or Prickle2 induces neurite outgrowth from neuroblastoma cells and this effect is repressed by over-expression of Dishevelled1. It is concluded that in Drosophila, Prickle promotes axon extension by inhibiting Dishevelled-mediated repression of axon growth, a hypothesis that could be tested by jointly manipulating levels of Prickle and Dishevelled expression in embryonic sensory neurons. Dishevelled has been implicated in regulation of neurite growth in other systems, but as part of a canonical Wnt signalling pathway that diverges downstream of glycogen synthase kinase (Gsk3β), leading to direct remodelling of microtubules in the growth cone. It will be interesting to see whether the same molecular components are employed downstream of Dishevelled in growing sensory axons in Drosophila (Mrkusich, 2011).

The site of sensory axon stalling in pk mutants -- the border between the CNS and the PNS -- has been previously recognised as a key choice point for growing axons in both insects and vertebrates. The entry point to the CNS is the most common stall point for dorsal cluster sensory axons in neuroglian mutants. In Drosophila, peripheral glial cells play an important role in enabling both efferent motor axons and afferent sensory to advance across this transition zone. Future studies should examine whether prickle mediates an interaction between axons and glial cells to promote their continued growth at this site (Mrkusich, 2011).

In conclustion, similarities in LOF mutant phenotypes and the presence of axonal growth defects in transheterozygous embryos provide evidence that flamingo and prickle interact in regulating sensory axon advance in the Drosophila embryo. The C-terminal cytoplasmic tail of Flamingo appears not to be necessary for this interaction. Overexpression experiments suggest that another core PCP gene, dishevelled, acts downstream of flamingo and prickle to regulate axon advance. Prickle may promote axon extension by inhibiting Dishevelled-mediated repression of axon growth. In summary, it appears that regulation of sensory axon advance in the Drosophila embryo is achieved by using some of the components of the PCP pathway in an altered molecular setting (Mrkusich, 2011).

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially, was analyzed. Components of the Wnt/Planar Cell Polarity (PCP) pathway were found to control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus (Ng, 2012).

Branching morphogenesis is a key feature in axons and dendrites. This study shows that Wnt/PCP proteins, known for their roles in tissue polarity, are involved in two aspects of axon branch formation: stereotyped extension and collective decision-making. Some PCP mutants have a preference for dorsal or medial branching phenotypes. As the genetic results suggest that Fz and Stbm functions are separable, this is distinct from some tissue polarity models that have been presented for Drosophila patterning. The results suggest that PCP proteins have complex cross-regulatory effects that include the spatial regulation of Pk. A balance of their functions is key to stereotyped branching and to axon growth and guidance (Ng, 2012).

An independent study recently reported similar results for Wnt/PCP components in MB axon branching, guidance and growth (Shimizu, 2011). When comparing the results with those described in this study, most of the data are consistent. Slight differences, such as the immunohistochemistry results and the role of Prickle, might be a result of differences in preparative methods, stages at which animals were analyzed and the genetic background of the animals used (Ng, 2012).

Branched axons and dendrites tend to follow a bifurcation motif. In MB axons, this corresponds to a single dorsal and a single medial projection. This study shows that PCP proteins control the extension of axon branches, rather than the initial branch formation. The working model is that growing axons can initially reach the axon peduncle and bifurcate independently of the Wnt/PCP pathway. Once the nascent branches have formed, Fz and Stbm direct the stereotyped extension of branches. Fz is the major determinant of dorsal projections. By contrast, Stbm predominantly controls medial projections. The results suggest that these are partly redundant systems; dorsal and medial projections also require minor contributions from Stbm and Fz, respectively. In the absence of both, branching errors are increased and randomized. In some cases, both extensions are lost. Axon branching relies mainly on mutual cooperation between Fz and Stbm. In some cases, antagonism between Stbm and Fz exists, although there is no evidence of Stbm inhibiting Fz signals. These results are reminiscent of observations in Drosophila showing that Fz and Stbm can exhibit redundant and cooperative PCP functions in the wing as well as in sensory organ precursor cell fate specification (Ng, 2012).

MB axons follow a branching mode that reflects collective behavior. The initial results suggest that PCP mutant phenotypes are recessive. Next to wild-type axons, PCP mutant axons tend to adopt a wild-type branched pattern. However, with the GFPstbm rescue paradigm, the results together suggest that it is the collective mix of mutant and non-mutant neurons that determines whether an axon branches or not. How is this regulated? One possibility is that as MB axons are fasciculated, cell-cell contacts can regulate collective branching behavior. Although axons are long and thin, it is unlikely that 1000 branched αβ neurons in each hemisphere are in contact with each other. It is more likely that, as in other Drosophila tissues, it is the behavior of cells that are widely interlinked together that is controlled by PCP factors. The non-autonomous results presented in this study differ significantly from previous PCP studies. Previous results in Drosophila wing and eye show that PCP mutations result in both cell-autonomous and non-autonomous tissue polarity defects. In this study PCP branch functions are not fully cell-autonomous. Furthermore, if domineering non-autonomy exists, it does not strongly affect wild-type axon branching (Ng, 2012).

Despite the uncertainty about its role, asymmetric organization of PCP proteins is a key feature in tissue polarity. However, there are variations to this theme. For example, although Fz and Stbm are often on opposite poles, in mouse eye lens fiber cells they appear in the same tissue pole. In zebrafish and ascidian models in which PCP signals control the convergence and extension of cells in the developing notocord, PCP protein localizations are variable. These and other observations have led to the suggestion that in motile cells, PCP proteins might be dynamically regulated. In short, despite PCP mutants having different preferences in dorsal or medial branch defects, no obvious PCP protein asymmetry can be found between axon branches. One possibility is that these proteins are regulated dynamically, therefore such differences are not readily observable in fixed tissue. Another possibility is that distinct branching fates might arise from differences in PCP effector functions between branches (Ng, 2012).

The observation that Pk axonal branch levels are reduced in fz and stbm mutants suggests that Pk regulation forms part of PCP branch functions. This is likely to be post-translational and axonal-specific as Pk localization in MB dendrites is unaffected by fz or stbm. This is similar to Drosophila wing patterning for which Fz loss also results in a cell-autonomous reduction of Pk. However, in the wing, Dsh, Fz and Stbm also regulate the intercellular accumulation of Pk at the cell cortex, along the pole vertices. Also, in contrast to some wing models, this study shows that Fz and Stbm branch functions can still occur independently of Pk (Ng, 2012).

Several results suggest that PCP proteins function early during axonal development. First, Pk expression is higher in the αβ core region, where newborn developing MB axons are located. Further evidence from the stbm rescue studies shows that GFPstbm expression in the αβ core alone is sufficient to rescue axon branching. Also, branching defects were apparent in PCP mutants at early stages of development. Lastly, Pk and Dsh αβ localization can also be detected at these stages (Ng, 2012).

How Wnt/PCP proteins interact has been studied extensively in Drosophila. This study shows that the MB axon phenotypes reveal some novel features of PCP signaling interactions. As already mentioned, Fz and Stbm play partly redundant, parallel roles. Although Pk controls axon branching and is regulated by Fz and Stbm, it does not account for all Fz and Stbm functions. This is also evident from other PCP studies. However, Pk does act additively to Fz and Stbm and has convergent as well as antagonistic functions with Dsh. Also, this study has shown that Dsh does not simply transduce Fz or Stbm signals. This was also reported in some studies. The working model is that both Fz and Stbm are likely to function in part through Dsh and Pk. Based on further experiments, their effects are likely to be complex and context-dependent on the activity levels of other PCP proteins (Ng, 2012).

Previous reports suggest that these factors interact physically with each other. There is evidence that the extracellular regions of Stbm and Fz can physically bind to each other, although this is debated. Physical associations between Stbm-Dsh and Fz-Dsh have been documented in vertebrate and fly proteins. Apart from Stbm, Pk also interacts physically with Dsh. Recent reports suggest that mouse Stbm (Vangl2) and Dsh (Dvl1) interact antagonistically to regulate Frizzled3 (Fzd3) phosphorylation. Whether these interactions affect axon branching is currently unknown. Nonetheless, novel Wnt/PCP interactions are involved in MB axon patterning (Ng, 2012).

In conclusion, this study demonstrates that Wnt/PCP proteins are key regulators in the stereotyped growth of axon branches. Given that branched growth is intrinsic to neuronal morphogenesis, Wnt/PCP components and their reported interactions might also be involved synapse and dendrite formation. Such highly cross-regulated programs are probably essential for the intricate patterning of the nervous system (Ng, 2012).

prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. This study shows that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. Enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, it was shown that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult Prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport (Ehaideb, 2014).

EVOLUTIONARY HOMOLOGS

Two new cDNAs have been cloned and sequenced that code for proteins carrying the related triple LIM domains (acronym of Lin-11, Isl-1, Mec-3) proteins. These LIM domains show good agreement to the LIM domain consensus sequence, but also exhibit some novel variations. The 1.36-and 2.8-kb cDNAs are probably splice variants of one gene and code for 42- and 50-kDa proteins, respectively. The larger transcript has a 900-nucleotide (nt) 3' untranslated region (UTR). High levels of the 2.8-kb transcript can be detected in many tissues, and all tissues show some level of expression of both transcripts, the larger transcript being more abundant. In adult testis there are very high levels of the 1.36-kb transcript and moderate levels of the 2.8-kb transcript. The wide tissue distribution and high levels of expression suggest an important role for these proteins in cellular function (Divecha, 1995).

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis. A noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila, participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, it is shown that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene, is an essential component for gastrulation cell movement. Xpk encodes an 835-amino acid protein with a single PET domain and three repetitive LIM domains in its N-terminal half. Both gain-of-function and loss-of-function of Xpk severely perturbs gastrulation and causes spina bifida embryos without affecting mesodermal differentiation. XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation (Takeuchi, 2003).

The possibility that XPK activates JNK was examined because JNK has been reported to act in the noncanonical Wnt pathway downstream of Dsh. To evaluate JNK activation, the phosphorylation of a target of JNK, c-Jun, was tested in HEK293T cells transfected with Xdsh, Xpk, or both cDNAs. Xpk alone failed to activate JNK, whereas Xdsh activates JNK in a dose-dependent manner. However, cotransfection of Xdsh with Xpk but not with ΔP/L (lacking the PET and LIM domains) cDNAs dramatically increase the level of c-Jun phosphorylation, even at Xdsh levels, which alone cannot activate JNK efficiently. This result suggests that XPK cooperates with Xdsh to activate JNK through its P/L domain. At a high level of Xdsh protein, Xdsh alone can activate JNK to a certain level, and interestingly, wild-type Xpk or ΔP/L but not P/L suppresses the JNK activation by Xdsh, suggesting that the part of XPK excluding the PET/LIM domain may act negatively to Xdsh-mediated JNK activation at high levels of Xdsh. This is consistent with the observation that ΔP/L and Xpk including the LIM domain counteract each other. These observations prompted a test of whether XPK interacts physically with Xdsh. To test this possibility, a GST-pull-down assay of tagged XPK and Xdsh was carried out in HEK293T cells. Xdsh and XPK are each efficiently precipitated with the other's GST fusion protein, indicating that XPK and Xdsh interacted physically with each other. The interaction between XPK and Xdsh is conserved among species; the Drosophila Prickle PET/LIM domain has been reported to bind Dsh. The yeast two-hybrid assay also demonstrates that the PET/LIM domains of XPK are sufficient to bind Xdsh (Takeuchi, 2003).

It is speculated that XPK might act as a scaffold for JNK activation, so whether XPK binds to JNK was tested. Neither the PET/LIM nor the ΔPET/LIM is sufficient to bind JNK, and only wild-type XPK can bind JNK significantly. Nevertheless, this further suggests that XPK forms a ternary complex with Xdsh and JNK. Although this possibility was examined, the formation of the ternary complex could not be demonstrated (Takeuchi, 2003).

It has been proposed that Prickle generates asymmetric Frizzled and Dishevelled localization in the Drosophila wing, through the suppression of Fz and Dsh localization at the proximal cell cortex. In this study, it is shown that XPK is a key component connecting Xdsh to JNK activation during Xenopus gastrulation. It has been predicted from Drosophila genetics that JNK is one of the downstream targets of the PCP pathway. These results reinforce the idea that the noncanonical Wnt (PCP) pathway regulates gastrulation cell movements in vertebrates through JNK activation. To further understand the pathway, attempts are currently being made to identify XPK-interacting components that regulate JNK activation (Takeuchi, 2003).

In addition to the canonical Wnt/ß-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila and the Wnt/calcium pathway in vertebrate embryos. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation. To further test this hypothesis, two zebrafish homologs were identified of the Drosophila PCP gene prickle (pk), both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/ß-catenin signaling. Like noncanonical Wnts, overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. These results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways (Veeman, 2003).

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterized by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. A noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. A zebrafish homolog of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP, has been isolated and functionally characterized. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localization of Dsh by downregulating levels of Dsh protein. Furthermore, pk1 is shown to interact genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway (Carreira-Barbosa, 2003).

Regulation of planar cell polarity by Smurf ubiquitin ligases

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. This study reports an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. This work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE (Narimatsu, 2009).

Mechanistic analysis of PCP signaling has revealed physical interactions between otherwise differentially localized PCP components that include interactions between Vangl, Prickle and Dvl. This study has mapped interactions between Smurfs and components of the Wnt signaling pathway using a systematic screen and identified an interaction between Smurf and Dvl that is dependent on phosphorylation of Dvl. Accordingly, a DEP domain mutant that blocked Dvl phosphorylation also blocked Smurf interaction, as did a DEP mutation that is PCP-specific in the fly. These findings are in agreement with key roles previously defined for the DEP domain in PCP signaling in flies and vertebrates. Constitutive interactions were identified between Par6 and Dvl, consistent with previous observations in neurons, as well as an interaction between Par6 and Prickle. Together, these results suggest a model in which Par6 is engaged in a trimeric complex with Prickle and Dvl that upon phosphorylation-dependent recruitment of Smurf to Dvl in response to Fzd signaling leads to Prickle ubiquitination and degradation. Consistent with this, it was found that Wnt5a induced Smurf-Par6 interactions and that Prickle bound to Par6 was subject to Smurf-dependent ubiquitination and turnover that was also dependent on the Dvl binding domain of Par6. Moreover, in Smurf mutants increased levels of Pk1 protein the following two observations were made: (1) loss of the local asymmetric distribution of Pk1 in the neuroepithelium, and in the cochlea, (2) Pk1 that was normally localized in a medial crescent in the OHCs, extended to the lateral side (Narimatsu, 2009).

Dvl has also been shown to interact with the transmembrane protein Vangl, which also binds Pk and Pk can antagonize Dvl function. Moreover, Diego, an ankyrin repeat containing protein has been shown to directly compete with Pk for Dvl binding and can promote Dvl function in the PCP pathway via recruitment to membranes. Whether Diego functions primarily on Dvl, or plays a role in antagonizing Pk coassembled with Par6, perhaps in cooperation with Smurfs is unknown, but altogether these findings suggest that Pk and Dvl assemble via unique signaling complexes that respond to different extrinsic cues during PCP signaling. Consequently, while all PCP components might physically interact with each other, their asymmetric distribution may be maintained through dynamic modulation of complex membership (Narimatsu, 2009).

Although PCP and CE have been linked via shared components and a key role for noncanonical Wnt signaling, the relationship of the molecular pathways governing tissue polarity to CE has been less clear. In particular, during CE the neuroepithelium and the underlying axial mesoderm do not display the highly organized structures typically associated with PCP and tissue polarity and while Vangl and Dvl show asymmetric distribution in the cochlea, there is no evidence for asymmetric distribution of either protein during CE in the mouse. Regardless, Par6-dependent asymmetric localization of endogenous Pk1 was clearly detected in neuroepithelial cells; this was manifest at the cellular level, and in Smurf DKO mutants this asymmetry was lost. Furthermore, Par6 can also control the stable mediolateral extension of cell protrusions during CE. Localized Fzd signaling may thus regulate the asymmetric distribution of specific PCP components such as Pk1 in the absence of asymmetric distribution of all PCP pathway components. Vertebrates may thus broadly employ PCP signaling to control polarity at the local and even cellular level. Consistent with this, noncanonical Wnt signaling, Dvl and Par6 have all been shown to play key roles in regulating directed cell motility and the polarity of neurons (Narimatsu, 2009).

These results demonstrate a noncanonical Wnt signaling pathway in which Smurf is recruited to Par6 via Dvl to regulate the degradation of PCP pathway components that in turn controls the asymmetric distribution of Pk1. Signal dependent degradation of PCP components may thus allow for dynamic use of the pathway in a local manner during convergent extension, as well as in the establishment of tissue polarity in organized epithelia such as the inner ear (Narimatsu, 2009).

Zebrafish Prickle1b mediates facial branchiomotor neuron migration via a farnesylation-dependent nuclear activity

The facial branchiomotor neurons (FBMNs) undergo a characteristic tangential migration in the vertebrate hindbrain. A morpholino knockdown approach has been used to reveal that zebrafish prickle1b (pk1b) is required for this migration. This study reports that FBMN migration is also blocked in a pk1b mutant with a disruption in the consensus farnesylation motif. It was confirmed that this lipid modification is required during FBMN migration by disrupting the function of farnesyl biosynthetic enzymes. Furthermore, farnesylation of a tagged Pk1b is required for its nuclear localization. Using a unique rescue approach, it was demonstrated that Pk1b nuclear localization and farnesylation are required during FBMN migration. The data suggest that Pk1b acts at least partially independently of core planar cell polarity molecules at the plasma membrane, and might instead be acting at the nucleus. It was also found that the neuronal transcriptional silencer REST is necessary for FBMN migration, and evidence is provided that interaction between Pk1b and REST is required during this process. Finally, it was demonstrated that REST protein, which is normally localized in the nuclei of migrating FBMNs, is depleted from the nuclei of Pk1b-deficient neurons. It is concluded that farnesylation-dependent nuclear localization of Pk1b is required to regulate REST localization and thus FBMN migration (Mapp, 2011).

PRICKLE1 interaction with SYNAPSIN I reveals a role in autism spectrum disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, studies were carried out in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. Mice with Prickle1 mutations were shown to exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, a yeast two-hybrid screen was performed with a human brain cDNA library and a peptide was isolated with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles (Paemka, 2013).

Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a

Ror2 gene defects, prompting an exploration of an association of Prickle1 with the Wnt pathway. This study shows that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. These studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome (Liu, 2014; PubMed).

Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth

PCP proteins maintain planar polarity in many epithelial tissues and have been implicated in cilia development in vertebrate embryos. This study examined Prickle3 (Pk3), a vertebrate homologue of Drosophila Prickle, in Xenopus gastrocoel roof plate (GRP). GRP is a tissue equivalent to the mouse node, in which cilia-generated flow promotes left-right patterning. Pk3 was shown to be enriched at the basal body of GRP cells but is recruited by Vangl2 to anterior cell borders. Interference with Pk3 function disrupted the anterior polarization of endogenous Vangl2 and the posterior localization of cilia in GRP cells, demonstrating its role in PCP. Strikingly, in cells with reduced Pk3 activity, cilia growth was inhibited and gamma-tubulin and Nedd1 no longer associated with the basal body, suggesting that Pk3 has a novel function in basal body organization. Mechanistically, this function of Pk3 may involve Wilms tumor protein 1-interacting protein (Wtip), which physically associates with and cooperates with Pk3 to regulate ciliogenesis. It is proposed that, in addition to cell polarity, PCP components control basal body organization and function (Chu, 2016z).

Wnt proteins can direct planar cell polarity in vertebrate ectoderm

The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. This study shows that in Xenopus early ectoderm, the Prickle3/Vangl2 complex (see Vang) was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists. In midgastrula embryos, Wnt5a, Wnt11, and Wnt11b, but not Wnt3a, acted across many cell diameters to orient Prickle3/Vangl2 complexes away from their sources regardless of their positions relative to the body axis. Planar polarity of endogenous Vangl2 in the neuroectoderm was similarly redirected by an ectopic Wnt source and disrupted after depletion of Wnt11b in the presumptive posterior region of the embryo. These observations provide evidence for the instructive role of Wnt ligands in vertebrate PCP (Chu, 2016b).

Vangl2 interaction plays a role in the proteasomal degradation of Prickle2

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, the regulation of Prickle2 protein levels was studied in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2; see Drosophila Van Gogh). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours (Nagaoka, 2019).

Spatial and temporal analysis of PCP protein dynamics during neural tube closure

Planar cell polarity (PCP) controls convergent extension and axis elongation in all vertebrates. Although asymmetric localization of PCP proteins is central to their function, little is understood about PCP protein localization during convergent extension. This study used quantitative live imaging to simultaneously monitor cell intercalation behaviors and PCP protein dynamics in the Xenopus laevis neural plate epithelium. Asymmetric enrichment of PCP proteins was observed, but more interestingly, tight correlation of PCP protein enrichment was observed with actomyosin-driven contractile behavior of cell-cell junctions. In addition to expected patterns of spatial asymmetry, PCP protein enrichment is tightly linked to cell-cell junction behavior: Prickle2 (Pk2) (Drosophila homolog: Prickle) and Vangl2 (Drosophila homolog: Van Gogh) were dynamically enriched specifically at shrinking cell-cell junctions and depleted from elongating junctions during cell intercalation. The turnover rates of junctional PCP proteins also correlated with the contractile behavior of individual junctions. All these dynamic relationships were disrupted when PCP signaling was manipulated. Together, these results provide a dynamic and quantitative view of PCP protein localization during convergent extension and suggest a complex and intimate link between the dynamic localization of core PCP proteins, actomyosin assembly, and polarized junction shrinking during cell intercalation in the closing vertebrate neural tube (Butler, 2018).

Prickle2 and Igsf9b Coordinately Regulate the Cytoarchitecture of the Axon Initial Segment

Prickle2 (see Drosophila Prickle) has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. In this study, because Igsf9 family proteins are associated with psychiatric diseases and seizures, the physiological interaction between Prickle2 and Igsf9b was studied. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy (Chowdhury, 2020).

REFERENCES

Search PubMed for articles about Drosophila prickle

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