InteractiveFly: GeneBrief

multiple wing hairs: Biological Overview | References

BIOLOGICAL OVERVIEW

The frizzled signaling/signal transduction pathway controls planar cell polarity (PCP) in both vertebrates and invertebrates. Epistasis experiments argue that in the Drosophila epidermis multiple wing hairs (mwh) acts as a downstream component of the pathway. The PCP proteins accumulate asymmetrically in pupal wing cells where they are thought to form distinct protein complexes. One is located on the distal side of wing cells and a second on the proximal side. This asymmetric protein accumulation is thought to lead to the activation of the cytoskeleton on the distal side, which in turn leads to each cell forming a single distally pointing hair. mwh has been identified as CG13913, which encodes a novel G protein binding domain-formin homology 3 (GBD-FH3) domain protein. The Mwh protein accumulates on the proximal side of wing cells prior to hair formation. Unlike planar polarity proteins such as Frizzled or Inturned, Mwh also accumulated in growing hairs. This suggested that mwh has two temporally separate functions in wing development. Evidence for these two functions also came from temperature-shift experiments with a temperature-sensitive allele. Overexpression of Mwh inhibits hair initiation, thus Mwh acts as a negative regulator of the cytoskeleton. These data argue early proximal Mwh accumulation restricts hair initiation to the distal side of wing cells and the later hair accumulation of Mwh prevents the formation of ectopic secondary hairs. This later function appears to be a feedback mechanism that limits cytoskeleton activation to ensure a single hair is formed (Yan, 2008).

The subcellular localization of Mwh is dynamic and unique for a protein involved in PCP. Mwh preferentially localizes to the proximal side of pupal wing cells prior to hair formation (e.g., 30-32 hr apf), in emerging hairs (at 32-34 hr), and later at the base of mature hairs (e.g., after 36 hr apf). Temperature-shift experiments argue that mwh has discrete functions before and after hair initiation. It is hypothesized that the early proximal accumulation of Mwh leads to hair initiation at the distal side of wing cells. This likely represents mwh's function as a downstream component in the fz pathway. It is further suggested that the inhibition of small ectopic hair formation that was detected by temperature shifts after hair initiation is mediated by Mwh that accumulates in the growing hair and in the base of the hair (Yan, 2008).

The observation that the overexpression of mwh around the time of hair initiation can lead to a delay in hair initiation and a small and multiple hair phenotype in adult wings argues that Mwh acts as a negative regulator of hair initiation. This is consistent with Mwh being localized to the proximal side of wing cells when hairs initiate on the distal side, and Mwh being localized to the hair when it functions to prevent the formation of small hairs after the normal hair has begun to grow (Yan, 2008).

The accumulation of Mwh in the hair is presumably due to its interacting with one or more hair components. The hair contains both peripheral actin filaments and centrally localized microtubules. Because of the small size of the hair it is difficult to distinguish between Mwh accumulating with either or both of these cytoskeletons. Therefore the localization of Mwh was examined in other cell types. In both bristles and salivary gland cells it was found that Mwh colocalizes with the actin cytoskeleton. Although Mwh contains a GBD-FH3 domain that is also found in formins it lacks the FH1 and FH2 domains that mediate formin binding to F-actin. This suggests Mwh does not interact directly with actin. Consistent with this hypothesis it was found that in larval muscle Mwh accumulates in a striated pattern that is offset from the bands of F-actin. From this it is concluded that Mwh does not bind directly to F-actin. The localization to what appeared to be the M line in muscle suggested Mwh interacts with a protein that regulates myosin assembly or activity in striated muscle and perhaps with a similar protein in epidermal cells (Yan, 2008).

The sequence similarity between Mwh and the regulatory domain of formins suggests several possible biochemical mechanisms that could be functionally important. In Diaphanous family formins the GBD binds Rho1, which results in the activation of the formin to promote actin polymerization. Rho1 has been implicated in fly planar polarity, making it a strong candidate for interacting with Mwh (Strutt, 1997). In preliminary studies both genetic and biochemical evidence has been obtained for such an interaction, but the interpretation of the data is complicated by the finding that Rho1 has multiple functions in wing hair development. The presence of the DD domain also suggests the possibility that Mwh might dimerize with a true formin and regulate the actin cytoskeleton in that way. An obvious candidate for such a function is the DAAM formin, which in Xenopus was found to bind to Dsh. However, studies on fly DAAM failed to reveal any function in planar polarity or hair morphogenesis. The diaphanous gene is another potential candidate. This gene has been well studied but there are no reports on its role in the differentiation of the adult epidermis. Intriguingly, Diaphanous localizes to growing embryonic denticles, which like hairs are actin-containing extensions of epidermal cells. As is the case for epidermal hairs, mwh mutations result in the formation of extra denticles suggesting Dia as a possible partner for Mwh. None of the other fly formins have been found to function in wing hair development or wing planar polarity but several have not been well studied and are expressed in pupal wings (e.g., CG10990, Formin 3, and Fhos). One of these could function with mwh (Yan, 2008).

There are several possible models as to how Mwh located on the proximal side of wing cells results in distal hair formation. One is that proximal Mwh stimulates hair initiation at the juxtaposed distal side of neighboring cells. However, mwh acts cell autonomously this model can be ruled out. An alternative is that Mwh, In and other PPE proteins formed a protein complex that organized the cytoskeleton to direct intracellular transport of hair-building components to the distal side leading to hair initiation there. This is plausible but no changes in the cytoskeleton prior to hair initiation have been seen in these mutants. A third possible model and one that is preferred is that proximal Mwh locally inhibits hair initiation by regulating the cytoskeleton. This would lead to the preferential activation of the cytoskeleton on the distal side of the cell where Mwh levels were lowest. The cytoskeleton would self assemble to refine the area for hair initiation to a small region near the distal vertex. As the cytoskeleton is activated distally and a new hair emerges, Mwh would be recruited to this region, perhaps by binding to a hair component and/or a protein that interacted with a cellular myosin. Mwh would once again function locally to inhibit new hair initiation events and hence ensure that only a single unbranched hair forms. This latter Mwh function is viewed as a type of feedback inhibition that limits the activation of the cytoskeleton (Yan, 2008).

The Drosophila planar polarity proteins inturned and multiple wing hairs interact physically and function together

The conserved frizzled (fz) pathway regulates planar cell polarity in both vertebrate and invertebrate animals. This pathway has been most intensively studied in the wing of Drosophila, where the proteins encoded by pathway genes all accumulate asymmetrically. Upstream members of the pathway accumulate on the proximal, distal, or both cell edges in the vicinity of the adherens junction. More downstream components including Inturned and Multiple Wing Hairs accumulate on the proximal side of wing cells prior to hair initiation. The Mwh protein differs from other members of the pathway in also accumulating in growing hairs. This study shows that the two Mwh accumulation patterns are under different genetic control with the early proximal accumulation being regulated by the fz pathway and the latter hair accumulation being largely independent of the pathway. Recruitment by proximally localized Inturned is also a putative mechanism for the localization of Mwh to the proximal side of wing cells. Genetically inturned (in) acts upstream of mwh (mwh) and is required for the proximal localization of Mwh. This study shows that Mwh can bind to and co-immunoprecipitate with Inturned. These two proteins can function in close juxtaposition in vivo. An In::Mwh fusion protein provided complete rescue activity for both in and mwh mutations. The fusion protein localized to the proximal side of wing cells prior to hair formation and in growing hairs as expected if protein localization is a key for the function of these proteins (Lu, 2010).

The frizzled (fz) signaling pathway regulates tissue planar cell polarity (PCP) in the epidermis of both vertebrate and invertebrate animals. PCP is dramatic in the cuticle of insects such as Drosophila, which is decorated with arrays of hairs and sensory bristles (Lu, 2010).

The genetic basis for tissue polarity has been most extensively studied in the fly wing. The Planar Polarity (PCP) genes of the fz pathway (also known as the core PCP genes), the planar polarity effector (PPE) genes and the multiple wing hairs (mwh) gene encode key components that regulate planar polarity in the wing. fz, disheveled (dsh), prickle/spiny leg (pk/sple), Van Gogh (Vang) (aka strabismus), starry night (stan) (aka flamingo) and diego (dgo) are members of the PCP group. A distinctive feature of these genes is that their protein products accumulate asymmetrically on the distal (Fz, Dsh, and Dgo), or both distal and proximal (Stan) sides of wing cells. These genes/proteins act as a functional group and are corequirements for the asymmetric accumulation of the others (Lu, 2010).

The PPE includes inturned (in), fuzzy (fy), and fritz (frtz) (Park, 1996; Collier, 1997; Collier, 2005). These genes are thought to function downstream of the PCP genes and the proteins encoded by these genes also accumulate asymmetrically in wing cells (Adler, 2004; Strutt, 2008). As is the case for the PCP genes, the PPE genes/proteins also appear to be a functional group and to be corequirements for the asymmetric accumulation of the others. Several observations support the hypothesis that the PPE genes are essential downstream effectors of the PCP genes. The earliest appreciation of this came from careful observations of the mutant phenotypes. A common feature of mutations in all of these genes is that they do not result in a randomization of hair polarity, but rather in a similar complicated and abnormal stereotypic pattern (Gubb, 1982; Adler, 2000). That the abnormal patterns were so similar suggested that these genes all functioned in the same process (Wong, 1993). The mutant phenotypes differed in that the vast majority of PCP mutant wing cells form a single hair, while many PPE mutant wing cells form two or three hairs. Mutations in PPE genes are epistatic to both loss- and gain-of-function mutations in PCP genes (Wong, 1993; Lee, 2002). Further evidence that the PPE genes function downstream of the PCP genes comes from the analysis of protein localization. PPE gene function is not needed for the proper asymmetric localization of PCP proteins but in contrast PCP gene function is essential for the asymmetric accumulation of PPE proteins (Adler, 2004; Strutt, 2008). Further, the PCP genes/proteins instruct the localization of the PPE proteins (Lu, 2010).

The multiple-wing-hairs (mwh) gene is thought to function downstream of both the PCP and PPE genes (Wong, 1993). This conclusion comes from analyses that are similar to those that established that the PPE genes function downstream of the PCP genes. The overall hair polarity pattern of mwh mutant wings shares the same complicated and abnormal stereotypic hair polarity pattern seen in PCP and PPE mutants. However, mwh cells differ by producing a larger number of hairs (typically three to four hairs) (Wong, 1993). mwh mutations are epistatic to mutations in both the PCP and PPE genes and mwh is not required for the asymmetric accumulation of either PCP or PPE proteins (Lu, 2010).

The mwh gene was recently determined to encode a novel G protein binding-formin homology 3 (GBD-FH3) protein with a complex accumulation pattern in wing cells (Strutt, 2008; Yan, 2008). Prior to hair initiation Mwh accumulates along the proximal side of wing cells and during hair growth Mwh accumulates in the growing hair. Temperature-shift experiments with a temperature-sensitive allele provided evidence for two temporally separate mwh functions and it was proposed that the two accumulation patterns were associated with the two temporal functions (Yan, 2008). This study shows that the early proximal accumulation of Mwh requires the function of the PCP and PPE genes (a result also seen previously by Strutt (2008), while the hair accumulation of Mwh is largely independent of these two groups of genes providing further genetic evidence for Mwh having two independent functions (Lu, 2010).

How does the Mwh protein accumulate proximally? An obvious possibility is that Mwh interacts directly with one or more of the upstream proteins and in this way is recruited to the proximal side. The PPE proteins are strong candidates to interact directly with Mwh, as they function genetically in between the PCP gene and Mwh (Wong, 1993). Consistent with this possibility it was found that In and Mwh interact in the yeast two-hybrid system and that these two proteins co-immunoprecipated from wing cells. This interaction was found not to be dependent on the function of the PCP genes consistent with the data from genetic studies that both in and mwh retain at least partial function in a fz mutant wing (Wong, 1993). The hypothesis that Mwh is recruited to the proximal side by interacting with In predicts that these two proteins function in close proximity to one another. Consistent with these expectations it was found that an In::Mwh fusion protein provided both In and Mwh function (Lu, 2010).

The data suggested that Mwh was recruited to the proximal side of wing cells by binding to proximally localized In. in function was found to be required for proximal Mwh localization, that the two proteins could be co-immunoprecipitated, and that they interact in the yeast two-hybrid system. However, these two proteins did not precisely colocalize with Mwh showing a somewhat broader accumulation pattern (Strutt, 2008; Yan, 2008). It is hypothesized that the lack of precise colocalization of In and Mwh was due to the dynamics of the system. For example, it is possible that the interaction is transient and cycles of binding and release leads to Mwh accumulating diffusely near the proximal edge of wing cells. It is also possible that the relative levels of the two proteins might not be compatible with all of each being in a common protein complex (Lu, 2010).

A fusion protein containing the complete amino acid sequences of both In and Mwh provides both in and mwh function. Prior to hair formation the fusion protein localizes to the proximal side of wing cells. This is seen for both In and Mwh so this is not a surprising result, although there is no way to be certain ahead of time that such a fusion protein will be functional. This early phase of proximal localization is presumably guided by the In part of the fusion protein as this is needed for in rescue activity and in acts upstream of mwh (Wong, 1993; Lee, 2002). These data established that prior to hair initiation it is sufficient for all of the Mwh to be closely juxtaposed to In (Lu, 2010).

The accumulation of Mwh in the hair cannot be due to binding to In as this is genetically independent of in function and In is not found in the hair (Adler, 2004). It is likely recruited to the hair by one or more constituents of the cytoskeleton. It was previously found that when expressed in striated muscle, Mwh accumulates in the region of the M band (Yan, 2008). Hence, myosin-interacting proteins are considered as candidates for interacting with Mwh. It was noted previously (YAN, 2009) the C-terminal half of Mwh is sufficient and necessary for it to accumulate in hairs. This part of the protein is not conserved outside of insects and it does not contain any recognizable domains so the molecular biology of Mwh has not provided hints as to what causes it to accumulate in growing hairs. The In::Mwh fusion protein accumulates in growing hairs in a manner similar to that of wild-type mwh. Thus, in the context of the fusion protein this Mwh accumulation pattern is epistatic to (acts downstream of) the In pattern. This is presumably mediated by the C-terminal part of Mwh in the fusion protein. The wild-type In protein is normally not found in hairs, but its presence as part of the fusion protein does not interfere with hair morphogenesis. A hypothesis for the function of In is that it functions as an inhibitor of hair initiation (Wong, 1993; Adler, 2004) (e.g., it can act as an inhibitor of actin polymerization). That its accumulation in growing hairs is of no consequence to hair morphogenesis argues against In being a general inhibitor of the cytoskeleton. An alternative hypothesis is that In functions to pull Mwh away from the region where hair initiation occurs. This hypothesis is consistent with the function of the fusion protein and the lack of consequence of In being present in growing hairs. Being bound to In might also be a requirement for the PPE-dependent phosphorylation of Mwh (Lu, 2010).

The general approach of making fusion proteins should allow one to test various models for the action of proteins involved in tissue planar polarity as long as the fusion proteins are functional. Published data have shown that fusions of proteins involved in tissue planar polarity to fluorescent proteins are compatible with function. Thus, there is reason to think that this approach will often be informative. The In::Mwh protein is the first example of a functional fusion between two components of the fz pathway. In the case of the In::Mwh fusion there was no way a priori to know how it would localize in wing cells. This will often be the case when a fusion protein contains tags that result in different localizations. For example, prior to doing the experiment it seemed possible that In would prevent the accumulation of Mwh in the hair after initiation. Such a result would have established that the In localization pattern is epistatic to that of Mwh. That result would have provided a test for the function of Mwh accumulation in the hair. Given the situation, alternative fusion strategies that were not subject to such uncertainty might be preferable although they often introduce alternative potential problems (Lu, 2010).

Immunostaining of wings bearing clones mutant for PPE or PCP genes show that the upstream genes are essential for both the recruitment of Mwh to the proximal part of the cell and for the accumulation of normal levels of Mwh. In PPE mutant cells a similar decrease is seen when overall and proximal edge accumulation is seen. In contrast, in PCP mutant cells a much greater decrease was seen for Mwh specifically localized at the proximal edge. In addition, the decrease in Mwh levels is substantially greater in PPE than in PCP mutant cells. These data suggest that PPE gene function is important for both the accumulation and localization of Mwh, while PCP gene function is primarily important for localization and not accumulation. Consistent with this hypothesis it was also found that Mwh and the PPE protein In can directly interact. It is suggested that this interaction normally leads to both the proximal recruitment and stabilization of Mwh. In a PCP mutant cell In and Mwh might still interact although this might not be restricted to the proximal edge of the cell. These proteins being spread throughout the cell would lower their local concentration and might make the interaction less favored. This could explain the decreased level of Mwh seen in PCP mutant cells. Consistent with this hypothesis it was found that In and Mwh still co-immunoprecipitated in a fz mutant cell. Further, on the basis of the multiple hair cell phenotype it has long been known that in and mwh retained some function in PCP mutants (Wong, 1993) as essentially all mwh and many in mutant cells produce more than one hair. In contrast, about 98% of wing cells form a single hair in PCP mutants (Lu, 2010).

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 (Wong, 1993; Collier, 2005), 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 (Lawrence, 2004). 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 (Strutt, 1997; Winter, 2001). 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 (Strutt, 1997; Winter, 2001). 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 (Adler, 1994; Collier, 1997; Collier, 2005). 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 (Wallar, 2003). 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).

The lack of direct vertebrate homologues of Mwh may indicate that in insects the GBD/FH3 domain of a conventional formin has become separated from the rest of the molecule but retained its function in inhibiting formin-mediated actin nucleation. Nevertheless, it is plausible that the core polarity proteins would use similar regulatory mechanisms to promote local changes in cytoskeletal structure in vertebrate cells as seen in the Drosophila wing. Importantly, vertebrate homologues of both Fuzzy and Inturned have been shown to be involved in regulating apical actin assembly and thus specifying the orientation of cilia (Park, 2006). By analogy to the current findings, it is suggested that core polarity proteins in vertebrates are likely to localise Fuzzy/Inturned activity within cells, and regulate formin activity via phosphorylation and/or Rho GTPase activation (Strutt, 2008).

Combover/CG10732, a novel PCP effector for Drosophila wing hair formation

The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, Combover (Cmb)/CG10732 was identified as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. These data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh (Fagan, 2014).

Rho kinase, a member of the AGC kinase family which also includes PKC and Akt was originally identified as a RhoA effector reorganizing the cytoskeleton by promoting the formation of actin stress fibers. In Drosophila, Rok was shown to act downstream of Fz and Dsh in the non-canonical Wnt/Planar Cell Polarity pathway causing ommatidial rotation and structural defects in the eye and multiple hairs cells in the wing. This study has identified Combover/CG10732 as a novel substrate of Rok. A cmb protein null allele lacking both Cmb protein isoforms that is homozygous viable. Homozygous cmb mutants display no visible phenotype in the wing or in sections of the adult eyes. As a reduction or an excess of actin polymerization can cause MHCs, this study assessed the overexpression phenotype of Cmb. Indeed, overexpression of either Cmb isoform caused a multiple hair cell phenotype that is strongly dominantly enhanced by rok and the fy/in/mwh PCP effectors, validating the in vitro screening approach to identify PCP effectors. Importantly, the cmb mutation suppresses the MHC phenotype of mwh in double mutants. The data thus indicate that Cmb, while not essential for wing hair formation, nevertheless promotes trichome formation in vivo (Fagan, 2014).

It has been noted that known phosphorylation sites of Rok targets such as ERM proteins, Vimentin, Myosin regulatory light chain, or Adducin, often follow the consensus site [R/K]XX[S/T] or [R/K]X[S/T]. Of the five Rok sites identified in in vitro kinase assays followed by MS analysis, only S300 is preceded by a basic residue at position [-2] (RT[S]). In all other cases, no basic amino acid is found at position [-1] or [-2]. However, T46, T206, T368, and T370 are all followed by a Proline, more typical of MAP kinase phosphorylation sites. Nevertheless, mutation of these sites strongly reduced Cmb phosphorylation in vitro (Fagan, 2014).

In rok mutants, multiple hairs form at the distal end of wing cells. Similarly, overexpression of either Cmb isoform causes MHCs that originate at the distal end of cells, distinct from the in/fy group of PCP effectors and mwh, which form MHCs around the periphery of the cells (note that in mwh mutants, actin patches are initially even formed all over the apical cell surface). Importantly, reduction of rok activity by the removal of one gene dose (by two different alleles or a deficiency) increases the number of MHCs, suggesting an inhibitory effect of Rok on Cmb. It was suggested that Myosin II, which is concentrated at the site of prehair initiation and whose activity is regulated by Rok via phosphorylation of its regulatory light chain (MRLC), must be within an optimal range to properly bundle actin and to ensure the formation of a single hair. Consistent with the genetic interaction between cmb and rok, it is possible that in addition to regulating MRLC, Rok might also inhibit a potential hair promoting activity of Cmb, although it cannot be excluded that Rok/MRLC activity acts in parallel to the effect Cmb exerts on wing hair formation (Fagan, 2014).

mwh and the in/fy group of PCP effectors all have been implicated in restricting actin hair initiation to the distal vertex of the cells by inhibiting proximal hair assembly. Core PCP signaling ensures proper proximal localization of In, Frtz, and Fy proteins (and thus inhibition of prehair formation) to the proximal end of wing cells leading to the formation of a single wing hair on the distal side (Fagan, 2014).

cmb-RA or cmb-RB cause the formation of MHC phenotypes upon overexpression with several wing drivers. Importantly, this overexpression phenotype is enhanced by the removal of one gene dosage of the PCP effectors fy, frtz, in, and mwh as well as deficiencies uncovering those loci. These genetic interactions suggest cmb could exert a positive effect on hair initiation, although such a function would play a supportive or redundant role as neither a lack or ectopic trichomes are found in cmb mutants (Fagan, 2014).

Significantly, this study showed that Cmb physically interacts with Mwh in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, while no vertebrate homologs of cmb have been identified, orthologs of both cmb and mwh were found outside of the insects in the genomes of the crustacean Daphnia magna and of the hard-bodied tick Ixodes scapularis. Ixodes is a member of the Chelicerata, the most basally-branching euarthropod clade that split from the remaining arthropod groups in the Cambrian. The presence of mwh and cmb in Ixodes may be indicative of an ancient protein-protein interaction that has been retained throughout arthropod evolution. Because both Ixodes and Daphnia lack wings, the Mwh/Cmb interaction likely performed different, possibly additional function in the ancestral arthropod. Consistent with this, mwh mutants cause other cuticular hair defects in other regions of the Drosophila body. Alternatively, the Mwh/Cmb interaction evolved much later than the appearance of both of these genes in the genome of the ancestral arthropod. The roles of and interactions between Cmb and Mwh proteins in more non-insect arthropods needs to be further explored (Fagan, 2014).

The presence of both mwh and cmb orthologs in the genomes of members of all holometabolous insect orders may indicate that the Mwh and Cmb interaction is also conserved in this insect clade. The retention of these two genes in members of the more basally-branching hemipteran orders, however, is less conserved. The conservation of mwh and cmb in Holometabolata may be due to their shared mode of wing development, i.e. via internal wing imaginal discs. This is in contrast to the mode of wing development in hemimetabolous insects by which the wings develop as buds outside of the body. Further study into the association of wing development and Mwh/Cmb interactions in other insect orders is needed to elucidate these findings (Fagan, 2014).

Interestingly, PCP effector mutations generally enhance each other. For example, the hypomorphic frtz3 allele is enhanced by weak alleles of in or fy in double mutants. Analogously, removal of a gene dosage of mwh in a fy or in background, enhances their MHC phenotype. In contrast, the MHC phenotype of mwh mutants was (partially) suppressed in mwh cmb double mutants, as significantly fewer cells formed additional hairs. This interaction is likely specific, because this study found it with the temperature sensitive mwh6 allele and with the spontaneous mwh1 allele, two alleles of independent origin unlikely to carry a similar second site mutation and thus further supporting the physiological function of Cmb as a PCP effector. cmb is the only gene reported so far to suppress mwh. Importantly, as mwh1 and cmb both are null alleles (cmbKO lacks expression of both protein isoforms), this result suggests that Mwh acts upstream of and normally antagonizes Cmb and that the derepression of Cmb thus may contribute to the MHC phenotype of mwh mutants (Fagan, 2014).

Unfortunately, tg Cmb antibodies do not detect endogenous Cmb protein in the developing pupal wing. Nevertheless, Cmb expressed in the posterior compartment of the wing under the control of en-Gal4 localizes apically in a punctate pattern. In cells that appear to express at a lower level, Cmb is enriched at the circumference of the cells, but shows no proximo-distal enrichment. Although it cannot be excluded that Cmb localization is an overexpression artifact, this appears unlikely, because it would be expected that Cmb would fill the cells rather than to localize specifically apically. Importantly, Cmb likely localizes to the area of wing cells where Mwh is present, as Mwh known to be initially enriched apically towards the proximal side, further supporting the model that a positive effect of Cmb as a novel PCP effector on wing hair formation may be restricted by Mwh (Fagan, 2014).

Search PubMed for articles about Drosophila Multiple wing hairs

Adler, P., Taylor, J. and Charlton, J. (2000). The domineering non-autonomy of frizzled and van Gogh clones in the Drosophila wing is a consequence of a disruption in local signaling. Mech. Dev. 96: 197-207. PubMed ID: 10960784

Adler, P. N., Zhu, C. and Stone, D. (2004). Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins. Curr. Biol. 14: 2046-2051. PubMed ID: 15556868

Collier, S., and Gubb, D. (1997). Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein. Development 124: 4029-4037. PubMed ID: 9374400

Collier, S., Lee, H., Burgess, R. and Adler, P. (2005). The WD40 repeat protein Fritz links cytoskeletal planar polarity to Frizzled subcellular localization in the Drosophila epidermis. Genetics 169: 2035-2045. PubMed ID: 15654087

Fagan, J. K., Dollar, G., Lu, Q., Barnett, A., Pechuan Jorge, J., Schlosser, A., Pfleger, C., Adler, P. and Jenny, A. (2014). Combover/CG10732, a novel PCP effector for Drosophila wing hair formation. PLoS One 9: e107311. PubMed ID: 25207969

Gubb, D. and Garcia-Bellido, A. (1982). A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster. J. Embryol. Exp. Morphol. 68: 37-57. PubMed ID: 6809878

Guild, G. M., et al. (2005). Actin filament bundles in Drosophila wing hairs: hairs and bristles use different strategies for assembly. Mol. Biol. Cell. 16: 3620-3631. PubMed ID: 15917291

Lawrence, P. A., Casal, J. and Struhl, G. (2004). Cell interactions and planar polarity in the abdominal epidermis of Drosophila. Development 131: 4651-4664. PubMed ID: 15329345

Lee, H. and Adler, P. N. (2002). The function of the frizzled pathway in the Drosophila wing is dependent on inturned and fuzzy. Genetics 160(4): 1535-47. PubMed ID: 11973308

Lu, Q., Yan, J. and Adler, P. N. (2010). The Drosophila planar polarity proteins inturned and multiple wing hairs interact physically and function together. Genetics 185(2): 549-58. PubMed ID: 20351219

Park, W. J., Liu, J., Sharp, E. J. and Adler, P. N. (1996). The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein. Development 122: 961-969. PubMed ID: 8631273

Park, T. J., Haigo, S. L. and Wallingford, J. B. (2006). Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat. Genet. 38: 303-311. PubMed ID: 16493421

Strutt, D. and Warrington, S. J. (2008). 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. Development 135(18): 3103-11. PubMed ID: 18701542

Strutt, D. I., Weber, U. and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signalling. Nature 387: 292-295. PubMed ID: 9153394

Turner, C. M. and Adler, P. N. (1998). Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila. Mech. Dev. 70(1-2): 181-92. PubMed ID: 9510034

Wallar, B. J. and Alberts, A. S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13: 435-446. PubMed ID: 12888296

Winter, C. G., et al. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar polarity signalling to the actin cytoskeleton. Cell 105: 81-91. PubMed ID: 11301004

Wong, L. L. and Adler, P. N. (1993). Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells. J. Cell. Biol. 123: 209-221. PubMed ID: 8408199

Yan, J., et al. (2008). The multiple-wing-hairs gene encodes a novel GBD-FH3 domain-containing protein that functions both prior to and after wing hair initiation. Genetics 180(1): 219-28. PubMed ID: 18723886

date revised: 3 November 2010

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