Van Gogh/strabismus


REGULATION

Protein Interactions

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 pksple1 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).

Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells

Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk), which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Galphai complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. It is proposed that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity (Bellaïch, 2004).

Planar polarization of the pI cell occurs prior to division and is required, upon entry into mitosis, to direct the Dlg-Pins-Galphai and Baz-Par6-aPKC complexes at the anterior and posterior cortex, respectively. The localization of Pins at the anterior cortex is regulated positively by the Stbm-Pk complex and negatively by Dsh. (1) Loss of stbm activity results in a delay in the cortical localization of Pins during prophase; (2) concomitant expression of Stbm and Pk leads to a broadening of the cortical crescent of Pins at prophase; (3) loss of dsh PCP activity similarly results in an extended Pins crescent at prophase. Moreover, analysis of the defective partitioning of Pon::GFP suggests that the Stbm-Pk complex acts antagonistically to Dsh to localize at the anterior cortex a centrosome-attracting activity. It is proposed that the Stbm-Pk complex organizes the anterior cortex and recruits the Dlg-Pins-Galphai complex as well as molecules regulating spindle positioning (Bellaïch, 2004).

Cortical localization of Pins is a novel read-out of PCP signaling in the pI cell that is distinct from the ones previously identified in wing and eye cells. In wing epidermal cells, Fz promotes the formation of a polarized actin cytoskeleton via a pathway that possibly involves a direct interaction between Dsh and a Daam1-Rho complex and a Rho Kinase-dependent phosphorylation of cytoplasmic myosin. Whether Dsh also regulates microfilament assembly in pI cells remains to be studied. In photoreceptor cells, the read-out for PCP signaling is the transcriptional regulation of the Delta gene in R3. Thus, the conserved core of PCP signaling molecules have different, cell-type specific read-outs (Bellaïch, 2004).

How does Stbm direct the localization of Pins to the anterior cortex? One hypothesis is that Stbm directs the anterior localization of Pins via the regulated assembly of a Stbm-Dlg-Pins complex. The anterior accumulation of Pins depends on its interaction with Dlg. Evidence is provided that Stbm may bind Dlg. (1) In vitro binding studies indicate that Stbm interacts with Dlg. It is noted, however, that PDZ-containing proteins other than Dlg may also bind Stbm in this assay. (2) The localization of Stbm overlaps with the distribution of Pins and Dlg in dividing pI cells. (3) The PBM motif of Stbm appears to regulate the re-localization of Stbm in pI cells. The data are therefore consistent with a model in which, upon mitosis, the binding of Stbm to Dlg in turn promotes the binding of Pins to Dlg and, hence, localization of Pins at the anterior cortex where Stbm and Dlg accumulations overlap. This model predicts that the PBM of Stbm should be required for the anterior localization of Pins. It was found, however, that StbmDeltaPBM is fully functional and that Pins is properly recruited at the anterior cortex in stbm6c mutant pI cells expressing StbmDeltaPBM. One interpretation of this result is that Stbm regulates the localization of Pins not only via the PBM-dependent assembly of the Dlg-Pins complex but also via a second PBM-independent mechanism. Since Dsh acts redundantly with Baz to localize Pins asymmetrically, it is suggested that this second mechanism may involve Dsh. Accordingly, in stbm6c mutant pI cells, uniformly distributed Dsh activity would prevent Pins cortical localization. By contrast, since the PCP function of stbm does not depend on its PBM, the activity of Dsh should be restricted to the posterior cortex in stbm6c mutant pI cells expressing StbmDeltaPBM. Dsh should therefore restrict Pins localization to the anterior cortex in this mutant background. Another interpretation of the correct localization of Pins in stbm6c mutant pI cells expressing StbmDeltaPBM is that Stbm recruits Pins via a mechanism that does not involve an interaction with Dlg (or any other PDZ-containing proteins). Future studies will address how the Stbm-Pk complex regulates the localization of Pins in the pI cell (Bellaïch, 2004).

Different mechanisms appear to cooperate to maintain Pins asymmetric localization. baz is required for the asymmetric localization of Pins in the absence of dsh PCP activity. This indicates that Baz can regulate the maintenance of Pins asymmetric localization at prometaphase. The loss of asymmetric localization of Pins in dsh baz mutant pI cells suggests that Dsh may also contribute to maintain Pins asymmetric localization at prometaphase. Dsh does not merely act by excluding Stbm, a positive regulator of Pins localization in prophase, because Pins localizes asymmetrically in baz stbm double mutant pI cells. The mechanisms by which Baz and Dsh regulates Pins localization are not known. However, because Pins regulates its own localization via a Gß13F-dependent positive feedback loop, one hypothesis is that Baz and/or Dsh negatively regulates Gß13F signaling activity (Bellaïch, 2004).

One of the best examples of PCP in mammals is the stereotyped planar orientation of the stereociliary bundles that are located at the apical cortex of each mechanosensory hair cell within the cochlea. In these cells, the first sign of polarization is the stereotyped movement, at the luminal surface of the cell and along the neural-abneural axis, of the kinocilium, the single tubulin-based cilium, from the center towards the abneural pole of the cell. Recently, a mutation in a stbm homolog, Vangl2, has been shown to result in the defective orientation of the stereociliary bundles. This planar cell polarity defect appears to result from the randomly oriented center-to-periphery movement of the kinocilium. Because LGN, a mammalian homolog of Pins, is known to regulate microtubule stability, it is tempting to speculate that Vangl2 may regulate via LGN a microtubule-dependent process regulating kinocilium movement along the neural-abneural axis. Future studies will reveal whether the regulation of Pins/LGN cortical localization is a conserved read-out of PCP (Bellaïch, 2004).

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).

The Frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/β-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz- patches reorient toward the fz- cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Un vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, biochemical and physical interactions have been demonstrated between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). This function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. The data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field (Wu, 2008).

The data suggest that the Fz ECD, including the CRD, acts as a Vang/Stbm ligand in nonautonomous signaling. How do these data and interpretations fit with other existing results and models? The Fz CRD is clearly dispensable for canonical Wg signaling in vivo. Previous studies have shown that it is essential for PCP signaling; however, the specifics of when and where have been controversial. A recent paper suggests that the CRD is not strictly required for PCP establishment, as FzΔCRD can partially rescue fz mutant phenotypes in the wing. However, some PCP defects remain. It is also worth noting that in experiments where the multiple wing hair phenotype of fz wings were assayed, this serves as a marker for late stage cell-autonomous Fz functions and does not address whether FzΔCRD is functional in intercellular nonautonomous communication. The experiments indicate that FzΔCRD does not fully rescue fz PCP phenotypes and does not affect domineering nonautonomy of fz mutant clones. Thus, it is concluded that the CRD of Fz is necessary for cells to send polarizing signals to neighboring cells (Wu, 2008).

Genetic and physical interaction data suggest that at the early PCP signaling stage (14-24 hr APF), Vang/Stbm functions as a receptor for FzCRD. As such, it would appear that Vang/Stbm senses how much Fz (activity) is present on adjacent cells and relays this information, causing a cell to orient toward the neighboring cell with lower Fz level/activity. The conclusion that Fz 'signals' and Vang 'receives the signal' is consistent with previous models, in that a fz cell at the clone boundary will orient toward the center of the mutant area as it compares levels of its two neighboring cells (one of which is the wild-type cell adjacent to the clone). Similarly, it has been shown that Vang is not needed in the 'sending' cell, which is consistent with the result that fz Vang double mutant clones behave like fz clones. How do these observations fit with the nonautonomous behavior of Vang clones? In Vang mutant cells, all Fz protein accumulates at the membranes abutting wild-type cells; wild-type cells at the clonal border would therefore presumably detect more Fz in Vang cells. The model would predict that this relocalization of Fz causes these cells to orient away from the Vang neighbors. This interpretation is also consistent with the Vang phenotype being suppressed in fz Vang double mutant clones, suggesting that the nonautonomous effect is mediated largely through Fz (Wu, 2008).

Fz-Vang/Stbm interactions are dependent on the presence of the Fmi (also known as Stan) protein but are independent of the core PCP factor Dsh. Similarly, they are independent of Pk, which mediates the cell-autonomous requirement for Vang/Stbm. It is important to note that Vang/stbm mutants affect fz nonautonomy differently from pk mutants: Vang/stbm backgrounds suppress the domineering nonautonomy of fz clones (consistent with the model), whereas pk mutants enhance the nonautonomous effects. These data suggest that during early nonautonomous PCP signaling, Fz-Vang/Stbm effects are independent of Pk. It is thus likely that there are two distinguishable phases of Fz-Vang/Stbm interactions: the nonautonomous phase addressed here (14-24 hr APF and a later autonomous phase involving Dsh and Pk (Wu, 2008).

The simplest interpretation of the data suggests signaling from Fz to Vang/Stbm during nonautonomous signaling. It cannot excluded, however, that the interaction is bidirectional and that Fz activity is also influenced by binding to Vang/Stbm (in a Dsh-independent manner). Nevertheless, comparing the gain-of-function data of Fz and Vang/Stbm, the effects of Fz in repolarizing neighboring cells are always robust, whereas those with Vang/Stbm are milder and more cell autonomous. Despite this observation, bidirectional signaling is possible, either through the Fz-Vang/Stbm interaction or through their links to the atypical cadherin Fmi as suggested in several models. The data do not exclude an instructive role for a Fmi-Fmi interaction as proposed earlier. Indeed, this latter idea is supported by the observation that nonautonomous fz clonal phenotypes are not completely suppressed in a Vang mutant background, as some nonautonomy is still observed (~25% of fz clones still display weak nonautonomy in a Vang background) (Wu, 2008).

Fmi has recently been shown to associate with Fz, and thus the homophilic cell adhesion behavior of Fmi could also contribute to an instructive directional signal. The observations that Fz can associate extracellularly with Vang/Stbm (this work) and within the membrane with Fmi suggest a complex scenario. A cross-cell interaction mediated by the homophilic Fmi interaction could display asymmetric properties, as Fmi-Fz and/or Fmi-Vang complexes could have different qualities and signal in either direction. However, fmi null clones show little nonautonomous behavior (a 1 cell wide effect), while the fz and fz Vang clones with widespread nonautonomy are nevertheless striking. Fmi causes significant nonautonomy when overexpressed, and this effect seems not to depend on the presence of Fz or Vang in the overexpressing clone. Multiple parallel mechanisms are thus likely to exist that contribute to cell-cell communication in transmitting the polarity signal (Wu, 2008).

In conclusion, this study provides molecular evidence for a mechanism of nonautonomous Fz signaling through direct interactions with Vang/Stbm on neighboring cells. It remains unclear how the levels of the initial Fz-Vang/Stbm interaction are established in wild-type. Both Fz and Vang/Stbm are expressed evenly and their initial subcellular localization is not polarized. Thus, in wild-type, the generation of a polarized Fz-Vang/Stbm interaction (across cells) must be mediated by other factors that modify Fz, Vang/Stbm, or their interaction in a graded manner (Wu, 2008).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Nemo kinase phosphorylates β-catenin to promote ommatidial rotation and connects core PCP factors to E-cadherin-β-catenin

Frizzled planar cell polarity (PCP) signaling regulates cell motility in several tissues, including ommatidial rotation in Drosophila melanogaster. The Nemo kinase (Nlk in vertebrates) has also been linked to cell-motility regulation and ommatidial rotation but its mechanistic role(s) during rotation remain obscure. This study shows that nemo functions throughout the entire rotation movement, increasing the rotation rate. Genetic and molecular studies indicate that Nemo binds both the core PCP factor complex of Strabismus-Prickle, as well as the E-cadherin-β-catenin (E-cadherin-Armadillo in Drosophila) complex. These two complexes colocalize and, like Nemo, also promote rotation. Strabismus (also called Vang) binds and stabilizes Nemo asymmetrically within the ommatidial precluster; Nemo and β-catenin then act synergistically to promote rotation, which is mediated in vivo by Nemo's phosphorylation of β-catenin. These data suggest that Nemo serves as a conserved molecular link between core PCP factors and E-cadherin-β-catenin complexes, promoting cell motility (Mirkovic, 2011).

The data suggest that Nmo connects the core PCP Stbm-Pk complex to the activity of E-cad-β-cat. Consistent with this, mutations in stbm and pk enhance not only the nmoP rotation defects but also rotation defects of hypomorphic shg (E-cad) backgrounds. As the presence of the Stbm-Pk complex seems to increase the amount of Nmo at R4 membranes and junctional complexes, it is hypothesized that a rise in Stbm levels would increase the ability of sev>Nmo to cause an over-rotation phenotype. This is indeed the case. These data indicate that Nmo serves as a link from PCP factors to the E-cad-catenin complexes. The data are consistent with a model in which the Stbm-Pk complex helps to recruit and/or stabilize Nmo at membrane regions (where the PCP factors partially overlap with E-cad-β-cat complexes (Mirkovic, 2011).

The effect of Nmo on E-cad-β-cat complexes could be mediated either through the dynamics of lateral clustering (for example, formation or disassembly of higher-order E-cad-β-cat complexes) or through changes in the interaction of β-cat with other associated proteins. An E-cad::β-cat fusion protein (which bypasses a β-cat requirement and provides stable adhesion is not influenced by Nmo, suggesting that once β-cat is part of the E-cad-catenin complex Nmo cannot affect their activity. It is thus possible that phosphorylation of β-cat by Nmo affects the E-cad-β-cat complex activity (as an ArmS10AAA isoform with the Nmo target sites mutated no longer cooperates with Nmo) and this phosphorylation may also modulate interactions of the complex with other binding partners, such as β-cat. The interactions of adhesion and planar polarity during the early 'convergence-extension' rearrangements in the fly embryo suggest a mechanism in which a polarized pattern of junction remodeling drives cell intercalation. Polarized activity of RhoA and Myosin II (encoded by zipper) regulates adherens junction disassembly along the anterior-posterior axis, primarily by regulating lateral cadherin clustering without affecting surface levels of cadherins. The specific effect of RhoA on rotation, along with the interaction of nmo with zipper, supports the idea that actin-myosin contractility is downstream of Nmo. Loss of maternal contribution or Nmo overexpression in the embryonic epidermis phenocopies shg alleles or ArmS10 cuticle defects, respectively. Thus, Nmo may be generally required in epithelia undergoing morphogenetic movements, where it modulates polarized remodeling of adherens junctions in response to local asymmetries created by, for example, the activity of PCP signaling complexes (Mirkovic, 2011).

In conclusion, this study defines a framework in which Nmo serves as a link between PCP (Stbm) and the regulation of adhesive cell behavior at the level of adherens junction complexes. Although Nmo is recruited and/or maintained apically by the Stbm-Pk complex, other factors must affect Nmo activity or localization as well, because the set of cells requiring nmo (all outer R-cells) is broader than the set of cells requiring stbm (R4). First, the Nmo could regulate rate of rotation independently of the PCP complexes through Notch (N) signaling and/or Egfr signaling, as suggested by genetic data: N- alleles strongly suppress sev>Nmo, and N functions in all R-cells. Second, an asymmetric input or localization of Nmo by Stbm would provide a direction to rotation. Thus, a Notch-Nmo interaction in all cells and an asymmetric Stbm effect in R4 could combine to regulate both rate and direction of rotation. The observation that zebrafish Nlk enhances the PCP-specific Wnt11 cell migration defects in prechordal plates supports a general Nmo-mediated mechanism in PCP-associated cell movements (Mirkovic, 2011).

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

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

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

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

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

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

Kermit interacts with gαo, vang, and motor proteins in Drosophila planar cell polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue - the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen Kermit, previously implicated in G protein and PCP signaling, was unvcovered as a novel binding partner of Go. Through pull-down and genetic interaction studies, it was found that Kermit interacts with Go and another PCP component Vang (Strabismis), known to undergo intracellular relocalization during PCP establishment. It was further demonstrated that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI (Jaguar). The results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment (Lin, 2013).

At the top of the signaling hierarchy in PCP lies a G protein-coupled receptor Fz. The heterotrimeric Go protein emerged as an immediate transducer of Fz in Drosophila as well as other organisms. One of the mediators of Go signaling in PCP is the endocytic GTPase Rab5 required for the proper Fz internalization and relocalization. During PCP establishment, Fz concentrates at the distal apical position of wing epithelia. This study describes identification of Kermit as another transducer of Go in PCP. kermit downregulation suppresses the Gαo-overexpression phenotypes, and Gαo and kermit co-overexpression results in a prominent synergism in PCP malformations (Lin, 2013).

Kermit and its mammalian homolog GIPC, through their PDZ domain, are known to interact with a number of proteins in various organisms. Observations in Xenopus and mice indicated that Kermit/GIPC could interact with members of the Fz and RGS protein families -- Fz3, Fz7, and RGS19 (De Vries, 1998; Tan, 2001). Since Go also binds Fz and RGS proteins, it was hypothesized that a quaternary complex consisting of Fz, Go, Kermit, and RGS19 could form in Drosophila PCP, with Kermit as a potential organizer of these interactions. However, Drosophila Kermit was found not interact with Fz. Similarly, no binding between Kermit and the Drosophila RGS19 homolog could be seen. Thus Kermit is unlikely to act as a scaffold in Fz-Go signaling, and another mode of action of Kermit in transducing Go signal exists in PCP (Lin, 2013).

In a recent study using mouse genetics and cellular assays, a role of GIPC1 in regulating Vangl2 (a murine homolog of Drosophila Vang) intracellular trafficking has been revealed (Giese, 2012). In Drosophila PCP, Vang relocalizes to the site opposite to Fz at the proximal apical tip of wing epithelia. This study provides genetic evidence placing Vang downstream from Kermit in Drosophila PCP, suggesting that the Kermit-Vang connection is conserved from insects to mammals (Lin, 2013).

kermit expression is strongly upregulated in the developing wing during PCP establishment, and kermit overexpression induces strong PCP phenotypes (Djiane, 2010). In Xenopus, both up- and down-regulation of kermit lead to defective Fz3-dependent neural crest induction. It is thus surprising that Drosophila kermit loss-of-function alleles were homozygous viable and did not reveal PCP phenotypes. It is proposed that Kermit may regulate Drosophila PCP redundantly with some other PDZ domain-containing proteins, such as Scribble or Patj, which genetically interact with PCP components but on their own also produce only mild phenotypes; of those Scribble has been shown to interact with Vang both in Drosophila and mammals. In general, up to 75% of genes Drosophila are estimated to be phenotypically silent in loss-of-function due to redundancy, and the significance of gain-of-function analysis in discovery of novel important pathway components has been highlighted in a recent large-scale Drosophila-based assay. Kermit, based on the presented overexpression and genetic interaction studies, can thus be considered as an important regulator of Drosophila PCP (Lin, 2013).

A genetic and physical interaction between Kermit and the unconventional actin-based motor MyoVI has been described. This study confirmed that the dominant UAS-kermit PCP phenotypes critically depend on the MyoVI activity. MyoVI has been previously shown to mediate removal of endocytic vesicles away from the cell's periphery. The excessive activity of Kermit or MyoVI may thus result in removal of Vang-containing vesicles from the apical membrane, contributing to mislocalization of Vang and appearance of the PCP defects. In contrast, microtubule-based transport along the apical microtubule cables, polarized below the apical plasma membrane in wing epithelia, mediates the correct relocalizations of Fz and Vang in PCP. It is probable that a competition between the actin-based and microtubule-based motors may exist for the endocytic vesicles containing PCP components, and that excessive Kermit activity unbalances this competition in favor of the actin-based transport. Thus whether reduction in the levels of the microtubule-based transport system would further aggravate the dominant UAS-kermit PCP phenotypes was tested. And indeed, reduction in either the minus end-directed motor dynein or the plus end-directed motor kinesin significantly enhances the UAS-kermit effects (Lin, 2013).

The following model is proposed to collectively explain the results. It is speculated that endocytic vesicles containing PCP components can be transported in a planar manner, along the microtubule meshwork underlying the apical plasma membrane -- the mode of transport required for the proper apical relocalizations of these components. Alternatively, the vesicles can be trapped by the actin cables and transported away from the apical membrane, removing them from the active pool of PCP components. In the case of Vang, the choice between these decisions is regulated by the Kermit protein, which favors the actin-based transport (Lin, 2013).

These findings and model shed new light on the mechanisms of complex inter-regulations ensuring the robust epithelial polarization, likely conserved across the metazoans (Lin, 2013).

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 pksple 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

Embryonic

stbm plays a role in early embryonic development, as many homozygous embryos die between 0-4 hours following egg laying. The role for stbm during these early developmental events has not been explored (Wolff, 1998).

Pupal

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

The initial asymmetrical enrichment of Fmi in both R3 and R4, and the subsequent enrichment in R4 only, raised the question of in which cell(s) of the precluster is fmi required for PCP establishment. Interestingly, the analysis of mosaic clusters revealed a requirement for fmi in both R3 and R4. An ommatidium always adopts the correct orientation when both R3 and R4 are fmi+. When either R3 or R4 (or both) are fmi-, the ommatidium selects chirality randomly or stays symmetrical. Significantly, all ommatidia with wrong or no chirality had fmi- R3 and/or R4 cells. Loss of fmi function in any other R cells in any combination has no effect on ommatidial polarity. These data indicate that fmi is necessary and sufficient in both the R3 and R4 photoreceptor precursors for normal polarity establishment (Das, 2002).

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

Since Fmi is initially expressed in both cells of the pair, its inhibitory role on Dl can only be allowed in R4 and thus needs to be regulated. How is this achieved? Diego (Dgo) is a good candidate for this role. The cytoplasmic Dgo protein depends on Fmi for membrane association and generally colocalizes with Fmi at all membranes in the eye disc. The genetic interactions with sev --> Fmi (resulting in Fmi overexpression in the R3/R4 pair) identify dgo as a strong enhancer, suggesting that it is suppressing Fmi function in this context. Mosaic analysis of dgo shows that it is required in R3 and thus might keep the inhibitory function of Fmi off in R3. Since Fmi is necessary, but not sufficient, for Dgo membrane recruitment, other factor(s) are also required. Since Dgo and Fmi colocalize also in R4, a factor is needed there to antagonize Dgo function. Strabismus (Stbm), since it is required in R4, is a candidate. Since fmi mutants are enhancers of an Stbm overexpression phenotype, Stbm could serve this function in R4 (Das, 2002).

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

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

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

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

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

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

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

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

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

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).

Mathematical modeling of planar cell polarity to understand domineering nonautonomy

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Effects of Mutation or Deletion

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, legs 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, as 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. 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).

Strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila

In stbm mutant eyes, the normally parallel rows of ommatidia are not properly aligned, giving the eyes a rough appearance. Tangential sections through adult eyes indicate that the vast majority of stbm ommatidia are correctly assembled and that the misaligned eye lattice is a consequence of aberrant orientation of ommatidial units. stbm ommatidia show several orientations: they are either normal, reversed dorsoventrally, anteroposteriorly, or both dorsoventrally and anteroposteriorly. In addition, while most ommatidia rotate the normal 90°, many undergo either partial or no rotation. Finally, in a small percentage of ommatidia, photoreceptors R3 and R4 are bilaterally symmetrical so the chirality of the ommatidium is abolished. The arrangement of rhabdomeres in these achiral ommatidia is rectangular rather than trapezoidal (Wolff, 1998).

To determine the relative proportions of the various ommatidial forms, polarity of genetically mutant ommatidia was quantitated in clones of homozygous stbm6cn tissue, a molecular null allele. The equator in the surrounding wild-type tissue provide a reference for a mutant ommatidium's dorsal vs. ventral position in the eye. Of 181 ommatidia scored, 46% showed normal orientation, 35% showed D/V pattern reversals, 7% displayed A/P reversals, 7% were reversed both dorsoventrally and anteroposteriorly, 3% failed to rotate and 2% were achiral. As a consequence of ommatidial misorientation, the pigment cell lattice is disrupted. stbm eyes contain a normal complement of primary pigment cells, bristles and cone cells, although occasional ommatidia are missing one cone cell. Secondary and tertiary pigment cell numbers are often correct, except at the vertices of partially or unrotated ommatidia, where there is an excess of these cell types (Wolff, 1998).

The position of the future equator is evident in the third instar eye disc prior to the start of ommatidial rotation. New rows emerging from the furrow are initiated at the D/V midline (the site of the future equator) and grow laterally as ommatidia are added to each end in a symmetrical manner. This `center-lateral' growth of a row and ommatidial rotation are both affected in frizzled mutants (Zheng, 1995), suggesting these two events may be linked. However, while the adult phenotypes of fz and stbm are similar, the center-lateral growth of a row is not altered in stbm mutants. This observation indicates that the two events are genetically separable and that stbm may act downstream of fz, or in a parallel pathway, in its role in orientation but not in its role in the center-lateral growth of new ommatidial rows (Wolff, 1998).

stbm affects the polarity of multiple tissues in Drosophila. In stbm, the polarity of many of the bristles and hairs is abnormal. In wild-type animals, the thoracic bristles and hairs are aligned in parallel rows with each bristle pointing toward the posterior of the animal while the leg bristles lie flat against the leg surface and point distally. In stbm homozygotes, many thoracic bristles have aberrant polarity and the leg bristles are oriented perpendicular to the leg. Hair polarity is disrupted throughout the body, for example surrounding the eye and on the wings and thorax. stbm also plays a role in setting up the cuticular polarity of the legs. In stbm legs, extra tarsal segments or partial duplications frequently occur in tarsal segments 3 and 4 and occasionally in tarsal segment 2. Finally, the wings of stbm flies are held out. This widespread requirement for stbm in various tissues is not uncommon among tissue polarity mutants; for example, fz, dsh, prickle (pk) and spiny legs (sple) affect the polarity of various structures, including bristles, hairs and the leg (Wolff, 1998).

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

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

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

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

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

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

Prickle mediates feedback amplification to generate asymmetric planar cell polarity signalling

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).

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).

The planar polarity pathway promotes coordinated cell migration during Drosophila oogenesis

Cell migration is fundamental in both animal morphogenesis and disease. The migration of individual cells is relatively well-studied; however, in vivo, cells often remain joined by cell-cell junctions and migrate in cohesive groups. How such groups of cells coordinate their migration is poorly understood. The planar polarity pathway coordinates the polarity of non-migrating cells in epithelial sheets and is required for cell rearrangements during vertebrate morphogenesis. It is therefore a good candidate to play a role in the collective migration of groups of cells. Drosophila border cell migration is a well-characterised and genetically tractable model of collective cell migration, during which a group of about six to ten epithelial cells detaches from the anterior end of the developing egg chamber and migrates invasively towards the oocyte. The planar polarity pathway promotes this invasive migration, acting both in the migrating cells themselves and in the non-migratory polar follicle cells that they carry along. Disruption of planar polarity signalling causes abnormalities in actin-rich processes on the cell surface and leads to less-efficient migration. This is apparently due, in part, to a loss of regulation of Rho GTPase activity by the planar polarity receptor Frizzled, which itself becomes localised to the migratory edge of the border cells. It is concluded that, during collective cell migration, the planar polarity pathway can mediate communication between motile and non-motile cells, which enhances the efficiency of migration via the modulation of actin dynamics (Bastock, 2007).

This study used the Drosophila ovary to study the control of coordinated cell movements by the planar polarity pathway, taking advantage of its relative simplicity and the ability to precisely manipulate gene function in individual cell populations. Activity of the core polarity genes facilitates invasive migration of the border cell cluster through the nurse cells. Of particular interest is the observation that migration of the border cells is enhanced by planar polarity activity in the non-migratory epithelial polar follicle cells, suggesting a key role for interactions between migratory and non-migratory cell types (Bastock, 2007).

In the Drosophila wing, the planar polarity pathway regionalises cells via the formation of proximal and distal domains at the level of the adherens junctions. The distal domain contains Fz, which acts via the downstream factors Dsh and RhoA to ensure local production of a single actin-rich trichome, while, in the proximal domain, Stbm recruits factors that locally inhibit trichome formation. During border cell migration, the coordinated movement of the non-migratory polar follicle cells and the migratory border cells is achieved in part by the border cells retaining epithelial character in the region contacting the polar follicle cells, but also having an actin-rich partly mesenchymal migratory region. Taking these observations together, it is proposed that, in border cells, localised Fz in the migratory region and localised Stbm in the junctional region might promote the production of actin-rich structures, which, in turn, would increase the motility both of individual cells and the cluster as a whole (Bastock, 2007).

Mosaic analyses suggest a mechanism for how this localised Fz and Stbm activity is established within the border cells. Fz and Stbm mediate intercellular communication between the polar cells and the border cells via the production of junctional complexes. Because contact with an Fz-expressing polar cell enhances the migration of border cells, it is surmised that Fz in each polar cell interacts with Stbm in the contacting border cell. Junctionally localised Stbm in the border cell can then act as a cue to indirectly promote actin-rich protrusion formation in the migratory region, at least in part via the localisation of Fz (Bastock, 2007).

Although the planar polarity pathway has been known for some years to promote cell rearrangements during vertebrate gastrulation, surprisingly little is understood about its mechanisms of action in cell movement and the particular roles of this pathway in cell-cell communication. This study has demonstrated that Fz/Stbm-mediated intercellular communication can enhance the invasive migration of a group of cells. Migration of groups of cells, sometimes including both motile and non-motile types, is important for many processes in animal morphogenesis and in disease processes, such as cancer metastasis. This work provides evidence that planar polarity pathway function could be generally important in coordinated cell migration, providing a mechanism by which cells within a group can communicate and establish the proper regional production of actin structures required for efficient movement (Bastock, 2007).

Planar polarity genes in the Drosophila wing regulate the localisation of the FH3 domain protein Multiple Wing Hairs to control the site of hair production

The core planar polarity proteins play important roles in coordinating cell polarity, in part by adopting asymmetric subcellular localisations that are likely to serve as cues for cell polarisation by as yet uncharacterised pathways. This study describes the role of Multiple Wing Hairs (Mwh), a novel Formin Homology 3 domain protein, which acts downstream of the core polarity proteins to restrict the production of actin-rich prehairs to distal cell edges in the Drosophila pupal wing. Mwh appears to function as a repressor of actin filament formation, and in its absence ectopic actin bundles are seen across the entire apical surface of cells. The proximally localised core polarity protein Strabismus acts via the downstream effector proteins Inturned, Fuzzy and Fritz to stabilise Mwh in apico-proximal cellular regions. In addition the distally localised core polarity protein Frizzled positively promotes prehair initiation, suggesting that both proximal and distal cellular cues act together to ensure accurate prehair placement (Strutt, 2008).

Activity of the core planar polarity proteins is required in cells of the Drosophila pupal wing to specify prehair initiation at the distal vertex (Wong, 1993). This study presents evidence that core polarity protein localisation at both proximal and distal cell edges provides redundant cues for specifying distal prehair initiation (Strutt, 2008).

Regarding the mechanistic basis of the proximal cue, this and previous work provide evidence for a plausible model. The downstream effectors In, Fy and Frtz all colocalise at the proximal cell edge with Stbm and in a Stbm-dependent manner. Activity of In, Fy and Frtz is required for Mwh phosphorylation and its subapical subcellular localisation, which is thus concentrated towards the proximal side of the cell. Genetic studies have shown that loss of fy, in, frtz or mwh activity leads to excess prehair initiation, and this study found that the initial defect in mwh is excess actin bundling across the entire apical face of cells. Thus, proximal restriction of Mwh activity in the cell results in actin bundling and prehair initiation specifically in distal regions (Strutt, 2008).

Additional evidence for the sufficiency of a Stbm-dependent cue for prehair initiation at opposite cell edges comes from experiments in the abdomen. It was reported that cells lacking fz activity, but juxtaposed to cells with fz activity, could produce polarised trichomes, as has also been observed in the first row of cells within a fz clone in the wing (Strutt, 2008).

Less information is available regarding the distal cue. Its existence is based upon two pieces of evidence. First, if prehair initiation were entirely dependent on Stbm-mediated localisation of Mwh activity, then prehairs should show no bias in their site of initiation in cells lacking stbm activity. In fact, stbm mutant cells with Fz localised at one cell edge show a strong bias towards initiating prehairs at this edge. Second, if prehair initiation is controlled only by a Stbm-dependent repressive cue, then in the absence of stbm activity, Fz would have no influence over prehair initiation. Instead, in a stbm background, fz activity still weakly promotes prehair formation. Taken together these data support the view that distally localised Fz acts as a prehair promoting cue (Strutt, 2008).

A possible mechanism of action of the distal cue would be if localised Fz were able to repress Mwh activity in distal cell regions, possibly via its known effectors RhoA and Drok. Alternatively Fz could promote prehair initiation in a Mwh-independent fashion, either via RhoA/Drok or other effectors (Strutt, 2008).

It is notable that absence of fz activity results in a delay in prehair formation, and a greater tendency for prehairs to form in the cell centre rather than towards a cell edge, than loss of stbm. It is surmised that in fz mutant cells, there is no Fz-dependent prehair promoting cue, and the Stbm-dependent repressive cue is evenly distributed around the cell edge, resulting in delayed prehair initiation in the cell centre. Conversely, in stbm mutant cells, there is no change in the activity of the repressive cue, but the Fz-dependent prehair promoting cue is localised to cell edges, albeit more thinly spread than in the wildtype situation. This results in approximately normally timed prehair initiation near cell edges (Strutt, 2008).

An unexplained observation is that within stbm mutant tissue, the site of prehair initiation appears to be biased towards that seen in neighbouring cells. Thus in the first rows of cells within a clone, prehairs tend to point towards the adjacent wildtype tissue. This phenomenon is presumably independent of core protein asymmetric localisation, and may depend upon some mechanical linkage between cells. In this context, there is already evidence that the microtubule cytoskeletons of adjacent cells may be linked and that this could coordinate cell polarity (Turner, 1998). An alternative core protein-independent mechanism to align wing hairs, relying on the activities of Gliotactin and Coracle has also been reported (Strutt, 2008).

Loss of in, fy, and frtz results in a similar phenotype to loss of mwh with multiple ectopic prehairs at the cell edge preceded by excess apical actin bundling. As In, Fy and Frtz are all required for the apical punctate distribution of Mwh within cells, and also appear to stabilise each other, this suggests that In, Fy and Frtz act together to activate Mwh and promote apical localisation. Conversely, while Stbm plays a role in localising Mwh within the cell, it is not required for its activity, as loss of stbm does not phenocopy mwh mutants in which increased apical actin bundling is observed. This role of Stbm in localising but not regulating Mwh activity is most simply explained by Stbm acting to localise, but not regulate In, Fy and Frtz activities. This is supported by the observation that whereas loss of fz or stbm has a strong effect on the distribution of Frtz to the apicolateral junctions, it has a negligible effect on the apparent phosphorylation state of Mwh (Strutt, 2008).

The regulation of Mwh activity appears to be largely post-translational; although the subcellular distribution of Mwh changes dramatically in frtz mutant cells, total levels of Mwh are not similarly altered. Further evidence that In, Fy and Frtz regulate Mwh activity by a mechanism largely independent of Mwh protein levels comes from the observation that Mwh overexpression in the wing produces no effect on trichome formation, rather than repressing trichome formation as might be predicted if protein levels were the main determinant of activity (Strutt, 2008).

The data are strongly suggestive that Mwh activity may be regulated by phosphorylation. Treatment of cell extracts with phosphatase results in increased mobility of Mwh. A similar increase in mobility is observed when frtz activity is removed, but not when stbm or fz activities are removed. Thus, at the least, Mwh phosphorylation correlates with Mwh activity and apical punctate localisation. Hence it is proposed that the rôles of In, Fy and Frtz may be to activate, or bring into proximity with Mwh, a kinase or kinases responsible for activating Mwh. Similarly, Fz could locally promote the dephosphorylation of Mwh to induce prehair initiation, although any such effect would have to be small, as Mwh phosphorylation is not obviously altered in the absence of Fz (Strutt, 2008).

Definitive proof that phosphorylation of Mwh is important for its activity would require the identification of particular phosphorylation sites which were required for specific molecular functions and/or identification of a kinase critically required for Mwh activity (Strutt, 2008).

An alternative regulatory mechanism for Mwh, via analogy to Diaphanous family formins, would be via RhoA GTPase activity. The FH2 domain of such formins promotes actin nucleation, an activity which is autoinhibited by interaction with the GTPase binding domain (GBD). Upon interaction of the GBD with GTPase-bound Rho GTPases, this autoinhibition is released. Notably, genetic interaction data suggest that Fz/Dsh can activate RhoA activity. This is consistent with a model whereby in the proximal cell Rho GTPase activity is low and Mwh inhibits prehair initiation, and in the distal cell activated RhoA alleviates the inhibitory activity of Mwh (Strutt, 2008).

Notwithstanding the evidence for post-translational regulation of Mwh activity in the normal context of the pupal wing, in cultured cells no effect is seen of Mwh overexpression on the actin cytoskeleton. This seems likely to be due to the much higher levels of expression that can be achieved in transfected cells as opposed to cells in the living organism, and hence the result should be treated with caution, but may suggest that S2 cells express a factor able to constitutively activate Mwh (Strutt, 2008).

The results also indicate that Mwh levels are influenced by temperature, which provides a plausible explanation for why in, fy and frtz phenotypes are stronger at 18°C. It is suggested that loss of in, fy and frtz reduces Mwh activity, and lower temperatures additively reduce Mwh levels, resulting in lower overall Mwh activity (Strutt, 2008).

What is the molecular function of Mwh? As already noted, the FH3 domain of conventional formins is thought to be involved in targeting the protein to particular cellular sites, whereas the GBD domain is involved in inhibition of the actin nucleating function of the FH2 domain. A plausible model is that Mwh acts as a dominant negative by binding via its GBD domain to other FH2 domain containing formins that are involved in the nucleation of actin filaments and inhibiting their activity. Notably, this dominant negative activity of Mwh could then be inhibited distally in the cell by Fz-mediated activation of RhoA GTPase activity (Strutt, 2008).

Electron microscopy studies suggest that prior to prehair initiation the apical cell surface is covered in electron-dense 'pimples' that are normally only activated at the distal cell edge and serve as foci for actin filament formation (Guild, 2005). It is proposed that at around 32 hours of pupal development, cells receive a general signal for pimple activation which results in actin nucleation, and that Mwh activity is required to inhibit this activation away from the distal cell edge (Strutt, 2008).


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Van Gogh/strabismus: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2016

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