InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

dachsous encodes a protein that controls imaginal disc morphogenesis in Drosophila and is a member of the cadherin superfamily. Two loci, dachsous(ds) and fat(ft), both members of the cadherin superfamily, have long been known to play important roles during imaginal disc development and morphogenesis. The genetic interaction between ds and ft suggested that these two genes might function in the same pathway. A spontaneous mutation at the ds locus, ds1 was discovered in 1917 by Calvin Bridges. The first recessive and dominant mutations of ft, known as ft1 and Gull, respectively, were isolated two years later; similarities between their phenotypes and that of ds1is the basis for the model suggesting that ds and ft function in the same genetic pathway. Consistent with this model, ds1 was shown by Mohr in 1929 to suppress the Gull phenotype, since one copy of ds1 causes a weak suppression of Gull, and two copies cause a strong suppression (Clark, 1995 and references).

Dachsous and Fat differ from the classic cadherins, and from another cadherin protein of Drosophila, Shotgun, mainly due to the much larger number of extracellular cadherin domains in Ds and Fat. In addition, Shotgun and Fat have EGF- and laminin A G-domain-like repeats in their extracellular domains that are absent in Ds and the classic cadherins. Based on the structures of Ds and Fat, and based on their genetic interaction, a model is presented which accounts for the physical interaction between Ds and Fat. It is proposed that Ds and Fat mediate cell-cell adhesion by homo- and heterophilic interaction of the cadherin domains and transmit signals regulating morphogenesis and cell proliferation via their cytoplasmic domains to the cell interior and nucleus. The morphogenetic signals transmitted by Ds and Ft are not necessarily the same, although the two proteins might cooperate. Only Ft (but not Ds), mediates signals controlling cell proliferation through its specific extracellular EGF-like and laminin A G-domain-like repeats, which act as receptors. Both of these processes, (1) control of cell proliferation and (2) morphogenesis, are intimately linked by coupled equilibria between homophilic and heterophilic associatons of the Ds and Fat cadherins. The likelyhood that Dachsous forms a heterodimeric association with Fat on adjacent cells is used to explain the suppression of Gull phenotype by dachsous mutation (Clark, 1995).

Flies mutant for dachsous exhibit a tissue polarity phenotype. At least some of the tissue polarity phenotypes associated with ds resemble those seen in frizzled pathway mutants and seem unlikely to be due to an effect on cell adhesion. It has been known for some time that the function of ds relates to tissue polarity. Held (1986) found that ds mutations disrupt the polarity of bristles on the legs and that they cause leg joint abnormalities that are similar to those produced by fz and spiny legs. The overall morphological abnormalities caused by ds mutations are greater on the leg than in the wing, but it appears that the effects on the wing and leg may be parallel (Adler, 1998).

Dachsous acts in the frizzled pathway where it affects signaling involved in the formation of proper wing hair polarity. dachsous clones disrupt the polarity of neighboring wild-type cells, suggesting the possibility that dachsous affects the intercellular signaling function of frizzled. The function of the frizzled pathway is essential for the formation of a wing with normal distally pointing hairs. This pathway is thought to be composed of both an intercellular signaling system and an intracellular signal transduction system. The cell autonomous function of this pathway leads to prehair initiation being restricted to the vicinity of the distal vertex. Mutations in genes such as dishevelled, inturned, fuzzy and RhoA produce a tissue polarity phenotype by interfering with the intracellular transduction of the fz signal. Dachsous is not required for the transduction of the fz signal and consistent with this conclusion, it does not cause a polarity pattern that is typical of either a lack of or reduction in fz pathway function. Instead, ds appears to cause a tissue polarity phenotype by altering the direction of fz signaling. The correspondence between the direction of hair polarity (distal), the subcellular location for prehair initiation (in the vicinity of the distal vertex of the cell) and the direction of fz domineering nonautonomy (distal) as seen in wild-type wings is maintained in regions of ds wings, but notably, with reversed polarity. In such wing regions, proximally pointed hairs are formed in the vicinity of the proximal vertex, and fz clones display proximal domineering nonautonomy. This has led to the conclusion that the abnormal hair polarity in ds wings is a consequence of the abnormal direction of fz signaling and that ds cells in these regions are responding normally to an abnormal signal (Adler, 1998).

Two models have been proposed to account for the role of Fz in tissue polarity and to explain the distal domineering nonautonomy of fz clones (Adler, 1997). The cell-by-cell signaling model suggests that the binding of ligand at one side of a cell leads to the Fz receptor becoming unevenly activated across the cell. This leads to both prehair initiation and the relaying of the signal being localized at the distal edge of cells, which leads to hair polarity being coincident with the direction of signaling. In this model the domineering distal nonautonomy of fz clones was ascribed to a failure to receive the signal in cells distal to the clone. The secondary signal model suggests that the Fz receptor is activated in a gradient fashion along the proximal/distal axis of the wing by a long range gradient of a morphogen ligand. Fz activation leads to the proportional production of a secondary signal, which acts locally to polarize cells. In this model the nonautonomy of fz clones is due to a failure of clone cells to produce the secondary signal. A group of proximal cells presumably form the source for the gradient of Fz ligand. Either of these models can easily accommodate the observation that there are regions in a ds wing where the direction of fz signaling is reversed. dachsous mutations could result in a change in the fate of cells that then serve either as ectopic sources of the gradient morphogen or could alter locations that initiate cell-by-cell signaling. Such models might also be able to explain the altered wing shape and wing vein pattern as a consequence of a population of cells with altered cell fate. However, this hypothesis does not however, explain the need for ds function for tissue polarity development in all regions of the wing, as has been shown by an analysis of ds clones. Nor does it explain the enhanced domineering nonautonomy of fz that is observed in ds mutant wings. Therefore, a hypothesis is preferred wherein ds mutations produce their phenotypic effects by altering the function of the fz pathway in all regions of the wing. It is possible that fz signaling takes place at the adherens junction and that ds mutations alter the structure or composition of the junction in a way that alters fz signaling. For example, ds could promote the assembly of an Fz receptor complex at the junction. An 'incomplete' complex formed in a ds mutant might be unstable, leading to aberrant signaling (Adler, 1998).

Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression

Planar cell polarity (PCP) occurs when the cells of an epithelium are polarized along a common axis lying in the epithelial plane. During the development of PCP, cells respond to long-range directional signals that specify the axis of polarization. It has been proposed that with respect to Drosophila eye morphogenesis a crucial step in this process is the establishment of graded expression of the cadherin Dachsous (Ds) and the Golgi-associated protein Four-jointed (Fj). These gradients have been proposed to specify the direction of polarization by producing an activity gradient of the cadherin Fat within each ommatidium. In this report, the key predictions of this model were tested and confirmed by altering the patterns of Fj, Ds and Fat expression. It was shown that the gradients of Fj and Ds expression provide partially redundant positional information essential for specifying the polarization axis. It was further demonstrated that reversing the Fj and Ds gradients can lead to reversal of the axis of polarization. Finally, it was shown that an ectopic gradient of Fat expression can re-orient PCP in the eye. In contrast to the eye, the endogenous gradients of Fj and Ds expression do not play a major role in directing PCP in the wing. Thus, this study reveals that the two tissues use different strategies to orient their PCP (Simon, 2004).

The development of organized PCP requires cells to polarize in response to directional signals within the plane of the epithelium. The apparent absence of local cues has suggested that cells orient their polarity in response to long-range diffusible signaling molecules that form gradients across the tissue. It has been proposed that the role of the diffusible signals, such as Wingless produced at the poles of the eye disc, is to drive graded transcription of Ds and Fj. In this model, the resulting Ds and Fj protein gradients then regulate the function of the cadherin Ft, resulting in a Ft activity gradient, which in turn controls the pattern of Fz competition within each ommatidium. Crucial tests of the model have been precluded by an inability to alter the patterns of Ds and Ft expression. This study has analyzed the effects of altering Fj, Ds and Ft expression in the eye, and provides evidence supporting crucial features of the model. Most importantly, it has been demonstrated that the Fj and Ds expression gradients provide redundant directional information that together orient PCP. Furthermore, the data shows that it is the combination of both gradients that provides the robust directional cues needed to support the perfect fidelity of polarization in wild-type eyes. In addition, it has been shown that graded Ft expression can direct the pattern of ommatidial polarity, thus providing support for the role of Ft as a graded regulator of Fz signaling acting under the control of the Fj and Ds gradients (Simon, 2004).

In the proposed model, the consistent equatorial bias of Fz signaling results from more effective Ft action in each equatorial R3/4 precursor cell when compared with its adjacent polar counterpart. Since this Ft difference results from the action of the Fj and Ds gradients, a key question is how these gradients could control the level of Ft function. Important insight into this issue has come from studies of the wing that suggest that Ft and Ds form a complex in which the localization of Ft on the surface of one cell is promoted by binding to Ds on the surface of the neighboring cell. The dependence of Ft plasma membrane localization on Ds may account for the requirement for Ds function during planar polarization in the eye, even when sufficient directional cues are provided by the Fj expression gradient (Simon, 2004).

The existence of Ds:Ft intercellular dimers suggests several mechanisms by which Ds might regulate Ft. One simple possibility is that Ds merely controls the accumulation of Ft on the surface of the neighboring cell. Thus, the relatively higher level of Ds in the polar R3/R4 precursor, which results from the polar gradient of Ds expression, would lead to the accumulation of more Ft on the bordering surface of the equatorial cell. This would result in an asymmetry in Ft protein levels precisely along the border between the precursor cells where Fz/PCP competition occurs. Although no such gradient has been observed, it would certainly be very subtle and perhaps undetectable. A second possibility is that Ds binding to Ft regulates Ft activity rather than localization. A third possibility is that Ds could participate with Ft in binding to the extracellular domain of a downstream target (Simon, 2004).

Fj appears to play a more limited role than Ds during planar polarization of the eye. Unlike Ds, which both contributes a directional signal through its graded expression and plays an essential role in the interpretation of directional cues, Fj appears only to participate in PCP establishment via the directional information provided by its graded expression. This more limited role can be seen in the observations that either the absence or the ubiquitous expression of Fj yields equivalent phenotypes, and does not grossly disrupt the pattern of polarization unless the Ds gradient has been replaced with ubiquitous expression. How might graded Fj fulfill this role? One possibility is that Fj may regulate the ability of Ft and Ds to productively interact with each other. Thus, the higher expression of Fj in the equatorial cell of each ommatidium leads to more Ft:Ds dimers being formed with Ft in the equatorial cell than in the opposite orientation. Since Fj appears to function in the Golgi, this regulation may involve the direct modification of Ft or Ds (Simon, 2004).

It is important to note that one aspect of the data reported here requires reconsideration of a feature of a previous model. In previous work, it was proposed that Fj acts upstream of Ds, perhaps by modifying the Ds activity gradient. This placement was based on genetic experiments showing that strong differences in Fj activity between R3/R4 precursor cells can direct ommatidial polarization only when Ds is present. The identification of an essential gradient-independent function for Ds clearly complicates the interpretation of these epistasis experiments. As a result, it is no longer possible to infer whether the information provided by the Fj expression gradient acts upstream of Ds to modify the information provided by the Ds gradient. An equally plausible possibility is that Fj regulates the function of the Ds:Ft complexes by modifying Ft rather than Ds function (Simon, 2004).

The work presented here was designed to test specific predictions of the model proposed in an earlier study. However, alternate roles for Ft function have also been proposed. In one model, Ft regulates the production of an unidentified long-range signal that is secreted at the equator and that directly controls eye polarity. The existence of such an unidentified patterning signal, often called Factor X, has been invoked frequently to explain the 'domineering nonautonomy' phenomenon seen in both the wing and the eye near clones of cells lacking function of PCP genes such as Fz. In the alternate model, the role of Ft is to prevent production of this factor everywhere in the eye except at the equator where Ft activity is proposed to be inhibited by unspecified mechanisms, presumably involving Ds. An important distinction between the two models relates to the predicted effects of graded Ft expression. In the model, graded Ft activity provides the key PCP directional cues, and thus ectopic Ft expression gradients are predicted to have the potential to orient ommatidial polarity. In an alternate model, gradients of Ft activity do not provide directional cues. Instead, it is the lack of Ft activity in a sharp zone at the equator that leads to the production of the unidentified patterning factor. As a result, this second model predicts that subtle gradients of Ft expression should not orient polarity, especially in the polar regions of the eye where Ft activity is proposed to be uninhibited. Thus, the data presented in this report demonstrating the orienting ability of Ft expression gradients presents a challenge to this alternate model. In addition, the need for Factor X, whose putative existence has been a common feature of PCP models in both the wing and eye, has been challenged recently on both experimental and theoretical grounds. These reports suggested that domineering nonautonomy results from the tendency of neighboring cells to align their polarization rather than the existence of an additional polarizing signal (Simon, 2004).

The key roles of Ft and the Fj and Ds expression gradients in the eye naturally raised the question of whether similar mechanisms are used to provide directional cues in other tissues, such as the wing. That such conservation might exist was suggested by the existence of gradients of Fj and Ds in the wing. Additionally, it has been demonstrated recently that ectopic gradients of Ft and Ds expression in the wing can produce re-orientation of polarity in the wing. Given the redundant nature of the directional cues provided by the Fj and Ds gradients in the eye, the most rigorous way to evaluate the roles of the Ds and Fj expression gradients in the wing was to examine the consequences of removing the directional information of both gradients simultaneously. When this was done, the resulting wings displayed almost completely normal polarity. Thus, the Ds and Fj expression gradients do not play a major role in orienting PCP in most of the wing blade. One possibility is that there are additional directional signals that act redundantly with the Ds and Fj gradients. Another possibility is that these gradients exist for reasons unrelated to PCP. For example, they may serve to regulate the function of Ft as a regulator of cellular proliferation. Possible support for such a role comes from the observation that flies in which both graded Fj and Ds expression has been replaced with ubiquitous expression survive to adulthood at reduced frequencies, and often display defects in the size and shape of their legs, wings and eyes (Simon, 2004).

The dispensability of the Fj and Ds gradients of expression during the polarization of the wing indicates that there must be currently unidentified directional cues directing wing PCP. Despite their mysterious nature, it is likely that their mode of action will involve the Ds:Ft complex. This inference can be drawn from the observation that animals lacking Ds function, or clones of cells lacking Ft or Ds activity, have substantial PCP defects in the wing. Importantly, clones of ft mutant cells in the wing appear not to read directional cues and instead align their polarity with that of their neighbors. Thus, whatever the nature of the unidentified signals, they appear not to function effectively in the absence of Ds and Ft. Since neither Ft nor Ds is directly required for the Fz PCP signaling at cell-cell junctions, the dependence of these unidentified signals on Ds and Ft suggests that they may act by asymmetrically modifying the action of the Ds:Ft complexes at cell-cell junctions engaged in PCP signaling. Thus, the elegant regulation of polarity in the eye by graded Fj and Ds expression may represent only one of a number of ways to modulate the action of Ft. Further analysis of the mechanisms by which Ft and Ds regulate the pattern of Fz/PCP signaling will undoubtedly aid in the identification of these unknown signals and their mode of action (Simon, 2004).

Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field

Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the 'core' Frizzled system, are still the subject of investigation. Examining the deployment of the Ds-Fat system in different tissues of Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, this study examined the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds-Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex (Lawlor, 2013).

Recent studies in the Drosophila wing and other tissues suggest that polarity may initiate at a localized signalling boundary. In this analysis of the denticle field, the examination of Ds and Fat accumulation and their loss-of- function phenotypes has suggested that a signaling boundary might also be involved. Indeed, creating an ectopic Ds focus supported that notion. Thus, across the denticle field it appears that Ds signaling from the anterior edge of column 5 cells generates an asymmetry in Fat enrichment with high levels along the posterior edge of neighboring cell column 4. This asymmetric deployment would need to be propagated to each column interface anterior (and posterior) to this. The distribution of Four-jointed coupled with its known influences on Ds-Fat binding could support this propagation. For example, since Fj is more highly expressed in column 3 compared with column 4 (see also Marcinkevicius and Zallen, 2013), that would increase the affinity of Fat from column 3 to bind Ds presented from the anterior of column 4 cells (Lawlor, 2013).

However, one difficulty in invoking a role for Four-jointed is that no phenotype was observed in fj mutants. An alternative explanation for propagation might be that since this cell field is rather small, if a strong enough bias existed that presented Ds from the column 5 side of the 4/5 interface, passive propagation could perhaps account for spread of that bias to other interfaces. The 4/5 interface indeed has special properties in several regards. First, across this interface there are signaling asymmetries in EGF receptor and Notch pathway activation. Second, tendon cell specification in column 5 cells nonautonomously influences the shape of denticles on column 4 cells. Finally, during column alignment of denticle field cells, the 4/5 interface aligns earlier and relies partly on a mechanism distinct from the columns anterior and posterior to it (Simone, 2010; Marcinkevicius, 2013). Thus, it is speculated that the 5 side of the 4/5 interface might present Ds in a manner that is unique from other interfaces and thereby sets polarization. The idea that initial polarization starts at a signaling boundary suggests a common theme between the embryonic epidermis and the much more expansive imaginal disk epithelia. Recent work in disks suggests that polarization needs to occur only over a few cell widths, and that, once established, this incipient polarization can be grown through morphogenesis, rather than continually developed by long range gradients. Thus, studying polarization in tissues that are small in scale, such as the embryonic epidermis, may contribute to understanding of the initial polarizing events that occur also in expansive tissues (Lawlor, 2013).

Prior analysis in the later larval epidermis showed that Ds and Fat acted to polarize a restricted domain of the denticle field. This idea was nicely extended by the observation that the precursor to denticles, the f-actin based protrusions (ABPs), were misplaced in the embryonic epidermis in ds and fat mutants. In this study, using quantitative analysis of ABP placement, the spatial requirement for Ds-Fat could further be characterized. Scatter plot analysis, which records the relative position of each individual ABP position, revealed that there exists a graded retention of polarity in the mutants. For instance, ds mutants exhibit a severe loss of polarity in column 5 with ABP placement appearing more and more correctly polarized as one moves anterior toward column 2. This strongly supports the idea that a second polarizing input remains in place in ds or fat mutants. That input is likely to be the Fz system, which has been shown to affect the anterior region of the denticle field. In fact, again using quantatative analysis, it was showm previously that removing fz in ds mutants leads to more severe mis-polarization of larval denticle columns 2 and 3 (Donoughe, 2011). Thus, the denticle field is polarized using input from each pathway, with the Fz system largely responsible over the anterior domain, and the Ds-Fat system responsible over the posterior. A corollary of this is that the Fz and Ds pathways provide separate inputs to planar polarity (Lawlor, 2013).

In considering how ABP assembly might be polarized several pieces of data enter into consideration. First, the ABPs did not simply exhibit binary states of 'membrane polarized', or not. Rather, the graded retention of polarization observed in mutants suggests that ABPs can be stably formed at different coordinates along the apical face of the cell. Furthermore, in cases where two or more ABPs are made by a single cell, they almost always both exhibit a similar polarity value. Thus, it will be important to understand what constitutes the cortical apical structures that capture the ABPs. Those structures must interact with the effector circuit for Ds-Fat signaling, and, since they appear to come under the influence of Fz system in ds or fat mutants, they must also interface with the Fz system effector circuitry. Nevertheless, since the Fz and Fat receptors are so dissimilar from a molecular standpoint, their immediate effectors are likely to be quite distinct. Only by identifying the immediate and downstream effectors can it be understood how this polarized output occurs (Lawlor, 2013).

The situation is more complex as there are distinct polarization outputs to account for, just considering the Ds-Fat circuit. These will likely require distinct effector circuits. Studies in the Drosophila thorax, eye and wing already showed that asymmetric Ds and Fat accumulation leads to alterations of polarity evident as changes in the enrichment of the myosin Dachs. However, Dachs cannot be the sole effector in the embryonic epidermis as ABPs are correctly placed in its absence. Strong evidence for distinct Fat effectors also derives from elegant work showing that Fat affects junctional polarity, and is important for columnar cell alignment within the denticle field (Marcinkevicius, 2013). However, junctional polarity is most severely affected over a domain distinct from that exhibiting the most striking ABP placement defects (Marcinkevicius, 2013). And, while the role in junctional reorganization is most clearly defined among denticle field cells, it appears to apply across the smooth field also (Marcinkevicius, 2013). In contrast, the current study showed that polarization of ABPs can only occur over the denticle and not smooth field. In fact the role of Fat in alignment appears genetically separable from that in ABP placement (Marcinkevicius, 2013). Finally, different labs have identified distinct critical regions of the Fat intracellular domain necessary for polarity signaling. Thus, the mechanisms underlying polarity signaling through Ds- Fat await the identification of these different effector circuits (Lawlor, 2013).

The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen

The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. The Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. It is suggested that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair (Mangione, 2018).

PCP pathways act as key coordinators in the makeover of planar epithelial tissues during development by modulating adhesive interactions and mechanical forces. However, the regulatory means that these pathways use to direct the topographical organization of epithelial cells are far from being clear. By applying in vivo analyses to the morphogenesis of the adult abdominal epidermis of Drosophila, this study has characterized a new mechanism for the stepwise long-range cobblestone organization of the tissue. The organization of the abdominal epithelial landscape was found to be the result of an axially oriented adhesiveness mediated by the Ds/Ft/Fj pathway. The directional cues dictated by this pathway put the epithelial cells on the right track, orienting their otherwise changing shapes along the A/P axis (Mangione, 2018).

Global tissue changes may involve many different activities: coordinated cell-cell rearrangements triggering tissue reorientation or convergent extension,or spatially controlled proliferation and growth, division orientation, and death. The uniform axially oriented alignment of histoblasts emerges in a precise spatiotemporal manner through coordinated changes in cell shape orientation. Histoblasts constitute a highly proliferative tissue with much room for expansion. In this scenario, which is very different from that of epithelial tissues constrained in their dimensions like the fly notum, to reach uniformity in cell alignment orientations, changes in cell shape and area would be preferred over cell intercalations. When the expressions of ds, ft, or fj are affected, the relative orientations of cell alignments are severely disturbed without alterations in tissue differentiation (Mangione, 2018).

It is not known which positional cues the Ds/Ft/Fj pathway interprets to dictate the stereotyped uniform anisotropic polarization of the abdominal epidermis along the A/P axis. Some tips may come from analyses on the regulation of the components of the core Fz-PCP system. This system is a required partner of the Ds/Ft/Fj pathway for proper planar polarity acquisition. Both pathways rely on primary long-range global cues to coordinate short-range cell polarity. During the development of the eye and wing in Drosophila, the secreted factor Wingless (Wg/Wnt1) modulates intercellular interactions in both systems. In the Ds/Ft/Fj pathway, Ds and Ft form heterodimers, whereas in the Fz-PCP system, there are Fz-Vang heterodimers and Stan-Stan homodimers. In the eye and the wing discs, Wg binds to Fz, which affects Fz-Vang interactions and the activity of the Fz-PCP system. As a result, cells orient toward the source of Wnt expression (Wg and Wnt4) at the compartment margins. Wg/Wnt4 could be playing an equivalent role during the axial uniform alignment of oriented cells in the abdomen. While Wg specifies the tergite and sternite cell fates, how it could regulate the graded expression of ds, ft, or fj or influence the uniform axial orientation of histoblasts remains poorly explored (Mangione, 2018).

It is known that differential adhesive properties between neighboring cells prevent intermingling, as they tend to minimize their contacts. In clones, this leads to smooth borders. Major differences were found in roughness, perimeter, and, to a lesser extent, roundness in mutant clones for members of the Ds/Ft/Fj pathway. These differences strongly support a role for the Ds/Ft/Fj pathway and, in particular, the opposing graded expressions of the atypical cadherins Fat and Ds, in generating directional information at cell contacts. Mutant clones generate planar conflicts between cells with different adhesive properties and smooth borders at specific edges, and the directional information is lost (Mangione, 2018).

The evolving functional pattern delineated by the Ds/Ft/Fj pathway arises as an elegant and efficient way to dictate directional order across developmental fields. Several pieces of evidence point to it as a key element modulating similar processes in different organisms. Mitral valve prolapse (MVP) is a common cardiac valve disease, the genetic etiology of which has remained elusive. Recently, it has been shown that MVP could be traced back, both in mice and fish models, to developmental errors in valve morphogenesis. The epicardial-derived cells fail to uniformly align into the posterior leaflet, and this failure correlates to a missense mutation in the DCHS1 gene, the human homolog of ds. Similarly, the establishment of polarized arrays of aligned chondrocytes in the zebrafish developing pharyngeal arch requires Fat3 and Dchs2 cadherins. During postnatal stages in mammals, hair follicles progressively get in line, both locally and globally, precisely with the A/P axis. This process of postnatal refinement has been suggested, although not proved, to be the result of instructive functions originating from the Ds/Ft/Fj pathway. Whether vertebrate Ds/Ft/Fj signaling is essential to propagate polarity at a distance, is linked to molecular gradients, or may interact with other polarizing signals remains unknown (Mangione, 2018).

The subcellular polarization of the atypical myosin Dachs (D) in response to the Ds/Ft/Fj pathway appears to be uncoupled from the uniform orientation of cell alignments; D polarity is reoriented but sustained in ds, and its loss did not affect cell alignments uniformity. These data led to a hypothesis that a bias in contractility mediated by D at the cell cortex may be not critical for uniform cell orientation. Moreover, contractile anisotropy as a factor directing the axial uniformity of histoblasts is unsupported by the observed isotropic distribution of vinculin at cell vertices. In this scenario, asymmetric adhesiveness through heterodimeric interactions between Ds and Ft appears to be a more plausible element directing the uniform orientation of histoblasts (Mangione, 2018).

Assuming that cells and tissues tend to minimize their surface free energy, contacts through adhesion molecules and contractile activities at the cell cortex would be key determinants of cell and tissue shape. Adhesiveness will promote cells to spread their shared surface, while contractility will counterbalance the adhesive forces. Differential adhesive properties within histoblasts would introduce anisotropic tension affecting cell-cell contacts and the capacity to coordinately orient shape changes in the tissue plane. This anisotropic tension, which is revealed by laser microsurgery, is lost in ds mutants. Anisotropic tension is also uncovered in clones, in which contact angles and lengths between histoblasts adjust to their conflicting genotypes and their relative location. Differential adhesiveness at cell junctions would have direct input into surface tension. Tensile patterns rather than cell positions would therefore play instructive roles in the acquisition of uniform order. At any given point in time, they will reflect the recent developmental history of the tissue. Along this line, during the expansion and remodeling of the histoblasts, the expression pattern of Ft modulated by Ds and fj evolves into an A/P gradient spanning whole compartments. This expression refinement will result in the spreading throughout the epithelium of a counterbalanced adhesion share between Ft and Ds that will delineate the axially oriented surface tension landscape that will instruct uniform cell alignments (Mangione, 2018).

Will the final arrangement of the cells be physically stable (minimal energy) upon completion or will a secondary event be necessary to stabilize it? In Caenorhabditis elegans, the epidermal cells elongate during development and subsequently attach to the cuticle to fix their shape. Thus, the collagenous exoskeleton secreted by the apical surface of the epidermis seems to be indispensable. The histoblasts use their apical surface to attach to the overlaying pupal cuticle very early on. These contacts are very dynamic during the period of expansion and become stabilized by the end of tissue remodeling. Whether they fulfill a hardening role on the tissue landscape is an open question (Mangione, 2018).

The role this study has uncovered for the Ds/Ft/Fj pathway implementing the uniform orientation of the alignment of histoblasts does not relate to any developmental function in patterning, cell specification, and/or differentiation. The directional evolution of the expression of the different Ds/Ft/Fj pathway elements sets a spatially and temporally controlled directional adhesive partnership between Ds and Ft. This provides the basis for the establishment of a dynamic adhesiveness pattern unfolding over time that directs cell shape changes, ultimately guiding the uniform alignment of the epithelial cells across the tissue (Mangione, 2018).

REGULATION

Protein interactions

Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains

The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. This study shows that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed is essential for its kinase activity in vitro and for its biological activity in vivo. These results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi (Ishikawa, 2008).

The Fat and Hippo signaling pathways intersect at multiple points and influence growth and gene expression through regulation of the transcriptional coactivator Yorkie. Fat signaling also influences planar cell polarity (PCP). Fat acts as a transmembrane receptor, and is a large (5147 amino acids) atypical cadherin protein, with 34 extracellular cadherin domains. Dachsous (Ds) is also a large (3503 amino acids) transmembrane protein with multiple cadherin domains and is a candidate Fat ligand because it appears to bind Fat in a cultured cell assay, acts non-cell autonomously to influence Fat pathway gene expression, and acts genetically upstream of fat in the regulation of PCP. A second protein, Four-jointed (Fj), also acts non-cell autonomously to influence Fat pathway gene expression and acts genetically upstream of fat in the regulation of PCP. However, Fj is a type II transmembrane protein that functions in the Golgi. Thus, Fj might influence Fat signaling by posttranslationally modifying a component of the Fat pathway (Ishikawa, 2008).

To investigate the possibility of modification of Fat or Ds, FLAG epitope-tagged fragments of their extracellular domains together were coexpressed with Fj in cultured Drosophila S2 cells. When the first 10 cadherin domains of Ds (Ds1-10) were coexpressed with Fj, a shift in mobility was observed. A common posttranslational modification of secreted and transmembrane proteins as they pass through the Golgi is glycosylation. Most glycosyltransferases contain a conserved sequence motif, Asp-X-Asp (DXD; X, any amino acid), which is essential for their activity. Because a related sequence motif [Asp-Asn-Glu (DNE) at amino acids 490 to 492] is present in Fj and its vertebrate homologs, a mutant form of Fj was created in which DNE was changed to GGG (Fj^GGG; G, glycine). The expression levels and Golgi localization of Fj^GGG appear normal, but Fj^GGG expression did not shift Ds1-10 mobility (Ishikawa, 2008).

To identify modified cadherin domains, smaller fragments of Ds1-10 were expressed. The smallest fragments whose mobility was shifted in cells expressing Fj were two-cadherin-domain polypeptides: Ds2-3, Ds5-6, and Ds8-9. Ds2-3 and Ds5-6 appeared to be stoichiometrically modified in cells expressing Fj, whereas Ds8-9 was only partially modified. Fat4-5 was also partially shifted by Fj coexpression. The mobility shifts of these two-cadherin-domain polypeptides were not observed with Fj^GGG. To identify potential sites of modification, their sequences were aligned. This identified four sites at which a Ser or Thr residue was conserved, whose hydroxyl groups could potentially be sites of posttranslational modification. To evaluate their influence, each was mutated in turn to Ala within the Ds2-3 polypeptide. Three of the four mutants had no effect; however, one, Ds2-3^S236A (mutation of Ser²³⁶ to Ala), completely eliminated the Fj-dependent mobility shift. Introduction of an analogous mutation into Ds8-9 also eliminated its mobility shift. Thus, a Ser reside at a specific location within the second of the two cadherin domains was essential for the Fj-dependent mobility shift. This amino acid was a Ser in each of these dicadherin domains, but Thr was also compatible with the Fj-dependent modification. In a structurally solved cadherin domain, this Ser is the seventh amino acid and predicted to be located on the surface near the middle of the cadherin domain (Ishikawa, 2008).

To identify posttranslational modifications associated with this mobility shift, Ds2-3 was purified from S2 cells expressing or not expressing Fj, the proteins were digested with trypsin, andthe resulting peptides were analyzed by mass spectrometry. One peptide from Fj-expressing cells was stoichiometrically shifted by 80 daltons relative to the same peptide from cells not expressing Fj, and it also eluted earlier on high-performance liquid chromatography (HPLC). Mass and tandem mass spectrometry (MS/MS) fragmentation patterns identified this peptide as amino acids 215 to 237 of Ds and refined the site of modification to within amino acids 232 to 237. The mass of the equivalent peptide from Ds2-3^S236A was not altered by Fj expression. Most of the peptides corresponding to Ds2-3 cadherin domains were identified, and none of the others were detectably modified in cells expressing Fj. Thus, the Fj-dependent modification of Ds2-3 comprises an addition of 80 daltons, which is attached to Ser²³⁶. An 80-dalton mass does not correspond to that of any known glycans, but does correspond to the mass associated with addition of a phosphate group. Incubation of Fj-modified Ds fragments with either calf intestinal alkaline phosphatase (CIP) or Antarctic phosphatase (AnP) reversed the Fj-dependent mobility shifts of Ds2-3, Ds8-9, and Fat 4-5. Thus, Ds and Fat cadherin domains are subject to Fj-dependent phosphorylation at a specific Ser residue (Ishikawa, 2008).

To investigate whether Fj itself has kinase activity, a secreted, epitope-tagged Fj (sFj:V5) was purified from the medium of cultured S2 cells. Purified sFj:V5 was then incubated with affinity-purified Ds2-3 and [γATP (adenosine 5'-triphosphate)] in buffer. Transfer of ³²P onto Ds2-3 was observed in the presence of sFj, but not in its absence, and not when sFj^GGG was used as the enzyme. Moreover, Ds2-3^S236A was not detectably phosphorylated by sFj. The activity of Fj expressed in a heterologous system was also characterized by expressing a glutathione S-transferase:Fj (GST:Fj) fusion protein in Escherichi coli and partially purifying it on glutathione beads. GST:Fj, but not GST:Fj^GGG, catalyzed the transfer of ³²P onto Ds2-3. Thus, Fj is a protein kinase (Ishikawa, 2008).

The generic kinase substrates myelin basic protein and casein were not detectably phosphorylated by sFj. Thus, Fj appears to have a limited substrate specificity. Only a few proteins have been identified as being phosphorylated in the secretory pathway, and none of the responsible kinase(s) have been molecularly identified. A Golgi kinase activity, referred to as Golgi casein kinase, preferentially phosphorylates Ser or Thr residues within a S/T-X-E/D/S(Phos) consensus sequence. Because Fj does not phosphorylate casein, and the Ser residues within cadherin domains targeted by Fj do not conform to Golgi casein kinase sites, Fj is not Golgi casein kinase. Fj autophosphorylation was detected, but this reaction was weak compared to phosphorylation of Ds2-3. The autophosphorylation reaction is apparently unimolecular, because GST:Fj and sFj:V5 did not phosphorylate each other and the fraction of Fj phosphorylated was independent of concentration (Ishikawa, 2008).

Some cadherin domain polypeptides that include a Ser as the seventh amino acid were not detectably shifted, but the mobility shift on Ds2-3 might reflect a conformational effect. To examine the ability of Fj to phosphorylate other cadherin domains, in vitro kinase reactions were performed with [γ-³²P]ATP. This identified phosphorylation sites on polypeptides that were not gel shifted, including Fat2-3, Fat10-11, and Fat12-13. The in vitro kinase reactions also identified differences in the efficiency with which different cadherin domains were phosphorylated by Fj, with Ft3, Ds3, and Ds6 being the best substrates (Ishikawa, 2008).

If the presence of a Ser or Thr at the seventh amino acid of a cadherin domain is taken as the minimal requirement for Fj-mediated phosphorylation, there are nine potential sites in Ds and 11 in Fat. However, Fat10, Ds2, Ds11, Ds13, and Ds18 were not detectably phosphorylated, despite the presence of Ser or Thr at this position. Presumably, there are other structural features important for recognition by Fj. This was also emphasized by the detection of phosphorylation of the Ds2-3 polypeptide, but not the Ds3-4 polypeptide, even though both contain Ser²³⁶. The dicadherin constructs were based on published annotations, but in comparing Ds cadherin domains to structurally solved cadherin domains, it was realized that these misposition the intercadherin domain boundary, and consequently these constructs lacked three amino acids of the first cadherin domain. Addition of these amino acids, together with the intercadherin domain linker sequence, enabled phosphorylation of a Ds3 single-cadherin domain construct (Ishikawa, 2008).

A weak similarity between Fj and the bacterial kinase HipA, and between Fj and the mammalian lipid kinase phosphatidylinositol 4-kinase II (PI4KII), has been suggested previously on the basis of bioinformatic analyses in which HipA or PI4KII were used as the starting point for PSI-BLAST searches. Asp residues play critical roles in catalysis and in the coordination of Mg²⁺ in these and other kinases, and the loss of Fj kinase activity associated with mutation of the conserved DNE motif is thus consistent with the inference that Fj is related to other kinases. A single Fj ortholog, Fjx1, is present in a range of vertebrate species, including humans (Ishikawa, 2008).

To investigate the biological requirement for Fj kinase activity, the catalytically inactive fj^GGG mutant was assayed in vivo. A V5 epitope-tagged form of this gene was expressed in transgenic Drosophila. At the same time, V5-tagged wild-type fj was contructed. To ensure that both forms were expressed in similar amounts, site-specific integration was used to insert transgenes at the same chromosomal location. Immunostaining confirmed that Fj^GGG:V5 and Fj:V5 both exhibited normal Golgi localization and were expressed in similar amounts. Uniform overexpression of fj reduces the growth of legs and wings and interferes with normal PCP. Fj:V5 exhibited phenotypes consistent with previous studies, but Fj^GGG:V5 was completely inactive. Thus, mutation of the DNE motif in Fj abolishes its biological activity (Ishikawa, 2008).

The identification of Fj's cadherin domain kinase activity provides a biochemical explanation for the influence of Fj on Fat signaling and supports a model in which Fj directly phosphorylates Fat and Ds as they transit through the Golgi to influence their activity, presumably by modulating interactions between their cadherin domains. Because there was a substantial difference in the efficiency with which individual cadherin domains could be modified by Fj, both in cell-based and in vitro assays, it is also possible that differences in the extent of Fat and Ds phosphorylation normally occur in vivo and might differentially modify their binding or activity (Ishikawa, 2008).

Drosophila lowfat, a novel modulator of Fat signaling

The Fat-Hippo-Warts signaling network regulates both transcription and planar cell polarity. Despite its crucial importance to the normal control of growth and planar polarity, there is only a limited understanding of the mechanisms that regulate Fat. This study reports the identification of a conserved cytoplasmic protein, Lowfat (Lft), as a modulator of Fat signaling. Drosophila Lft, and its human homologs LIX1 and LIX1-like, bind to the cytoplasmic domains of the Fat ligand Dachsous, the receptor protein Fat, and its human homolog FAT4. Lft protein can localize to the sub-apical membrane in disc cells, and this membrane localization is influenced by Fat and Dachsous. Lft expression is normally upregulated along the dorsoventral boundary of the developing wing, and is responsible for elevated levels of Fat protein there. Levels of Fat and Dachsous protein are reduced in lft mutant cells, and can be increased by overexpression of Lft. lft mutant animals exhibit a wing phenotype similar to that of animals with weak alleles of fat, and lft interacts genetically with both fat and dachsous. These studies identify Lft as a novel component of the Fat signaling pathway, and the Lft-mediated elevation of Fat levels as a mechanism for modulating Fat signaling (Mao, 2009).

Recent studies have linked together the action of several tumor suppressors into a Fat-Hippo-Warts signaling network. These genes play a crucial role in growth control from Drosophila to mammals, as exemplified by the ever-increasing number of cancers that have been associated with mutations in pathway genes. Fat-Warts signaling regulates growth through a transcriptional co-activator protein, called Yorkie (Yki) in Drosophila and YAP in vertebrates. In addition, Fat influences a distinct planar cell polarity (PCP) pathway. Planar cell polarity is the polarization of cells within the plane of a tissue, and can include both polarized structures, like hairs and bristles, and polarized behaviors, such as cell division and cell intercalation (Mao, 2009).

Fat is a large member of the cadherin family, and acts as a transmembrane receptor. Fat influences the subcellular localization of both the unconventional myosin Dachs and the FERM-domain protein Expanded, and through these proteins ultimately regulates the kinase Warts. Warts then inhibits Yki by phosphorylating it: phosphorylated Yki is retained in the cytoplasm, but unphosphorylated Yki enters the nucleus to promote the transcription of target genes. The Fat PCP pathway is less well characterized, but it is partially dependent upon Dachs, and also involves Atrophin (Grunge), a transcriptional co-repressor that can bind to the Fat cytoplasmic domain (Mao, 2009).

The only Fat ligand identified is Dachsous (Ds), which like Fat is a large, atypical cadherin, and which influences the phosphorylation of Fat by Discs overgrown. ds mutants have phenotypes similar to, but weaker than, those of fat mutants, raising the possibility that there might be other ligands, or other means of regulating Fat. The Golgi kinase Four-jointed (Fj) also regulates Fat signaling, but presumably acts by modulating Fat-Ds interactions. Intriguingly, the two known Fat pathway regulators (ds and fj) are expressed in gradients in developing tissues. The vectors (directions) of these gradients parallel vectors of PCP, and experimental manipulations of ds and fj indicate that, at least in some tissues, their graded expression can direct PCP. The graded expression of ds and fj also influences the transcriptional branch of the pathway and wing growth, but in this case it is the slope rather than the vector of their gradients that appears to be instructive (Mao, 2009).

Although thus far most components of Fat signaling have been identified through genetic studies in Drosophila, protein interaction screens are an alternative approach with which to identify components of signaling pathways. A genome-wide yeast two-hybrid screen identified the product of the CG13139 gene as both a candidate Fat-interacting protein and a candidate Ds-interacting protein. This gene, which has been named lowfat (lft), encodes a small protein of unknown structure and biochemical function. It shares sequence similarity with two vertebrate genes, Limb expression 1 (Lix1) and Lix1-like (Lix1l;. Lix1 was first identified in chickens through a differential screen for genes expressed during early limb development. Subsequent analysis in mice revealed that Lix1 is actually expressed more broadly. Lix1l has been defined only by its sequence similarity to Lix1. The biological functions of these genes have not been described, although genetic mapping of a feline spinal muscular atrophy identified LIX1 as a candidate gene (Mao, 2009).

While a basic outline of Fat signaling has emerged, many steps remain poorly understood. This study shows that lft is a modulator of Fat signaling, and identified a cellular requirement for Lft in establishing normal levels of both Fat and Ds. These observations identify transcriptional regulation of lft as a potential mechanism for modulating Fat signaling through its post-translational regulation of Fat and Ds protein levels. It was also establish human LIX1L as a functional homolog of Lft, and LIX1 and LIX1L were shown to be Fat-interacting proteins, thus identifying a likely cellular function of vertebrate Lix1 genes as modulators of Fat signaling. This linkage raises the possibility that other Fat pathway components could be candidate susceptibility loci for spinal muscular atrophy (Mao, 2009).

lft mutants display decreased levels of both Fat and Ds protein staining, and presumably as a consequence exhibit a characteristic Fat pathway phenotype in the wing. In addition, lft can genetically interact with both fat and ds to cause more severe phenotypes. The lft mutant phenotype resembles weak mutant alleles of fat or ds, and lft mutants do not exhibit any additional phenotypes that could not be accounted for by effects on Fat signaling. The expression of lft itself is modulated by other signaling pathways, and differences in lft expression levels correlate with differences in Fat and Ds protein levels both in wild-type animals, and when lft levels are experimentally increased or decreased. Thus, transcriptional regulation of lft defines a mechanism for modulating Fat signaling (Mao, 2009).

Lft influences levels of both Fat and Ds. Because Fat and Ds in turn can influence levels of Lft, and because Fat and Ds also influence the localization of one another to the membrane, it is inferred that for any one of these three proteins, the influence that it has on the other two includes both direct effects, and indirect effects mediated through the third protein. In addition, the net effect observed for any one protein presumably also reflects the consequences of feedback regulation of its own levels via the other two proteins (Mao, 2009).

Given the substantial decrease in Fat staining in lft mutants, the phenotype appears surprisingly mild. This observation suggests that Fat is normally present in excess; for example, it could be that only a fraction of Fat is normally active, and that levels of Fat are not normally limiting for pathway activation. This hypothesis was supported by the observation of enhanced Fat pathway phenotypes in combination with fat¹, and would be consistent with the conclusion that Fat acts as a ligand-activated receptor, with only a fraction of Fat normally being present in the active form (Feng, 2009; Sopko, 2009). Complicating this simple explanation is the observation that the levels of the Fat ligand Ds are also reduced in lft mutants. However, because Fat signaling is influenced not only by the amount of Ds, but also by the pattern of Ds (i.e. is Ds expression graded, and how steeply), Ds can have positive or negative effects on Fat activity. Thus, it is suggested that the lft mutant phenotype might be relatively weak because decreased Fat and Ds levels, which would be expected to decrease Fat signaling, are partially offset by a flattening of the Fat and Ds expression gradients, which would be expected to increase Fat-Warts signaling (Reddy, 2008; Rogulja, 2008; Willecke, 2008; Mao, 2009 and references therein).

The observation that ds lft double mutants have more severe phenotypes than do ds or lft single mutants indicates that ds and lft can each independently influence Fat. lft and ds both influence Fat levels and localization, but even in the absence of these two genes, there was a visible difference in Fat protein staining between the wing pouch and the wing hinge. This implies that there are additional Fat regulators, and that the expression of these additional Fat regulators is differentially distributed between the wing pouch and the wing hinge. One additional Fat regulator that is differentially expressed between the pouch and the hinge is Fj, although as Fj is thought to act by influencing Fat-Ds interactions, it is not clear whether it could explain the differential Fat staining observed (Mao, 2009).

It appears that Lft is a major contributor to the normal levels of Fat. Since Lft binds to the Fat cytoplasmic domain, it presumably influences Fat protein levels through this direct binding. Different molecular mechanisms for how Lft might influence Fat (and Ds) levels can be envisioned. One attractive possibility, given that Fat and Ds are transmembrane proteins, and that Lft could co-localize with them at the sub-apical membrane, is an effect on endocytosis, but it is also possible that Lft affects them in some other way (Mao, 2009).

Because Lft is closely related to LIX1 and LIX1L, and indeed LIX1L is functionally homologous to Lft, these studies of Lft identify regulation of mammalian Fat and Ds homologs as the likely cellular functions of LIX1 and LIX1L. Consistent with this inference, these proteins could bind to the cytoplasmic domain of human FAT4, and a BLASTP search with a short sequence motif of Fat common to Ds and FAT4 (WEYLLNWGPSYENLMGVFKDIAELPD) identifies these three proteins plus the mammalian Ds homologs DCHS1 and DCHS2 as the five closest matches in protein databases. This sequence motif also exhibits weak similarity to a region of E-cadherin that has been identified as contributing to binding to β-catenin, but there is no obvious primary sequence similarity between Lft and β-catenin, and Lft did not detectably affect E-cadherin staining (Mao, 2009).

Functional studies of LIX1 and LIX1L in vertebrates have not yet been reported. However, feline LIX1 has been genetically linked to feline spinal muscular atrophy. Direct examination of human LIX1 in spinal muscular atrophy patients did not reveal any mutations. Nonetheless, the linkage of LIX1 and LIX1L to Fat signaling suggests that other members of the Fat signaling pathway should also be examined as potential candidate susceptibility loci for this debilitating disease. Murine Fat4 has been shown to be required for normal PCP in the ear and kidney; however, it is also highly expressed in the nervous system, as are murine Lix1 and Dchs genes, consistent with the expectation that these genes will interact in mammals, and might influence nervous system development (Mao, 2009).

Processing and phosphorylation of the Fat receptor

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. This study shows that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. These observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance understanding of the requirement for Dco in Fat signaling (Feng, 2009).

Activation of transmembrane receptors often involves post-translational modifications, such as phosphorylation or cleavage. To investigate potential modifications, Fat was examined by Western blot analysis. In lysates of wing discs, antisera raised against the Fat intracellular domain (anti-Fat ICD) detected a prominent band with a mobility of ~95 kDa (Ft-95), and a faint band with a mobility corresponding to a much larger polypeptide (Ft-565). fat is predicted to encode a 5,147 amino acid protein, with a calculated mass of 565 kDa. Thus, Ft-95 is too small to correspond to full length Fat. Nonetheless, examination of lysates from fat mutant discs confirmed that both Ft-95 and Ft-565 are fat-dependent (Feng, 2009).

To investigate this apparent cleavage of Fat, a C-terminally tagged Fat protein (Fat:FVH) was created. When Fat:FVH was transfected into cultured Drosophila S2 cells, a band with a high apparent molecular weight, consistent with full length Fat, was observed. However, most Fat was detected in lower molecular weight bands. One correlates with the 95-kDa fragment of endogenous Fat (after accounting for the C-terminal tags), but the other appears smaller, ~70 kDa (Ft-70). Although Ft-70 was not detected when endogenous Fat was examined in imaginal discs, it could be detected in discs when Fat:FVH was overexpressed from UAS transgenes. Expression of Fat:FVH under tub-Gal4 control also confirmed that Fat:FVH is functional, because it rescued fat mutant animals. The detection of Ft-95 and Ft-70 with C-terminal epitope tags supports the conclusion that Fat is proteolytically processed. Based on their mobility, the cleavage leading to Ft-95 occurs in or near the 2 extracellular laminin G-like domains, whereas the cleavage leading to Ft-70 occurs near the transmembrane domain. A Fat construct that excludes the cadherin and EGF domains but includes most of the laminin G domain region appears to be processed to the same cleavage products as is full-length Fat, whereas a smaller Fat construct that also lacks the laminin G domains (Fat-STI-4:FVH) yields a single major band, suggesting that it is not processed (Feng, 2009).

To further characterize Fat processing, an N-terminally tagged Fat (V5:Fat) was constructed. Examination of V5:Fat by Western blotting lysates of S2 cells identified 2 bands of high apparent molecular weight, and did not detect Ft-70 or Ft-95. Although the resolving power of the gel and the lack of suitable markers precluded precise determination of the size of these large bands, their mobility is consistent with the expected detection of both full-length Fat (Ft-565) and an approximate 470-kDa N-terminal product of proteolytic processing in the Laminin G domain region (Ft-470). Double staining V5:Fat with anti-Fat ICD and anti-V5 supported the conclusion that slowest mobility isoform is full-length Fat, whereas Ft-470 lacks the Fat ICD. To characterize cleavage of V5:Fat in vivo at endogenous expression levels, the V5 tag was incorporated into a fat⁺ genomic clone, and then phiC31-mediated recombination was used to insert this into the Drosophila genome. This genomic V5:fat⁺ construct rescued fat mutants. Western blotting lysates of imaginal discs revealed that Ft-470 is more abundant than Ft-565. Because these proteins are similar in size, this differential detection is unlikely to be due to differences in blotting transfer efficiency. Hence, it is concluded that the majority of Fat protein in vivo is processed (Feng, 2009).

To investigate the nature of Fat displayed on the cell surface, biochemical experiments were performed on cultured cells. S2 cells expressing V5:Fat were incubated with anti-V5 in the absence of detergent, and then cell surface Fat bound by anti-V5 antibodies was immunoprecipitated. As a control, Fat:FVH, which includes a cytoplasmic V5 tag that should not be accessible in intact cells, was expressed. Western blot analysis of the immunoprecipitated material with anti-Fat ICD antibodies confirmed that cell surface V5:Fat is processed. In addition, these experiments demonstrate that Ft-470 and Ft-95 remain stably associated after processing. By contrast, Ft-70 was not detected, indicating that it is not associated with Ft-470. Because coimmunoprecipitation of Ft-470 and Ft-95 could be observed under reducing conditions, the association between them does not require disulfide bonds (Feng, 2009).

Because Fat processing can occur in S2 cells, which do not express detectable levels of Ds and grow as isolated cells, and processing can occur on a truncated Fat polypeptide that lacks the cadherin and EGF domains (Fat-STI:FVH), it appears that Fat processing is part of its normal maturation, rather than a regulated event. In this regard, it appears analogous to the S1 cleavage that is involved in maturation of the Notch receptor, or to the apparent processing of the Starry night/Flamingo cadherin (Feng, 2009).

Under optimal conditions, Ft-95 from wing discs runs as doublet, with a prominent lower band, a weaker upper band, and a faint smear in between. Treatment of lysates with calf intestinal alkaline phosphatase (CIP) resulted in a single sharp band ~95 kDa, with a mobility similar to the fastest of the 95-kDa mobility isoforms in untreated samples. Thus, a fraction of Ft-95 in vivo is phosphorylated. Because Ft-95 is too C-terminal to include the cadherin domains, the phosphorylation detected presumably reflects a phosphorylation of the intracellular domain, rather than Fj-mediated phosphorylation of cadherin domains. To investigate the relationship between Ft-95 phosphorylation and Fat signaling, Fat was examined in lysates of wing imaginal discs in which its putative ligand, ds, was either mutant or overexpressed. Proteolytic processing of Fat was not Ds-dependent, because Ft-95 was observed at similar levels in all cases. Mutation of ds results in enlarged wings and wing discs, and lower levels of Wts protein, a phenotype similar to, although weaker than, that of fat. Western blot analysis of Fat from ds mutant wing discs revealed that levels of the faster mobility Ft-95 band are elevated, whereas the slower mobility band (Ft-95-P) is reduced. Ds overexpression reduces wing size. When Ds was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Ds levels of 10-fold. Strikingly, this overexpression of Ds increased the relative amount of Ft-95-P. These observations imply that the presence or absence of Ds modulates Fat phosphorylation. This was confirmed by the observation that phosphatase treatment of lysates from Ds-expressing discs collapsed the Ft-95 doublets into a single band. The visual impression that the presence of the slower mobility (Ft-95-P) isoform(s) was promoted by Ds was confirmed by quantitative line scanning of Western blot analyses (Feng, 2009).

Both mutation of fj and fj overexpression are associated with modest reductions in wing and leg size. When fj was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Fj levels of 100-fold. This overexpression of fj was associated with an increase in the relative amount of phosphorylated Fat, and when coexpressed with ds, the increase in phosphorylated Fat appeared even greater, consistent with the reductions in wing size. Mutation of fj had only subtle affects (Feng, 2009).

Altogether, these observations identify a correlation between the presence of the Fat ligand Ds, the level of signaling through Fat to regulate Warts levels and wing growth, and the phosphorylation of the Fat cytoplasmic domain. Thus, they suggest that activation of Fat by its ligand Ds is associated with Fat phosphorylation. From the relative levels of different mobility isoforms if is inferred that in the absence of Ds overexpression, a majority of Fat is in a hypophosphorylated form, whereas overexpression of Ds promotes the production of a hyperphosphorylated form. This identification of a posttranslational modification of Fat that is promoted by Ds is consistent with the hypothesis that Fat and Ds act as receptor and ligand in a signal transduction pathway, and identifies a molecular process that appears correlated with Fat activation. Constructs that lack most of the extracellular domain, and presumably can not interact with Ds, can rescue fat mutants. However, this rescue is only partial, and has only been observed when intracellular domain constructs are overexpressed. One possibility is that interaction with ligand triggers clustering of Fat, and that overexpression of the intracellular domain allows ligand-independent clustering. This could be analogous to other signaling pathways (e.g., TGF-β, receptor tyrosine kinase), in which ligand-mediated clustering promotes phosphorylation of the cytoplasmic domain of the receptor, and for which the requirement for ligand can sometimes be bypassed by receptor overexpression (Feng, 2009).

In considering kinases that might contribute to the Ds-promoted phosphorylation of Fat, the CKIδ/ε family member Dco was a logical candidate. Genetic epistasis tests positioned dco within the Fat pathway, upstream of dachs. At the same time, dco³ exerts cell-autonomous affects on the expression of Fat target genes, which implies that it acts within receiving cells. These observations suggested Dachs or Fat as potential substrates. Initial assessment of the ability of Dco to phosphorylate them was conducted by assaying for mobility shifts in S2 cells. Dco had no effect on Dachs. By contrast, when Dco was cotransfected together with Fat, a shift in the mobility of the C-terminal cleavage products was observed. A Dco-dependent mobility shift was also observed for both the Fat-STI:FVH and Fat-STI-4:FVH constructs. Confirmation that this mobility shift was due to phosphorylation of Fat was provided by the observation that it could be reversed by phosphatase. Overexpression of a Dco construct under UAS-Gal4 control could also increase phosphorylation of endogenous Fat in vivo (Feng, 2009).

If phosphorylation of Fat by Dco is relevant to the participation of Dco in Fat signaling, then the dco³ mutation, which causes loss of Fat signaling, should impair Fat phosphorylation. Sequencing of dco³ identified 2 distinct amino acid substitutions; these were introduced into a Dco:V5 expression construct. Dco³:V5 resulted in much less shift in the mobility of Fat in S2 cells than did wild-type Dco:V5. Thus, the same amino acid changes that cause overgrowth in vivo impair Dco-dependent phosphorylation of Fat in cultured cells. To investigate whether endogenous phosphorylation of Fat could also be influenced by mutation of dco, the mobility of Fat was examined in lysates from dco³ mutant wing discs. Unphosphorylated Fat (Ft-95) appeared slightly elevated, and a distinct Ft-95-P band was no longer visible, but rather a faint smear was detected. This change in Fat mobility was confirmed by line scanning. Thus, dco³ reduces levels of phosphorylated Fat in vivo (Feng, 2009).

To explore the relationship between the Ds-promoted phosphorylation of Fat, and the Dco-dependent phosphorylation of Fat, the mobility of Fat isolated from discs simultaneously overexpressing Ds and mutant for dco³ was examined. Direct examination of Western blots, as well as line scanning, revealed that Fat mobility in these lysates was similar to that in dco³ mutants. Thus, Ds-mediated phosphorylation can be influenced by Dco. dco³ mutant clones have no obvious effect on Fat protein staining in wing imaginal discs, suggesting that they do not affect its overall levels or distribution. Nor did dco³ noticeably affect processing of Fat (Feng, 2009).

The simplest explanation for Dco-promoted Fat phosphorylation, and for dco-dependent effects on Fat signaling, would be that Dco directly phosphorylates Fat. A purified mammalian homologue of Dco (CKIδ) phosphorylated the Fat intracellular domain in vitro, but with reduced specificity, because even greater mobility shifts than those observed in vivo could be induced. CKI's are Ser/Thr kinases, and the 538 amino acid Fat ICD includes 109 Ser or Thr residues. Three different kinase site prediction programs individually predict 7, 15, or 36 CKI sites, and cumulatively identify 46 potential CKI sites. This variation emphasizes the limited accuracy of kinase site predictions. It is also noted that distinct CKI sites could act redundantly, and that among the many potential CKI sites within the Fat ICD, phosphorylation sites responsible for the evident mobility shift on SDS-PAGE gels could be distinct from sites responsible for the influence of ds or dco³ on Fat activity. Thus, the identification of specific phosphorylation sites within the Fat ICD that are required for its biological activity will ultimately be essential for confirming the importance of Dco- and Ds-promoted phosphorylation of Fat to Fat signaling (Feng, 2009).

In contrast to the overgrowth associated with dco³ mutants, dco null mutants lack discs, and dco null mutant clones grow poorly. This could reflect the participation of dco in other processes. However, targets of Fat signaling, including Wingless (WG) in the proximal wing, and Diap1, are up-regulated in dco³ mutant clones, but not in dco null (dco^le88) mutant clones. The apparent absence of fat phenotypes in dco null alleles suggests that dco³ is an unusual allele (Feng, 2009).

Dco is also known as double time, because viable alleles were independently isolated as circadian rhythm mutants. This circadian phenotype reflects a role for Dco in phosphorylating, and thereby promoting the turnover, of the circadian protein Period. This activity of Dco can be reproduced in S2 cells. Notably, Dco³:V5 was as effective as wild-type Dco:V5 at promoting Period turnover in S2 cells, whereas a circadian rhythm mutant isoform, Dco^Dbt-AR, was less effective. Thus, dco³ is impaired in promoting Fat phosphorylation, but active on another substrate (Feng, 2009).

Analysis of the Dco-Period interaction revealed that Dco and Period can be stably associated, as assayed by their ability to be coprecipitated from cultured cells. Similarly, Dco and the Fat-ICD can be coprecipitated, and this association was not impaired by the Dco³ mutations. Because Dco³ can associate with Fat, but does not efficiently phosphorylate it, Dco³ might act as an antimorphic (dominant-negative) protein by competing with wild-type kinase. Indeed, although dco³ is recessive at endogenous expression levels, when dco³ was overexpressed, aspects of the dco³ phenotype, including wing overgrowth and the induction of a Fat pathway target gene could be reproduced. By contrast, overexpression of wild-type forms of Dco does not cause detectable overgrowth phenotypes. Instead overexpression of Dco modestly decreased wing growth and slightly reduced transcription of diap1, suggesting that Fat pathway activity might be increased (Feng, 2009).

In addition to having a CKIδ/ε homologue, Drosophila also have a CKIα homologue, and in some contexts they can act partially redundantly. A partial shift in Fat ICD mobility could be detected when CKIα was expressed in S2 cells or in wing discs. Thus, CKIα can promote phosphorylation of Fat, although it appears less effective than Dco. This observation, together with the dco³ phenotypes observed when Dco³ is overexpressed, and the observation that although dco³ is defective in Fat phosphorylation, dco null mutant cells do not appear to be impaired for Fat signaling, suggest that dco³ might act as an antimorphic, or dominant negative, mutation, failing to effectively phosphorylate Fat and at the same time interfering with an ability of CKIα to phosphorylate Fat. By contrast, it is hypothesized that in dco-null mutant cells, CKIα or other kinases could phosphorylate Fat without interference. Although dco³ could not be rescued with a UAS-CKIα transgene, different CKI transgenes are inserted in different chromosomal locations, and their specific activities on Fat might be distinct. Thus, it remains possible that Dco and CKIα could be partially redundantly for Fat signaling (Feng, 2009).

Dco also participates in other pathways and processes. To determine whether the tumor suppressor phenotype of dco³ can be accounted for solely by its influence on Fat signaling, advantage was taken of the observation that overexpression of Wts under the control of a heterologous promoter (tub-Gal4 UAS-Myc:Wts) could rescue the lethality and tumor suppressor phenotype of fat mutants. The lethality and overgrowth phenotypes of dco³ were also rescued by Wts overexpression (tub-Gal4 UAS-Myc:Wts), resulting in animals that, aside from some mild wing vein phenotypes, are indistinguishable from wild-type animals overexpressing Wts. Because they are rescued simply by elevating Wts expression, dco³ mutant animals are specifically defective in Fat signaling; other essential processes that Dco participates in are not impaired (Feng, 2009).

Although Wts overexpression rescued the overgrowth and lethality of fat mutants, these animals have obvious PCP phenotypes in multiple tissues, consistent with the conclusion that Wts functions specifically in a Fat tumor suppressor pathway, and not in a Fat PCP pathway. By contrast, Wts-rescued dco³ mutants appear to have normal PCP. The absence of an obvious PCP phenotype also indicates that the influence of Dco and CKIα on PCP through phosphorylation of Dishevelled is not affected by dco³ (Feng, 2009).

To confirm the lack of influence of dco³ on PCP, dco³ mutant clones were examined. fat mutant clones in the abdomen exhibit obvious disruptions in the normal posterior orientation of hairs and bristles, but dco³ mutant clones had no effect. In addition to affecting the canonical PCP pathway, studies of the relationship between Fat and its downstream effector Dachs revealed a form of PCP in which Fat signaling causes a polarized distribution of Dachs, which can be visualized by mosaic expression of a tagged form of Dachs, Dachs:V5. In the developing wing, Dachs:V5 is present on distal cell membranes, but not on proximal cell membranes. In clones of cells mutant for fat, Dachs:V5 is equally distributed on proximal and distal membranes. In clones of cells mutants for dco³, Dachs:V5 localization is still polarized. Thus, the regulation of Dachs localization by Fat does not appear to be affected by dco³, although a weak effect on Dachs localization cannot be excluded. The absence of visible Dachs relocalization in dco³ clones appears to conflict with the hypothesis that the influence of Fat signaling on Warts depends on its ability to polarize Dachs, and further studies will be required to resolve this (Feng, 2009).

The atypical cadherin Fat is a transmembrane receptor for pathways that control PCP and transcription. This study has identified 2 posttranslational modifications of Fat. First, Fat is proteolytically processed, resulting in the production of stably associated N- and C-terminal polypeptides. The functional significance of this processing is not known, but its discovery is a necessary precursor to further experiments aimed at this question. Processing appears to be constitutive rather than regulated. Nonetheless, processing may facilitate subsequent events that regulate Fat (Feng, 2009).

Phosphorylation of the Fat cytoplasmic domain was also discovered. Phosphorylation is promoted by the Fat ligand Ds, is influenced by the Fat pathway kinase Dco, and correlates with Fat pathway activity in ds or dco³ mutant animals, or when Ds or Fj are overexpressed. These observations suggest that phosphorylation of Fat is a key step in Fat receptor activation. When Dco or CKIα are overexpressed, the phenotypic effects appear mild compared with the evident increase in phosphorylation. However, because there could be multiple CKI sites within the Fat ICD, it is possible that the phosphorylation-dependent mobility shift of Fat is a general marker of the extent of Fat phosphorylation, rather than a precise marker of phosphorylation at a site or sites required for Fat activity. Nonetheless, the observation that dco³ can be completely rescued by Warts overexpression, together with the epistasis of dachs to dco³, indicates that the tumor suppressor phenotype of dco³ is due to an impairment of Fat-Warts signaling, which occurs at or upstream of the action of Dachs. Altogether, these observations implicate Fat as the likely target of Dco activity in the Fat pathway (Feng, 2009).

Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous

Two pathways regulate planar polarity: the core proteins (Warts-Hippo) and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Morphogens specify complementary expression patterns of Ds and Fj that potentially act as polarizing cues. It has been suggested that Ft-Ds-Fj-mediated cues are weak and that the core proteins amplify them. Another view is that the two pathways act independently to generate and propagate polarity: if correct, this raises the question of how gradients of Ft and Ds expression or activity might be interpreted to provide strong cellular polarizing cues and how such cues are propagated from cell to cell. This study demonstrates that the complementary expression of Ds and Fj results in biased Ft and Ds protein distribution across cells, with Ft and Ds accumulating on opposite edges. Furthermore, boundaries of Ft and Ds expression result in subcellular asymmetries in protein distribution that are transmitted to neighboring cells, and asymmetric Ds localization results in a corresponding asymmetric distribution of the myosin Dachs. The generation of subcellular asymmetries of Ft and Ds and the core proteins is largely independent in the wing disc, and additionally ommatidial polarity in the eye can be determined without input from the Ft-Ds-Fj system, consistent with the two pathways acting in parallel (Brittle, 2012).

The results demonstrate the importance of gradients and boundaries of Ds and Fj expression in the generation of cellular asymmetry. Previous reports have suggested that weak differences in Ft and Ds binding across cells could be amplified to produce asymmetric localization of downstream pathway effectors such as Dachs. This study reports significant asymmetry of both Ft and Ds localization, suggesting that physical polarization of these proteins is an important part of the mechanism by which Ft-Ds-Fj generate polarity. This study thus reveals the Ft-Ds-Fj system as a mechanism for converting long-range morphogen-induced gene expression patterns into planar polarity cues at the level of individual cells (Brittle, 2012).

In the wing disc, Dachs asymmetry is particularly prominent at the pouch-hinge boundary where a strong disparity in Ds levels exists. In this situation, the Ds boundary may contribute to the high level of asymmetry, for instance, via a feed-forward mechanism that suggests that Dachs asymmetry is produced by strong differences in Ds and Ft binding between neighboring cells, that is passed from cell to cell as the wing grows. However, strong asymmetry of Ds and Dachs were also detected in the eye disc, where there is no evidence for sharp disparities of Ds or Fj, consistent with expression gradients providing sufficient cues. Dachs asymmetry was also seen in 6 hr pupal wings consistent with Ft-Ds-Fj signaling continuing to provide polarizing cues after the third-instar stage (Brittle, 2012).

The ability of shallow expression gradients to produce observable asymmetry of Ft and Ds distribution is unexpected. A possible mechanism is that a weak asymmetry in activity or protein distribution across the cell is amplified by a feedback loop to produce an observable protein asymmetry, in a manner similar to that suggested for the generation of core protein asymmetry. Notably, Dachs does not seem to be part of any such amplification mechanism. Indeed loss of Dachs activity appears to promote Ft and Ds asymmetry. It may be that cell divisions, which are reduced in dachs mutants, disrupt the appearance of asymmetry, possibly explaining the high level of variance of asymmetry of Dachs, Ft and Ds in WT tissue. To understand further how the asymmetry of Ft and Ds is achieved, and whether this requires an amplification mechanism, it will be necessary to combine more detailed quantitative analyses together with computational approaches (Brittle, 2012).

The data suggest that Ft and Ds asymmetry leads directly to the observed Dachs asymmetry in both wing and eye discs. Although no direct interactions were detected between Ds and Dachs, the colocalization and the similar degree of subcellular asymmetry observed for these proteins support a model in which Ds recruits Dachs (Brittle, 2012).

Finally, this study reassessed the link between Ft-Ds-Fj and the core planar polarity proteins. In the wing, it was demonstrated that throughout much of the third-instar disc, both Ft-Ds-Dachs and the core proteins independently adopt PD-oriented subcellular localizations, most likely under the influence of the morphogen gradients that pattern the axes of the tissue. However, in the most proximal regions of the wing (adjacent to the pouch-hinge boundary in the disc), Ft-Ds-Fj appear to act via Dachs to ensure correct polarization of the core proteins. The mechanism behind Dachs regulation of the core needs further investigation, but because Dachs plays a role in orientated cell division and influences apicolateral junctional length [28], these factors may be involved (Brittle, 2012).

In the eye, Ft-Ds-Fj seem to play a more general role in polarizing the core proteins throughout the tissue, apparently independently of Dachs activity. Ft-Ds-Fj may also provide a Dachs-independent input to the core in the wing, but data presented in this study suggest that it is at best redundant. Even in the eye, Ft-Ds-Fj are not absolutely essential for the core to polarize, indicating that there are other unknown inputs (Brittle, 2012).

An important observation is that complete loss of ft or ds activity in the eye or wing results in very strong defects in core protein polarity, but when overgrowth is suppressed in these backgrounds via manipulation of Wts-Hpo pathway activity, then much milder defects are observed. On one hand, excessive cell division alone may disrupt the process of planar polarity establishment by the core proteins, possibly due to asymmetric localization being lost each time a cell undergoes mitosis. Alternatively, Ft-Ds-Fj-mediated polarity cues may constitute more important inputs to the core proteins in proliferating tissues. Finally, it is possible that other Wts-Hpo pathway transcriptional targets, not related to growth, contribute to the planar polarity phenotype (Brittle, 2012).

Overall, the data support a model in which the Ft-Ds-Fj system and core planar polarity proteins act independently to generate and propagate planar polarity through the asymmetric subcellular distribution of their protein components. No evidence was found that the core proteins can influence the asymmetry of the Ft-Ds-Fj system; however, in particular contexts, the Ft-Ds-Fj system can act through different effectors to influence core protein polarity (Brittle, 2012).

Collective polarization model for gradient sensing via Dachsous-Fat intercellular signaling

Dachsous-Fat signaling via the Hippo pathway influences proliferation during Drosophila development, and some of its mammalian homologs are tumor suppressors, highlighting its role as a universal growth regulator. The Fat/Hippo pathway responds to morphogen gradients and influences the in-plane polarization of cells and orientation of divisions, linking growth with tissue patterning. Remarkably, the Fat pathway transduces a growth signal through the polarization of transmembrane complexes that responds to both morphogen level and gradient. Dissection of these complex phenotypes requires a quantitative model that provides a systematic characterization of the pathway. In the absence of detailed knowledge of molecular interactions, this study took a phenomenological approach that considers a broad class of simple models, which are sufficiently constrained by observations to enable insight into possible mechanisms. Two modes are proposed of local/cooperative interactions among Fat-Dachsous complexes, which are necessary for the collective polarization of tissues and enhanced sensitivity to weak gradients. Collective polarization convolves level and gradient of input signals, reproducing known phenotypes while generating falsifiable predictions. A construction of a simplified signal transduction map allows a generalization of the positional value model and emphasizes the important role intercellular interactions play in growth and patterning of tissues (Mani, 2013).

The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila

The atypical cadherins Dachsous (Ds) and Fat (Ft) are required to control the size and shape of tissues and organs in animals. In Drosophila, a key effector of Ds and Ft is the atypical myosin Dachs, which becomes planar polarised along the proximal-distal axis in developing epithelia to regulate tissue size via the Hippo pathway and tissue shape via modulating tension at junctions. How Ds and Ft control Dachs polarisation remains unclear. This study identified a ubiquitin ligase, FbxL7, as a novel component of the Ds-Ft-Dachs system that is required to control the level and localisation of Dachs. Loss of FbxL7 results in accumulation of Dachs, similar to loss of Ft. Overexpression of FbxL7 causes downregulation of Dachs, similar to overexpression of the Ft intracellular domain. In addition to regulating Dachs, FbxL7 also influences Ds in a similar manner. GFP-tagged FbxL7 localises to the plasma membrane in a Ft-dependent manner and is planar polarised. It is proposed that Ft recruits FbxL7 to the proximal side of the cell to help restrict Ds and Dachs to the distal side of the cell (Rodrigues-Campos, 2014).

How animal cells cooperate to build tissues of particular forms remains a fundamental unsolved problem in biology. One molecular system that controls tissue size and shape in animals is the Dachsous (Ds)-Fat (Ft) cadherin system. Ds and Ft encode large atypical cadherins that interact heterotypically to form cell-cell junctions in epithelia and are required to control tissue form in both Drosophila and mice. The Ds-Ft system is known to induce a molecular polarity in the plane of the epithelium, and this planar polarity has at least three distinct consequences, including control of tissue growth via regulation of the Hippo signalling pathway, control of tissue morphogenesis by modulating tension at cell-cell junctions, and control of the orientation of hairs, bristles and eye ommatidia in Drosophila, in part by modulating the Frizzled system of planar cell polarity (Rodrigues-Campos, 2014).

One important effector of Ds and Ft is the atypical myosin Dachs, which is thought to bind to the Ds intracellular domain and becomes planar polarised towards the distal side of each cell in the developing Drosophila wing or eye epithelium. Ds and Ft can also themselves become planar polarised, which may contribute to the polarisation of Dachs itself. Dachs then generates tension at distal cell-cell junctions to orient cell shapes, cell divisions or cell-cell rearrangements to drive tissue elongation along the proximal-distal axis of various fly epithelia. In addition, Dachs can signal to the nucleus via the Hippo pathway effector Yki (YAP/TAZ in mammals) to promote cell proliferation and tissue growth. Notably, Dachs appears to be dispensable for planar polarisation of the Frizzled system, and the ability of Ds and Ft to polarise hairs and bristles, a process that may instead depend on microtubules. This study focused on the Dachs-dependent roles of Ds and Ft in controlling tissue size and shape in Drosophila (Rodrigues-Campos, 2014).

The global cues that orient Dachs polarisation along the proximal-distal axis are known: Dachs localises distally in response to graded expression of the Ds cadherin and also of Four-jointed (Fj), a kinase that modulates Ds-Ft interactions. The gradients of Ds and Fj are opposing, such that Ds is highly expressed at the proximal end of the tissue and Fj is highly expressed at the distal end of the tissue. Yet, how epithelial cells read the slope of these gradients and translate this information into a planar polarised localisation of Dachs is still unknown. This study identified the ubiquitin ligase FbxL7 as a novel component of the Ds-Ft system that is crucial to control Dachs levels and localisation at apical cell-cell junctions (Rodrigues-Campos, 2014).

The results suggest a close relationship between the function of the Ft intracellular domain, Dco and FbxL7. Therefore whether phosphorylated Ft intracellular domain might recruit FbxL7 to the plasma membrane was tested. The localisation of GFP-tagged FbxL7 expressed in clones was examined and found that FbxL7-GFP localises to apical cell-cell junctions. By contrast, when FbxL7-GFP is expressed in ft mutant clones, it localises to the cytoplasm in a punctate pattern. A similar punctate pattern is observed when FbxL7-GFP is co-expressed with dominant-negative Dco3. Notably, the loss of Dachs that is normally induced by expression of FbxL7-GFP fails to occur when it is not recruited to the membrane by Ft and Dco. These findings support the notion that phosphorylated Ft recruits FbxL7 in order to downregulate Dachs. This model predicts that FbxL7 itself should be planar polarised to the proximal side of cells, where Ft is thought to be most concentrated and active, whereas Dachs localises to the distal side of cells away from FbxL7 and in a complex with Ds. Accordingly, low-level expression of FbxL7-GFP with ms1096.G4 reveals a planar polarised localisation, presumably to the proximal side of wing epithelial cells where Ft is known to concentrate (Rodrigues-Campos, 2014).

The above findings identify the FbxL7 ubiquitin ligase as a novel component of the Ds-Ft-Dachs system. FbxL7 is recruited to the membrane by Ft, where it then acts together with Ft and the Dco kinase to promote degradation or removal of both Dachs and Ds. The effect of FbxL7 loss and gain of function on Dachs levels are particularly strong and the phenotypic consequences in adult Drosophila closely resemble gain and loss of Dachs function, respectively. In vitro ubiquitylation assays suggest that FbxL7 can directly ubiquitylate Dachs, which is predicted to lead to its proteolytic degradation. In addition, it was observed that FbxL7 can also ubiquitylate the Ds intracellular domain in vitro and can modulate the level and localisation of Ds in vivo. It remains possible that FbxL7 acts indirectly by stabilising or activating Ft, which then acts via a different mechanism to degrade or remove Ds and Dachs proximally. The direct model is favored because of its simplicity and because ubiquitylation is generally thought to promote degradation, rather than stabilization, of proteins (Rodrigues-Campos, 2014).

These observations suggest a model in which Ft, which has been reported to localise proximally, recruits FbxL7 to the proximal side of the cell to help restrict Dachs and Ds to the distal side of the cell. These results also suggest that polarised Ds may also promote degradation or removal of Ft on the distal side so that Ft concentrates proximally, thereby assisting polar Ds-Ft bridge formation. Thus, there appears to be mutual antagonism between Ds and Ft within the same cell, as well as heterotypic Ds-Ft bridge formation between neighbouring cells, an event that then leads to loss of Dachs proximally and recruitment of Dachs distally. Such a mechanism might explain how this system can become planar polarized; however, it is still unclear how the system is able to read the slope of the Ds and Fj gradients continuously, rather than switch to a more permanently polarised state (Rodrigues-Campos, 2014).

Notably, the degree of Dachs polarisation (and the strength of its effect on Hippo signalling and tissue growth) correlates with the steepness of the Ds and Fj gradients, indicating that cells can obtain both vectorial information and a measure of steepness at the same time from the Ds-Ft system. These features of the Ds-Ft system match very well with those proposed for the hypothetical gradients originally conceived following surgical manipulation of insect development and regeneration. This identification of FbxL7 as a key player in this system will help enable further work to understand how the system can translate the steepness of the gradient into the degree of Dachs polarisation (Rodrigues-Campos, 2014).

Vamana couples fat signaling to the Hippo pathway

The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933; FlyBase name Dachs ligand with SH3s or Dlish) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat⁶¹ and fat^sum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs¹ phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).

DEVELOPMENTAL BIOLOGY

Embryonic

The first dachsous transcripts are detectable during gastrulation in a pair-rule pattern of six weak epidermal stripes and in a prominent stripe at the amnioproctodeal invagination. During germ-band extension, DS mRNA accumulates in a segmentally repeated pattern of 14 stripes and in the procephalon. The most pronounced expression of ds is observed during the extended germ-band stage, mainly in the forming tracheal pits. At the beginning of head involution, DS mRNA appears in the anterior spiracles and again in stripes of the segmental groves and buds, while it remains weakly expressed in the remnants of the tracheal pits. DS mRNA is first detected in the primoridal leg discs,which form in the ventral posterior part of each throracic segment. At late stage 14, ds is expressed strongly in the nearly fused labial buds and at invaginations of the maxillary segment while it continues to be expressed in the leg disc primordium, along the segmental folds, and probably in the apodemes (muscle attachment sites of the ectoderm). After dorsal closure, expression persists only in the apodemes and in the head region (Clark, 1995).

The atypical cadherin Dachsous controls left-right asymmetry in Drosophila

Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. This study proposes a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. It was further shown that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry (Gonzalez-Morales, 2015).

This work has revealed the existence of an hindgut-specific LR organizer having transient activity. LR information is transferred non-autonomously from this organizing center to the target tissue, involving a unique MyoID-Ds interaction taking place at a PCP signaling boundary (the H1/H2 boundary). Propagation of this initial LR information to the developing hindgut requires both Ds/Ft global and core Fz PCP signaling. Notably, these results suggest that MyoID can act as a directional cue to bias planar cell polarity (Gonzalez-Morales, 2015).

So far, only a role for the core PCP pathway in cilia positioning and LR asymmetry had been reported in mouse, chick, and Xenopus. This study revealed a role of the Fat/Ds PCP pathway in LR asymmetry. The atypical cadherin Ds is essential for early LR planar polarization of hindgut precursors and later on for looping morphogenesis. Ds has a cell-non-autonomous function, allowing transfer of LR information from the H1 domain to H2 hindgut precursor cells. Ds, therefore, represents a critical relay factor acting at the boundary between, and linking, a LR organizer and its target tissue (Gonzalez-Morales, 2015).

In addition to a MyoID-dependent function in H1, the mislooped phenotype induced upon Ds silencing in the H2 domain suggests that Ds also has a MyoID-independent activity in H2 cells, likely through interaction with other PCP genes. Indeed, reducing the activity of PCP global or core gene functions reveals that the two pathways are important in the H2 region for adult hindgut looping. However, the results reveal important differences in the way these pathways control hindgut asymmetry. First, although the adult phenotype is similar upon silencing of one or the other pathway, the early polarization of H2 cells in pupae (10 hr APF) is only affected when knocking down the activity of Ds, Ft, and Fj. These results show that the Ds/Ft pathway, but not the core pathway, is required for establishing early LR polarity. Second, the phenotype is quantitatively different, since silencing of the Ds, Ft, or Fj PCP gene led to a consistent and very strong phenotype, while reducing Fz PCP signaling had a significantly less penetrant one. These data suggest a partly overlapping function of both PCP signaling pathways for late hindgut morphogenesis. Therefore, the following sequential model is proposed: in H1 cells, MyoID interacts with the Ds intracellular domain, which becomes 'biased' toward dextral through a currently unknown mechanism. This initial LR bias is then transmitted across the H1/H2 boundary through Ds/Ft heterophilic interaction. Then, boundary H2 cells relay the initial bias and spread it to the remaining H2 cells through classical Ds/Ft PCP. It is interesting that the local signaling boundary suggested by this model is consistent with recent studies showing that Ds can propagate polarity information in a range of up to eight cells, a distance that is consistent with the size of the H2 domain at 10 hr APF. Once initial polarity has been set up through the Ds/Ft pathway, this is further relayed to and/or amplified by the core pathway. Notably, a similar two-step mechanism has also been proposed for the wing and could apply to other tissues (Gonzalez-Morales, 2015).

The discovery of a coupling between the MyoID dextral factor and Ds is a nice example of crosstalk between existing signaling modules. In the simplest crosstalk model, the role of MyoID would just be to bias or tilt Ds function toward one side, possibly through Ds localization and/or activity polarization along the LR axis. Using both in vitro and in vivo assays, this study has shown that interaction between Ds and MyoID requires Ds intracellular domain, supporting a cytoplasmic interaction between the two proteins. These results, along with recent findings, suggest that Ds may represent a general platform for myosin function in different tissues. In particular, the intracellular domain of Ds was found to bind to the unconventional myosin Dachs, controlling Dachs polarized localization, which is important for subsequent cell rearrangements underlying thorax morphogenesis. However, in contrast to thoracic Dachs, MyoID is not obviously polarized in H1 cells, suggesting that the interaction between myosins and Ds may involve different mechanisms. Additionally, no LR polarized localization of MyoID or Ds was observed in H1 cells, although the existence of subtle asymmetries undetectable by available tools cannot be excluded. Nevertheless, alternative means to generate the LR bias in H1 include: (1) LR polarized expression of an unknown asymmetric factor or (2) LR asymmetric activity of Ds. These interesting possibilities are consistent with recent work showing that some type I myosins can generate directed spiral movement of actin filaments in vitro. It is tempting to speculate that, similarly, MyoID putative chiral activity could be translated into Ds asymmetrical function along the LR axis. Future work will explore this possibility as well as others to unravel the molecular basis of MyoID LR biasing activity in the H1 organizer (Gonzalez-Morales, 2015).

The identification of the H1 domain as a specific adult tissue LR organizer demonstrates the existence of multiple independent tissue and stage-specific LR organizers in flies. This situation echoes what is known in other models, including vertebrates, in which at least two phases of asymmetry establishment can be distinguished. A first pre-gastrula phase, as early as the four-cell stage in Xenopus, involves the generation of asymmetric gradients of ions. Then, a second phase takes place at gastrulation and involves Nodal flow and asymmetric cell migration, eventually leading to asymmetric expression of the nodal gene in the left lateral plate mesoderm. In Drosophila, some interesting common and specific features can be drawn out by comparing the hindgut and terminalia organizers. The first major common feature is the fact that both organizers rely on MyoID function, showing the conserved role of this factor in Drosophila LR asymmetry. Second, the two organizers show temporal disconnection, acting much earlier than LR morphogenesis, which is expected of a structure providing directionality to tissues per se (24 hr for terminalia and ~72 hr for hindgut looping). Such temporal disconnection of MyoID function with late morphogenesis is also observed in the terminalia where a peak of MyoID activity precedes terminalia rotation by 24 hr. Time lag in MyoID function requires LR cue transmission and maintenance in developing tissues until directional morphogenesis. The finding of a role of Ds and PCP in hindgut LR asymmetry provides a simple mechanism by which initial LR information is maintained and transmitted across tissue through long-range PCP self-propagation (Gonzalez-Morales, 2015).

Notably, the two organizers also show distinct features. In terminalia, MyoID has a cell-autonomous function in two adjacent domains. In addition, the terminalia organizer is permanent, developing as an integral component of the adult tissue. In contrast, MyoID in the imaginal ring has a cell-non-autonomous function. Indeed, a striking feature of the hindgut organizer is its transience as it detaches from the hindgut precursors 50 r before full looping morphogenesis prior to its degradation and elimination; hence, the need to transfer LR information to the H2 hindgut primordium. An interesting question then is whether the MyoID-Ds/PCP interaction is conserved in terminalia. This study has shown that the terminali rotation requires the activity of DE-cadherin; however, invalidation of the atypical cadherins Ds or Ft or core PCP signaling in the terminalia organizer did not affect asymmetry. The fact that PCP does not have a general role in Drosophila LR asymmetry is not altogether surprising, as MyoID cell-autonomous function in terminalia and organizer persistence does not require that LR information be transferred to and stored in other parts of the tissue, as is the case in the hindgut. Therefore, despite conservation of the MyoID-dependent upstream dextral cue, significant differences in downstream morphogenetic pathways imply alternative cellular mechanisms controlling cue transmission and maintenance (Gonzalez-Morales, 2015).

The LR signaling module, comprising the dextral determinant MyoID and the still-unknown sinistral determinant, can therefore be coupled to distinct morphogenetic modules, including PCP, as shown in this study. It is suggested that coupling between LR asymmetry and PCP might be observed in processes requiring long-distance patterning of tissues and organ precursors, both in invertebrate and vertebrate models. Understanding organ LR morphogenesis clearly requires studying diverse and complementary models. In this context, the multiplicity of LR organizers discovered in Drosophila represents a powerful model to study the diversity in the coupling of LR organizers with downstream programs responsible for late tissue morphogenesis. In particular, the Drosophila hindgut represents an invaluable model for studying the genetic basis and molecular mechanisms coupling LR asymmetry with PCP patterning (Gonzalez-Morales, 2015).

Larval

In third-instar larvae, ds transcripts are found in the imaginal discs and the brain. In the supraesophageal ganglion (brain), ds is expressed in two areas of the areas of the optic lobe and in a region that might belong to the mushroom body. In imaginal discs, strong ds expression occurs frequently along folds separating the anlagen of distinct imaginal structures. In the antennal disc, ds is expressed in the arista and first and second antennal segment anlagen; in the eye disc, ds transcripts are abundant along the folds of the future bristle region of the head capsule. Expression of ds is also observed in humeral, genital and labial discs. In leg discs, ds is expressed strongly in the anlagen of the tarsal joints and in particular, the most proximal leg segment. Similarly, ds expression is strongest in the pleural, dorsal, hinge, and prescutal regions of the wing disc, whereas the anlage of the future wing blade is virtually free of DS mRNA. In contrast, the haltere disc exhibits high levels of DS transcripts in the capitellum, pedicle, and scabellum, while expression in the notum remains relatively low (Clark, 1995).

Homothorax and Extradenticle are expressed in the proximal domain of the leg (the Hth domain) : all the other factors studied (Wingless, Decapentaplegic and Distalless) are expressed in more distal regions (the Dll domain). Dachsund (Dac) is expressed in an intermediate domain, dorsal and lateral to the more distal Dll domain (the Dac domain). What follows is a more complete description of these domains. The expression of several targets of the signaling molecules Wg and Dpp were examined in relation to the hth expression domain. dpp expression in the leg disc at the early third larval instar stage consists of a sector that originates at the center of the disc, extends dorsally to the periphery and shows extensive overlap with Hth. omb, a target of the Dpp-signaling pathway, is expressed in a dorsal sector that, in contrast to dpp, extends dorsally only to abut, but not overlap with, the hth domain. wg expression consists of a ventral sector of cells that extends from the center to the periphery of the disc, whereas H15, an enhancer trap line that requires wg signaling for its activation, is largely not transcribed in the hth domain. The restriction of these Wg and Dpp target genes to non-hth-expressing cells suggests that hth restricts signaling by these two molecules. By the late third larval instar stage, there is a small degree of overlap between hth and omb expression as well as between hth and H15. This expression corresponds to the trochanter domain where gene activation can occur independent of the Wg- and Dpp-signaling pathways. Unlike omb and H15, the Dll and dac genes require input from both the Dpp and Wg signal transduction pathways to be activated in leg discs. Dll encodes a homeodomain protein present in the central portion of leg discs, and its activation requires the highest concentrations of Wg and Dpp. dac encodes a nuclear protein and a putative transcription factor whose expression is repressed by high concentrations, and activated by intermediate concentrations, of Wg and Dpp. By performing triple stains for the dac^P-lacZ reporter gene, and Dll and Hth proteins at the early third larval instar stage, it was found that the leg disc is defined by three non-overlapping domains of gene expression. The distal-most domain of the leg disc contains Dll protein (the Dll domain). Dorsal and dorsolateral, but not ventral, to the Dll domain are cells that express dac (the Dac domain). The proximal-most cells of the disc, which surround the dac and Dll domains, express hth (the Hth domain). At the mid 3rd larval instar stage (~96 hours after egg lay, or AEL), the distal-most cells express only Dll and are surrounded by a ring of cells that express both Dll and dac. At this stage, there is also a dorsal patch of cells that express dac but not Dll. hth expression remains limited to the proximal-most cells of the disc and shows no overlap with dac or Dll. By the late 3rd larval instar stage (~120 hours AEL), hth is still not co-expressed with dac or Dll, with the exception of a thin band of cells corresponding to the trochanter domain, where all three genes are co-expressed. Gene expression in the trochanter domain is likely to represent secondary patterning events, because it is not dependent on Wg- or Dpp-signaling. At this stage dac expression also surrounds and partially overlaps the Dll expression domain. It is proposed that the Dll and Dac domains, where hth transcription is off and Exd is cytoplasmic, are Dpp- and/or Wg-responsive domains, as demonstrated by the ability of these cells to respond to these signals by activating the target genes Dll, dac, omb and H15. In contrast, the hth domain, where hth is active and Exd is nuclear, is a Wg- and/or Dpp-non- responsive domain, where these signals are present but cannot activate these targets (Abu-Shaar, 1998).

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).

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 process involves four genes (stan, fz, Vang and pk) that probably act downstream of ds, ft and fj (Lawrence, 2004).

dachsous is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of Wingless during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis (Rodríguez, 2004).

In second instar larvae, ds-lacZ expression is essentially confined to the distal part of the wing disc, but is absent in those distal A cells in which Wg strongly accumulates. This Wg expression constitutes the earliest marker for the nascent wing pouch. Soon thereafter, when Wg expression is expanded to the adjacent P cells, ds-lacZ expression fades away and becomes confined to a ring of cells around the prospective wing pouch. At this stage, most of the hinge cells located between the prospective notum and wing pouch express ds-lacZ at high levels, as revealed by the Iro and Nub markers. A weak expression of ds-lacZ overlaps with the periphery of the Nub domain and marks the region that will become the proximal wing. At third instar, ds-lacZ expression is also observed within the lateral regions of the prospective notum. An antibody directed against the cytoplasmic region of Ds protein reveals a Ds protein distribution similar to the ds-lacZ expression pattern and an apical location at the plasma membrane. From these results, it is concluded that ds-lacZ expression is one of the earliest and most specific markers of the prospective hinge during the second and early third larval instar (Rodríguez, 2004).

During patterning and growth of the wing blade, Wg distribution has been proposed to signal to distant cells in a concentration-dependent manner. Several mechanisms, such as the interaction of Wg with heparin sulfate-containing proteoglycans, as well as regulated endo- and exocytosis, are involved in shaping the gradient and delimiting the range of signaling. Wg protein is predominantly located at the apical surface in the producing cells, and in the embryo it has been demonstrated that this sub-cellular location is essential for its signaling activity. When the hinge territory is already specified at early third instar, wg is activated in these cells and acts as a cell proliferation signal necessary for the development of most structures. Wg expression within the hinge can be described as two rings, the 'inner ring’ (IR) and the 'outer ring’ (OR), which overlap with the areas of low (ring I) and high expression of ds-lacZ (ring II), respectively. In this context, since Ds is also apically located, it was of interest to determine whether Ds has a role in Wg-producing cells. To test this, the distribution of Wg was examined in large clones of ds mutant cells. Two results were obtained from these experiments: (1) the level of Wg in the producing cells was slightly increased with respect to neighbouring wild-type cells, and (2) the Wg gradient within mutant tissue appears to be broader. These results should imply a higher signaling capacity of Wg in ds mutant cells; however, that was not the case. In ds clones, Wg accumulation was less marked apically and was relatively more abundant in the baso-lateral region than in the wild-type cells. Interestingly, this phenomenon seems not to be strictly cell autonomous, because adjacent wild-type cells also displayed a similar abnormal sub-cellular localization of Wg protein. This effect could be due to basal Wg protein diffusing more rapidly to adjacent cells than apical protein does, as has been observed in the embryo. Taken together, these observations suggest that Ds protein contributes to the apical localization of Wg protein at the plasma membrane. It is thought unlikely that this function of Ds is responsible for the early PD patterning defects in DNW discs, since ds and wg are expressed in complementary domains during early larval development (Rodríguez, 2004).

Whether Ds is required for Wg-mediated patterning in imaginal discs other than the wing disc was examined by analyzing the genetic interactions between ds and several components of the Wg pathway during leg development. Homo- and hetero-allelic combinations of ds cause a reduction of the segment size and fusion of the tarsal segments, with partial elimination of the tarsal joints. This phenotype resembles some defects associated with the loss of function of pangolin/dTCF, and legless/BCL9. Therefore, the levels of Wg signaling were manipulated in mid-strength heteroallelic combinations of ds. The loss of one wild-type copy of dsh enhanced the fusion of leg tarsi and shortened the leg segments. By contrast, the leg phenotype of ds showed a complete recovery of the tarsal joints and an increase in the length of the segments when one dose of the nkd gene, an antagonist of the Wg pathway, was eliminated. Taken together, these findings support a more general role for ds in Wg-mediated patterning processes (Rodríguez, 2004).

The wing primordium is specified as a few anterior cells that express wg at the distal-most part of the wing imaginal disc at second larval instar. Slightly later, wg is also expressed in P cells and these cells are recruited into the wing fate. In 'double-notum-winglet' (DNW) ds strongly hypomorphic mutant discs, the level of Ds protein is highly reduced and only the initial anterior group of Wg-expressing cells becomes specified into the wing fate. The levels of this initial Wg expression seems not to be affected in DNW discs. However, neither the P cells abutting the initial anterior Wg domain nor the surrounding cells of this early wing primordium are able to respond to Wg, leading to the formation of a wing pouch composed exclusively by A cells. Moreover, the activation of Wg target genes (such as hth, required for the specification of hinge cells) fails in DNW discs, and, consequently, the proximal wing and hinge structures do not develop. The significantly reduced rings of ds-lacZ, hth and zfh2 expression in DNW discs most likely reflect the residual Ds activity retained in the ds^38k mutant. Cells close to the Wg source might thus still be able to respond to high Wg levels during early stages of wing development. However, under null conditions for ds (ds^D36) the expression of zfh2 is eliminated (Rodríguez, 2004).

Thus, in addition to its function in PCP, ds plays a role in early patterning when the specification of the different territories along the PD axis takes place in response to Wg. Initially, ds facilitates the recruitment of P cells into the wing fate in response to Wg. Subsequently, Ds promotes the activation of Wg target genes in the surrounding cells to specify the hinge. Note that once the hinge cells have been specified in response to Wg signaling, ds seems to be dispensable for global wing disc patterning, as the ‘classical’ ds^38k phenotype shows. In this case, only mild defects such as slight tissue overgrowth or polarity defects were observed, suggesting additional functions of ds related to cell adhesion (Rodríguez, 2004).

Ectopic expression of Dpp in wing cells of DNW discs restores both the formation of the AP border and cell proliferation within the wing pouch, indicating that both Wg and Dpp orchestrate these events. Only cells previously committed to the wing fate by Wg are able to proliferate in response to Dpp, as the UAS-dpp/dpp-Gal4 and UAS-dpp/omb-Gal4 experiments suggest. In the ds mutant background, omb is expressed in anterior wing cells, albeit in the absence of the AP border/Dpp source within the wing pouch, suggesting that this initial omb expression might not be Dpp dependent. Similar results were observed for spalt (sal), another known target gene of dpp. It is proposed that Ds primarily regulates Wg signaling in the initial recruitment of P cells into putative wing territory. Once this initial recruitment has occurred, Dpp expression is established and Dpp signaling can contribute to the further recruitment of P cells. Expression of UAS-dpp in anterior wing pouch cells of ds mutant discs using omb-Gal4 can bypass the initial requirements for Wg in P cell recruitment, leading to the observed wing pouch rescue (Rodríguez, 2004).

In vertebrates, during telencephalon formation, the organization into different structures requires the expression of different cadherins in adjacent regions to maintain a compartment boundary based on differential cell affinity features. It has been suggested that the expression pattern of each of these cadherins is under the control of specific signaling cascades (Rodríguez, 2004).

In Drosophila, during imaginal disc development, indirect evidence has suggested that cell adhesion might be under the control of the same signaling pathways that control cell proliferation and patterning. The smooth borders of clones mutant for thick vein (tkv), the receptor of Dpp, or smoothened (smo), a downstream component of the Hedgehog (Hh) signaling pathway, indicate that mutant cells change their affinity properties and therefore try to minimise the contact with surrounding wild-type cells. Nevertheless, little is known about the molecules involved in these adhesiveness differences. Recent work has proposed that both tartan and capricious (caps), two transmembrane proteins regulated by ap, are putative candidates to maintain the affinity boundary between dorsal and ventral cells. However, whereas clones ectopically expressing tartan and caps in V cells tend to contact D cells, the elimination of tartan and caps in clones from D cells had no effect on DV boundary formation (Rodríguez, 2004).

In the DNW phenotype, the ectopic notum develops from cells of the hinge territory. According to the proposed subdivision into concentric rings (I to III), cells from the outermost ring III expressing Tsh and Ds will give rise to that part of the body wall that is excluded from the notum region. In DNW discs, the absence of Ds produces an expansion of notal-specific iro-C expression to more distal positions to fill up the Tsh domain. These distal cells acquire a notum fate, generating an ectopic notum similar to wg¹ mutant flies (Rodríguez, 2004).

Thus, Ds protein contributes to hinge/notum boundary formation by means of an affinity border. This process would occur at early second instar when Iro-C expression is capable of specifying the notum fate. This finding provides the first evidence that a cadherin is able to maintain the cell boundary between two adjacent territories in Drosophila (Rodríguez, 2004).

How does ds participate in Wg signaling? Several findings point out a specific role of Ds in the modulation of Wg signaling: (1) the elimination of zfh2 expression in ds mutant clones; (2) the genetic interactions of ds alleles with several components of the Wg signaling pathway, and (3) the rescue of the DNW phenotype by increasing Wg levels. It has been shown that Ds is associated with adherens junctions at the apical surface of the imaginal cells, to mediate cell-cell adhesion. A major step of the cell adhesion mechanism requires interaction of the cytoplasmic tail with Arm/ß-catenin to connect the cadherin-catenin complex to the actin cytoskeleton. Thus, the phenotype could reflect changes in the balance between cytoplasmic Arm versus Arm anchored to the plasma membrane. If this were the case, then a reduction of ds function would increase Wg signaling; however, the results presented above indicate that loss of ds decreases Wg signaling. Moreover, sequence analysis has shown that the ß-catenin binding motifs in the Ds protein, which have to be in tandem to be functional, are separated by a stretch of amino acids, further discarding the possibility that Ds binds directly to Arm to modulate its cytoplasmic levels (Rodríguez, 2004).

Alternatively, the apical plasma membrane acts as a structural center that contains crucial components that modulate the Wg pathway, such as Dsh, E-APC and Axin. Axin and E-APC, promote the degradation of cytoplasmic Arm, the main effector of the Wg cascade. Previous work has shown that, upon binding of Wg in the receiving cells, the Axin/E-APC complex becomes anchored to the plasma membrane to prevent Arm degradation. In this context, Ds protein, as part of the adherens junctions, could be the cadherin required to anchor this degradation complex to the plasma membrane. In ds mutant cells, the cytoplasmic levels of the Axin/E-APC complex would be higher and, therefore, Wg signaling would decrease. In agreement with this hypothesis, mild ds phenotypes are enhanced when a copy of dsh gene is eliminated. Still, Ds could act at the level of Wg reception, by increasing the Fz/Wg-binding affinity or by recruiting Fz molecules to the apical plasma membrane, as has been demonstrated for the cadherin Fmi in the PCP processes (Rodríguez, 2004).

Early anterior Wg activity initiates specification of the PD axis in the wing disc: To date, the current model explaining the specification of the territories along the PD axis assumes that the initial anterior Wg expression at second instar is required only for cells to acquire the wing fate. It is only later, when wg is expressed in two concentric rings that its function is required to specify the hinge territory (Rodríguez, 2004).

Wg has been shown to be required for the development of the hinge. In contrast, Wg activates downstream genes such as hth or zfh2 to specify the hinge fate. In contrast, Wg controls cell proliferation when it is expressed from early third instar into the IR and OR rings. It has been established that the specification of the hinge takes place later than the wing; however, the data show that an early and timely limited depletion of Wg activity causes a failure in hinge specification. This is mainly based on the observation that only early-induced ds clones abolish zfh2 expression required for hinge formation. In ds mutant clones induced later, hinge development is unaffected, although a perdurance of ds activity in these clones cannot be excluded. Still, the rescue of hinge development in DNW discs that ectopically express Wg under dpp-Gal4 further support an early specification of the hinge. In these discs, ectopic Wg expression stays confined to the AP border. At early stages, the AP border must be located close enough to the nascent wing primordial to allow the spreading of Wg into regions destined to become hinge territory. At late stages, the narrow stripe of ectopic Wg expression can no longer account for the maintenance of the whole hinge territory. It is rather the Wg within the IR and OR that maintains hth expression and, with it, the specification of the hinge fate. At this stage, either Wg works independently of ds or its requirements for ds are lower. Thus, if hinge specification is not initiated early upon ds and wg activities, wg expression cannot be established and the development of the hinge is aborted (Rodríguez, 2004).

The present results provide insights that help in the understanding of how the PD axis is established in the wing disc. The initial event in this process would be the early activity of Wg. When Wg is expressed at the distal part of the wing disc in a small group of anterior cells, it not only promotes the activation of target genes like vg, nub or scalloped (sd) in the wing cells, but also the expression of hth and zfh2 to specify the hinge. At the same time, Wg would repress tsh or vein (vn) at the distal part of the wing disc to separate the proximal wing and hinge regions from the body wall where Egfr signaling activates notum-specific genes like iro-C. Thus, in cooperation with dpp, wg establishes the AP and PD axis in the prospective wing and hinge regions (Rodríguez, 2004).

In DNW discs, even though the Dpp source is distantly and asymmetrically located with respect to the wing pouch, anterior wing cells differentiate into distinct cell types in a mirror image disposition. This result suggests that specific positional information might be provided independently of dpp. Ap in combination with Wg might contribute to this initial AP positional information. Once P cells are recruited into the wing fate, Dpp takes over and promotes pattern formation along the AP axis, as well as proliferation within the wing pouch (Rodríguez, 2004).

The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like

The activity of different signaling pathways must be precisely regulated during development to define the final size and pattern of an organ. The Drosophila tumor suppressor genes dachsous (ds) and fat (ft) modulate organ size and pattern formation during imaginal disc development. Recent studies have proposed that Fat acts through the conserved Hippo signaling pathway to repress the expression of cycE, bantam, and diap-1. However, the combined ectopic expression of all of these target genes does not account for the hyperplasic phenotypes and patterning defects displayed by Hippo pathway mutants. This study identified the glypicans dally and dally-like as two target genes for both ft and ds acting via the Hippo pathway. Dally and Dally-like modulate organ growth and patterning by regulating the diffusion and efficiency of signaling of several morphogens such as Decapentaplegic, Hedgehog, and Wingless. These findings therefore provide significant insights into the mechanisms by which mutations in the Hippo pathway genes can simultaneously alter the activity of several signaling pathways, compromising the control of growth and pattern formation (Baena-Lopez, 2008).

A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth

During development, the Drosophila wing primordium undergoes a dramatic increase in cell number and mass under the control of the long-range morphogens Wingless (Wg, a Wnt) and Decapentaplegic (Dpp, a BMP). This process depends in part on the capacity of wing cells to recruit neighboring, non-wing cells into the wing primordium. Wing cells are defined by activity of the selector gene vestigial (vg) and recruitment entails the production of a vg-dependent 'feed-forward signal' that acts together with morphogen to induce vg expression in neighboring non-wing cells. This study identifies the protocadherins Fat (Ft) and Dachsous (Ds), the Warts-Hippo tumor suppressor pathway, and the transcriptional co-activator Yorkie (Yki, a YES associated protein, or YAP) as components of the feed-forward signaling mechanism; this mechanism promotes wing growth in response to Wg. vg generates the feed-forward signal by creating a steep differential in Ft-Ds signaling between wing and non-wing cells. This differential down-regulates Warts-Hippo pathway activity in non-wing cells, leading to a burst of Yki activity and the induction of vg in response to Wg. It is posited that Wg propels wing growth at least in part by fueling a wave front of Ft-Ds signaling that propagates vg expression from one cell to the next (Zecca, 2010).

During larval life, the Drosophila wing primordium undergoes a dramatic ~200-fold increase in cell number and mass driven by the morphogens Wg and Dpp. Focusing on Wg, it has been established that this increase depends at least in part on a reiterative process of recruitment in which wing cells send a feed-forward (FF) signal that induces neighboring cells to join the primordium in response to morphogen. The present results identify Ft-Ds signaling, the Wts-Hpo tumor suppressor pathway, and the transcriptional co-activator Yki as essential components of the FF process and define the circuitry by which it propagates from one cell to the next. This discussion considers, in turn, the nature of the circuit, the parallels between FF signaling and PCP, and the implications for the control of organ growth by morphogen (Zecca, 2010).

Several lines of evidence are presented that expression of the wing selector gene vg drives production of the FF signal by promoting a non-autonomous signaling activity of Ft. First, it was shown that vg acts both to up-regulate fj and down-regulate ds, two outputs known to elevate an outgoing, signaling activity of Ft in PCP. Second, it was demonstrated that experimental manipulations that elevate Ft signaling -- specifically, over-expression of Ft or removal of Ds -- generate ectopic FF signal. Third, and most incisively, it was shown that ft is normally essential in wing cells to send FF signal (Zecca, 2010).

Ft and Ds are both required in non-wing cells to receive the FF signal, functioning in this capacity to prevent the activation of vg unless countermanded by FF input. Notably, the removal of either Ft or Ds from non-wing cells constitutively activates the FF signal transduction pathway, mimicking receipt of the FF signal. However, the pathway is only weakly activated in this condition and the cells are refractory to any further elevation in pathway activity (Zecca, 2010).

Previous studies have defined a transduction pathway that links Ft-Ds signaling via the atypical myosin myosin Dachs (D) to suppression of the Wts kinase and enhanced nuclear import of Yki. Likewise, Ft and Ds operate through the same pathway to transduce the FF signal. Specifically, it was shown that manipulations of the pathway that increase nuclear activity of Yki (over-expression of D or Yki, or loss of Wts or Ex) cause non-wing cells to adopt the wing state. Conversely, removal of D, an intervention that precludes down-regulation of Wts by Ft-Ds signaling, prevents non-wing cells from being recruited into the wing primordium (Zecca, 2010).

To induce non-wing cells to become wing cells, transduction of the FF signal has to activate vg transcription. Activation is mediated by the vg QE and depends on binding sites for Scalloped (Sd), a member of the TEAD/TEF family of DNA binding proteins that can combine with either Yki or Vg to form a transcriptional activator Hence, it is posited that Yki transduces the FF signal by entering the nucleus and combining with Sd to activate vg. In addition, it is posited that once sufficient Vg produced under Yki-Sd control accumulates, it can substitute for Yki to generate a stable auto-regulatory loop in which Vg, operating in complex with Sd, sustains its own expression. Accordingly, recruitment is viewed as a ratchet mechanism. Once the auto-regulatory loop is established, neither FF signaling nor the resulting elevation in Yki activity would be required to sustain vg expression and maintain the wing state (Zecca, 2010).

Both the activation of the QE by Yki as well as the maintenance of its activity by Vg depend on Wg and Dpp input and hence define distinct circuits of vg auto-regulation fueled by morphogen. For activation, the circuit is inter-cellular, depending on Ft-Ds signaling for vg activity to propagate from one cell to the next. For maintenance, the circuit is intra-cellular, depending on Vg to sustain its own expression. Accordingly, it is posited that growth of the wing primordium is propelled by the progressive expansion in the range of morphogen, which acts both to recruit and to retain cells in the primordium (Zecca, 2010).

To date, Ft-Ds signaling has been studied in two contexts: the control of Yki target genes in tissue growth and the orientation of cell structures in PCP. Most work on tissue growth has focused on Yki target genes that control basic cell parameters, such as survival, mass increase, and proliferation (e.g., diap, bantam, and cyclinE). In this context, Ds and Ft are thought to function as a ligand-receptor pair, with tissue-wide gradients of Ds signal serving to activate Ft to appropriate levels within each cell. In contrast, Ft and Ds behave as dual ligands and receptors in PCP, each protein having intrinsic and opposite signaling activity and both proteins being required to receive and orient cells in response to each signal (Zecca, 2010).

This study has analyzed a different, Yki-dependent aspect of growth, namely the control of organ size by the regulation of a selector gene, vg. In this case, Ft appears to correspond to a ligand, the FF signal, and Ds to a receptor required to receive the ligand -- the opposite of the Ds-Ft ligand-receptor relationship inferred to regulate other Yki target genes. Moreover, as in PCP, evidence was found that Ft and Ds operate as bidirectional ligands and receptors: like Ds, Ft is also required for receipt of the FF signal, possibly in response to an opposing signal conferred by Ds (Zecca, 2010).

Studies of Ft-Ds interactions, both in vivo and in cell culture, have established that Ft and Ds interact in trans to form hetero-dimeric bridges between neighboring cells, the ratio of Ft to Ds presented on the surface of any given cell influencing the engagement of Ds and Ft on the abutting surfaces of its neighbors. These interactions are thought, in turn, to polarize the sub-cellular accumulation and activity of D. Accordingly, it is posited that vg activity generates the FF signal by driving steep and opposing differentials of Ft and Ds signaling activity between wing (vg^ON) and non-wing (vg^OFF) cells. Further, it is posited that these differentials are transduced in cells undergoing recruitment by the resulting polarization of D activity, acting through the Wts-Hpo pathway and Yki to activate vg (Zecca, 2010).

Thus, it is proposeed that FF propagation and PCP depend on a common mechanism in which opposing Ft and Ds signals polarize D activity, both proteins acting as dual ligands and receptors for each other. However, the two processes differ in the downstream consequences of D polarization. For FF propagation, the degree of polarization governs a transcriptional response, via regulation of the Wts-Hpo pathway and Yki. For PCP, the direction of polarization controls an asymmetry in cell behavior, through a presently unknown molecular pathway (Zecca, 2010).

FF propagation and PCP may also differ in their threshold responses to D polarization. vg expression is graded, albeit weakly, within the wing primordium, due to the response of the QE to graded Wg and Dpp inputs. Hence, a shallow differential of Ft-Ds signaling reflecting that of Vg may be sufficient to orient cells in most of the prospective wing territories, but only cells in the vicinity of the recruitment interface may experience a steep enough differential to induce Yki to enter the nucleus and activate vg (Zecca, 2010).

Finally, FF propagation and PCP differ in at least one other respect, namely, that they exhibit different dependent relationships between Ft and Ds signaling. In PCP, clonal removal of either Ft or Ds generates ectopic polarizing activity, apparently by creating an abrupt disparity in the balance of Ft-to-Ds signaling activity presented by mutant cells relative to that of their wild type neighbors. By contrast, in FF propagation, only the removal of Ds, and not that of Ft, generates ectopic FF signal. This difference is attributed to the underlying dependence of Ft and Ds signaling activity on vg. In ds^o cells, Ft signaling activity is promoted both by the absence of Ds and by the Vg-dependent up-regulation of fj. However, in ft^o cells, Ft is absent and Vg down-regulates ds, rendering the cells equivalent to ds^o ft^o cells (which are devoid of signaling activity in PCP). Thus, for FF propagation, the underlying circuitry creates a context in which only the loss of Ds, but not that of Ft, generates a strong, ectopic signal. For PCP, no such circuit bias applies (Zecca, 2010).

Morphogens organize gene expression and cell pattern by dictating distinct transcriptional responses at different threshold concentrations, a process that is understood conceptually, if not in molecular detail. At the same time, they also govern the rate at which developing tissues gain mass and proliferate, a process that continues to defy explanation (Zecca, 2010).

One long-standing proposal, the 'steepness hypothesis,' is that the slope of a morphogen gradient can be perceived locally as a difference in morphogen concentration across the diameter of each cell, providing a scalar value that dictates the rate of growth. Indeed, in the context of the Drosophila wing, it has been proposed that the Dpp gradient directs opposing, tissue-wide gradients of fj and ds transcription, with the local differential of Ft-Ds signaling across every cell acting via D, the Wts-Hpo pathway, and Yki to control the rate of cell growth and proliferation. The steepness hypothesis has been challenged, however, by experiments in which uniform distributions of morphogen, or uniform activation of their receptor systems, appear to cause extra, rather than reduced, organ growth (Zecca, 2010).

The current results provide an alternative interpretation (see The vestigial feed-forward circuit, and the control of wing growth by morphogen). It is posited that 'steepness,' as conferred by the local differential of Ft-Ds signaling across each cell, is not a direct reflection of morphogen slope but rather an indirect response governed by vg activity. Moreover, it is proposed that it promotes wing growth not by functioning as a relatively constant parameter to set a given level of Wts-Hpo pathway activity in all cells but rather by acting as a local, inductive cue to suppress Wts-Hpo pathway activity and recruit non-wing cells into the wing primordium (Zecca, 2010).

How important is such local Ft-Ds signaling and FF propagation to the control of wing growth by morphogen? In the absence of D, cells are severely compromised for the capacity to transduce the FF signal, and the wing primordium gives rise to an adult appendage that is around a third the normal size, albeit normally patterned and proportioned. A similar reduction in size is also observed when QE-dependent vg expression is obviated by other means. Both findings indicate that FF signaling makes a significant contribution to the expansion of the wing primordium driven by Wg and Dpp. Nevertheless, wings formed in the absence of D are still larger than wings formed when either Wg or Dpp signaling is compromised. Hence, both morphogens must operate through additional mechanisms to promote wing growth (Zecca, 2010).

At least three other outputs of signaling by Wg (and likely Dpp) have been identified that work in conjunction with FF propagation. First, as discussed above, Wg is required to maintain vg expression in wing cells once they are recruited by FF signaling, and hence to retain them within the wing primordium. Second, it functions to provide a tonic signal necessary for wing cells to survive, gain mass, and proliferate at a characteristic rate. And third, it acts indirectly, via the capacity of wing cells, to stimulate the growth and proliferation of neighboring non-wing cells, the source population from which new wing cells will be recruited. All of these outputs, as well as FF propagation, depend on, and are fueled by, the outward spread of Wg and Dpp from D-V and A-P border cells. Accordingly, it is thought that wing growth is governed by the progressive expansion in the range of Wg and Dpp signaling (Zecca, 2010).

Identification of Ft-Ds signaling, the Wts-Hpo pathway, and Yki as key components of the FF recruitment process provides a striking parallel with the recently discovered involvement of the Wts-Hpo pathway and Yki/YAP in regulating primordial cell populations in vertebrates, notably the segregation of trophectoderm and inner cell mass in early mammalian embryos and that of neural and endodermal progenitor cells into spinal cord neurons and gut. As in the Drosophila wing, Wts-Hpo activity and YAP appear to function in these contexts in a manner that is distinct from their generic roles in the regulation of cell survival, growth, and proliferation, namely as part of an intercellular signaling mechanism that specifies cell type. It is suggested that this novel employment of the pathway constitutes a new, and potentially general, mechanism for regulating tissue and organ size (Zecca, 2010).

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).

There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).

A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).

This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).

Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).

The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).

The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).

The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).

The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).

The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).

The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).

In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wg^spd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wg^spd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).

Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila

The dachs gene was first identified almost a century ago based on its requirements for appendage growth. This paper describes the phenotypes of strong dachs mutations, reports the cloning of the dachs gene, characterizes the localization of Dachs protein, and investigates the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in Imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. These results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development (Mao, 2006).

The observation that a hypomorphic mutation of dachs could suppress the effects of fat mutations on wing growth and Wg expression in the proximal wing has led to the suggestion that dachs might act as a downstream component of a Fat signaling pathway. This study provides two types of evidence that confirm this suggestion. First, dachs is epistatic to fat for multiple phenotypes in multiple tissues, including gene expression, growth and cell affinity. Indeed, with the notable exception of the influence of fat on tissue polarity, all known fat mutant phenotypes are completely suppressed by mutation of dachs. Second, it was found that expression of regulators of Fat, Fj and Ds, or of Fat itself, influence the localization or stability of Dachs protein at the membrane, thus providing a molecular link from Fat to Dachs (Mao, 2006).

The predicted structure of Dachs is unique within the myosin superfamily, and places Dachs in a new class of unconventional myosins. It has most similarity to myosins V, VII, and X. This is intriguing, as a mammalian protocadherin, Cdh23, has been functionally linked to myosin VIIa during the development of sensory hair cells in the inner ear (Mao, 2006).

Within the myosin head region, the major conserved domains are all present, suggesting that Dachs functions as a motor protein. However, it is also possible that Dachs serves a structural or scaffolding role. For example, in the Hedgehog pathway, a kinesin-related protein, Costal2, is thought to function largely as a scaffold that brings together crucial kinases with their substrates (Mao, 2006).

The d^GC2 mutation deletes part of the N terminal extension. As d^GC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. d^GC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele (Mao, 2006).

Characterization of new dachs alleles has provided an opportunity to define more clearly the requirements for dachs. dachs is required for normal wing and leg growth, although some appendage growth is dachs independent. Importantly, the identification of dachs as a downstream component of a Fat signaling pathway that influences growth implies that the reduced growth in dachs mutants is reflective of a normal role for a Fat pathway in growth promotion. That is, while fat is a gene whose normal role can be thought of as to restrain growth, as mutant tissue overgrows, it is suggested that inhibition of Fat occurs during normal development, and that this inhibition contributes to normal appendage growth, as defined by the reduced growth of dachs mutants. Normal inhibition of Fat activity would presumably be effected by the two known regulators of Fat, Fj and Ds (Mao, 2006).

Whether available dachs mutations fully define the normal involvement of the Fat pathway in growth promotion is not yet clear. The possibility cannot be excluded that dachs is partially redundant with other proteins (e.g. other myosins), although this seems unlikely given the complete suppression of all non-polarity phenotypes of fat by dachs. It is also possible that dachs is required only for peak Fat signaling. This explanation is suggested by the observation that expression of the Fat target genes wg, Ser and fj is only partially or transiently lost in dachs mutants, yet the elevated or ectopic expression of these genes in fat mutants is completely eliminated by mutation of dachs (Mao, 2006).

The relatively mild tissue polarity phenotypes of dachs mutants, and the inability of dachs mutation to completely suppress the influence of fat on tissue polarity, contrast with the absolute dependence of fat gene expression, growth and affinity phenotypes on dachs. These observations suggest that there are two distinct Fat pathways. One, crucially dependent on Dachs, influences gene expression, growth and cell affinity, and another, partially independent of Dachs, influences tissue polarity. Studies of the atrophin protein Grunge also support the suggestion that there is a distinct Fat polarity pathway, as Grunge interacts with Fat and influences tissue polarity, but does not exhibit other phenotypes observed in fat mutants. Thus, Dachs might act redundantly with another protein in a polarity pathway, but non-redundantly in a pathway that influences gene expression. It should also be noted that effects of dachs on gene expression might contribute to the polarity phenotypes of dachs mutants. For example, fj is regulated by dachs, and fj has polarity phenotypes (Mao, 2006).

The asymmetric localization of Dachs observed in wild-type wings, and the influence of Fj and Ds on Dachs localization, have important implications for tissue polarity. (1) The asymmetric localization of Dachs is itself a form of polarity, and its detection in third instar imaginal discs emphasizes that these cells are polarized well before core polarity proteins such as Frizzled and Dishevelled become asymmetrically localization in pupal wings. A similar conclusion can be drawn from the recent observation that fat and ds influence the orientation of cell divisions in third instar discs. (2) The observations identify an ability to induce asymmetric protein localization as a mechanism through which the Fat pathway might influence tissue polarity. Dachs is one target, but the Fat polarity pathway might similarly involve asymmetric localization of other myosins, or of other types of proteins, to affect tissue polarity (Mao, 2006).

Mutation of fat is associated with elevated Dachs staining at the membrane, and overexpression of Fat decreases Dachs staining at the membrane. Although this negative effect of Fat on Dachs is subject to the caveat that only tagged overexpressed Dachs:V5 can be detected, this tagged protein rescues dachs mutants, and the effects of Fat on Dachs staining are consistent with their opposite phenotypes and the epistasis of dachs to fat. Manipulations of the expression of Fat regulators provide further evidence that Fat regulates Dachs levels at the membrane, and altogether these observations implicate Dachs as a crucial intracellular component of a Fat signaling pathway (Mao, 2006).

The concomitant elevation of Fat staining and loss of Dachs staining observed at the perimeter of Fj-expressing clones is consistent with the conclusion that Fat can antagonize the localization or stability of Dachs at the membrane. Because the elevation of Fat is limited to the periphery of Fj-expressing clones, it is hypothesized that it results from an influence of Fj on Fat-Ds interactions, rather than the expression of Fj per se. Tissue polarity studies have implied that Fj and Ds have opposite affects on Fat. Although it has not yet been determined whether Fj can directly modify Fat or Ds, the simplest explanation for the elevated Fat staining at the edge of Fj-expressing cells would be to propose that Fj modifies Ds to inhibit its interactions with Fat. In this case, Fat protein within Fj-expressing clones would be predicted to prefer to bind to Ds outside of the clone, and hence to accumulate at the clone perimeter, where it would then downregulate Dachs (Mao, 2006).

The interpretation of the elevated Dachs staining at the perimeter of Ds-expressing clones is more complex. Although Fat is elevated at the clone perimeter, the depletion of Fat from neighboring cells suggests that the elevated Fat staining largely reflects Fat outside of the clone, rather than in Ds-expressing cells. Given that dachs and fat influence transcriptional targets cell autonomously, and dachs acts genetically downstream of fat, the link between elevated Fat in one cell and elevated Dachs in a neighboring cells must be indirect. It might be that Ds can also influence Dachs localization, and does so in opposite fashion to Fat. According to this scenario, the elevated Fat staining in cells neighboring the clone would be reflective of high levels of Ds engaged by Fat at the clone perimeter, which would then recruit or stabilize Dachs at the membrane. However, mutation of ds did not result in any noticeable decrease of Dachs:V5 staining. Alternatively, it might be that Fat antagonizes the accumulation of Ds within the same cell. High Fat accumulation at the edge of one cell could then result in low Fat accumulation at the edge of its neighbor through this hypothesized downregulation of Ds. In this case, the elevated Dachs accumulation at the edge of Ds-expressing clones would be a consequence of low levels of Fat. This model would also imply that asymmetric localization of Fat could be propagated from cell to cell, which could have important consequences for Fat pathway regulation. However, there is as yet no evidence that Fat is asymmetrically localized at wild-type levels of Fj and Ds expression (Mao, 2006).

Delineation of a Fat tumor suppressor pathway

Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).

Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).

Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).

In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).

dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco³, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).

Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).

Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).

The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco³, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco³, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).

The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco³ mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco³ also has weaker effects on Wg and fj expression; the weaker effects of dco³ could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco³ mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).

Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).

dachs is also epistatic to dco³ for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco³ implies that the overgrowth phenotype of dco³ is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco³, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).

Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs², consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).

When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco³ still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).

The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).

The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).

Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco³ homozygous mutant animals but was not diminished in fat or dco³ heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).

The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).

Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco³ is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco³, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).

In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco³ and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wts^P2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).

Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).

Morphogen control of wing growth through the fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).

Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).

Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).

The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).

Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).

In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or Tkv^Q-D is biased toward cells outside of clones (Rogulja, 2008).

The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).

The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).

Propagation of Dachsous-Fat planar cell polarity

The Fat pathway controls both planar cell polarity (PCP) and organ growth. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP, whereas their slope can influence growth. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling. This study shows that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, it was shown that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. A biochemical mechanism was identified that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat (Ambegaonkar, 2012).

The observation that differences in Fj or Ds expression can alter Fat PCP at a distance and that Ds, and to a lesser extent Fat, is polarized within the wing, together with other recent studies, imply that establishment of polarity in the Fat PCP system relies not just upon direct interpretation of Fj and Ds gradients but also upon amplification and propagation of PCP. To achieve this, PCP models incorporate both asymmetric intercellular signaling and antagonistic intracellular interactions between complexes that localize to distinct sides. Intercellular binding between Ds and Fat is well established, but on its own, this would not propagate polarity from cell to cell. However, incorporation of a local, intracellular antagonism of Ds by Fat activity could polarize Ds localization, which could then enable Fat-PCP to propagate. It is hypothesized that Fat regulates Ds by influencing production or stability of processed Ds isoforms (Ambegaonkar, 2012).

The propagation of polarity means that Fat-PCP is influenced not only by the local gradient but also by differential expression at a distance. Strong repolarization of Dachs was dependent upon having substantial differences in expression. Notably, strong differences in expression of both Fj and Ds normally occur in the proximal wing, and these differences have significant effects on Fat activity. Both measures of the range of Dachs repolarization and mathematical modeling suggest that the Fj/Ds expression boundary in the proximal wing would not be sufficient to direct Fat-PCP across 30 or more cells, as would be required at late third instar. However, at early third instar, when the developing wing is small, a mechanism that propagates PCP from an expression boundary for several cells could in principle be sufficient to establish PCP throughout the wing. Once established, the mechanisms that allow Fat-PCP to propagate could also help maintain Fat-PCP as the wing grows. In this case, the Fj and Ds boundaries at the edge of the developing wing would be the main drivers of polarity, rather than the shallow gradients of their expression within the wing itself (Ambegaonkar, 2012).

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

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

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

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

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

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

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

EFFECTS OF MUTATION

The adult phenotype of ds mutants is consistent with the high levels of ds transcript in imaginal discs. In all known alleles defects are seen with 100% penetrance in the legs, wings, and thorax. In contrast, eye defects, apparent as rough patches, occur at a low frequency. On ds wings, the anterior cross-vein is displaced distally, that is, closer to the posterior cross-vein; the legs are stubby, with a reduced number of tarsal joints in stronger alleles, and the thorax is broadened. These adult phenotypes are more pronounced in stronger mutant alleles, with the addition of duplicate bristles on the notum and wings stiffly held out, with broken and ectopic cross-veins. All ds mutant discs exhibit normal size range with no overgrowth. Therefore, in contrast to mutations in the fat gene, encoding a similar cadherin protein, ds mutations appear to alter imaginal disc morphogenesis exclusively without affecting cell proliferation (Clark, 1995).

The adult cuticular wing of Drosophila is covered by an array of distally pointing hairs that reveal the planar polarity of the wing. Mutations in dachsous disrupt this regular pattern, and do so by affecting frizzled signaling. Mutations in dachsous also result in a tissue polarity phenotype that at the cellular level is similar to frizzled, dishevelled and prickle, as many cells form a single hair of abnormal polarity. Although their cellular phenotype is similar to frizzled, dishevelled and prickle, dachsous mutant wings display a unique and distinctive abnormal hair polarity pattern, including regions of reversed polarity. The development of this pattern requires the function of frizzled pathway genes and suggests that in a dachsous mutant the frizzled pathway is functioning - but in an abnormal way. Genetic experiments indicate that dachsous is not required for the intracellular transduction of the frizzled signal. However, dachsous clones disrupt the polarity of neighboring wild-type cells, suggesting the possibility that dachsous affects the intercellular signaling function of frizzled. Consistent with this hypothesis it has been found that frizzled clones in a dachsous mutant background display enhanced domineering non-autonomy, and that the anatomical direction of this domineering non-autonomy is altered in regions of dachsous wings that have abnormal hair polarity. The direction of this domineering nonautonomy is coincident with the direction of the abnormal hair polarity. It is concluded that dachsous causes a tissue polarity phenotype because it alters the direction of frizzled signaling (Adler, 1998).

Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts an a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation (Buratovich, 1997).

Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Arm^over) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Arm^under). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Arm^over and/or Arm^under. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 2 consists of genes required for cell adhesion: This group includes shotgun (which encodes DE-cadherin), as expected. Also uncovered were fat (ft) and dachsous (ds). These two genes encode nonclassical cadherin characterized by a huge extracellular domain containing up to 35 cadherin repeats and a bipartite Arm binding site. Interactions with these two mutants are similar to those observed with shotgun (DE-cadherin), the only difference being that ft interacts more weakly than shg with Arm^over. In addition to genes encoding cadherins (classical and nonclassical), interactions have been observed with some of the genes known to be essential (directly or indirectly) for the assembly or maintenance of adherens junctions—stardust (sdt), discs-large (dlg), and crumbs (crb). These interact in the same direction as shg; however, the suppression of Arm^under is always weaker and only dlg^M52 enhances Arm^over to the same extent as zw3^M11 (Greaves, 1999).

The interaction with shotgun (encoding DE-cadherin) itself is not very illuminating since it is expected that the phenotype caused by an excess of intracellular cadherin domain will be suppressed by decreasing endogenous cadherin levels. Still, this interaction shows that the level of overexpression afforded by the Gal4p system is within physiological levels. Interaction with fat and dachsous suggests that these two nonclassical cadherins interact (maybe directly) with Arm. Initial analysis of the intracellular domain of Fat and Dachsous fail to identify an Arm/ß-catenin binding site homologous to that found in E-cadherin. However, subsequent sequence examination suggests the existence of a bipartite site. Genetic interactions with fat and dachsous strongly suggest that this proposed site is functional, and thus removing one copy of the fat or dachsous gene would release additional Arm to the cytoplasm and make it available for use in Wg transduction. Interactions with fat and dachsous in the eye confirm the ability of these genes to modify cytoplasmic Arm levels. It also indicates that these genes are expressed in the eye and may be functional there (Greaves, 1999).

Cadherin-N (CadN) binds to Arm. Therefore the failure of CadN to interact in this screen suggests that CadN may not be expressed to significant levels in the posterior compartment of wing imaginal discs or in eye precursors. In contrast, crumbs (crb) and stardust (sdt) do interact. The proteins encoded by these genes are not thought to participate in junctional complexes per se. Rather, they control the biogenesis of the junctions. It is suggested that decreasing the activity of crb or sdt has a quantitative effect on the number or size of adherens junctions and this would lead to more Arm being released from the membrane and made available for Wg signaling (Greaves, 1999).

The fat gene negatively controls cell proliferation in a cell autonomous manner. The Fat protein (with 5,147 amino acids) contains four major regions. Beginning by the N-terminus there are 34 cadherin-like domains, five EGF-like repeats interspersed with two laminin A-G chain motifs, a transmembrane domain and a novel cytoplasmic domain (Mahoney, 1991). Several cell behavior parameters of mutant alleles of fat ( ft) have been studied in Drosophila imaginal wing disc development. Mutant imaginal discs continue growing in larvae delayed in pupariation and can reach sizes of several times those of wild-type. Their growth is, however, basically allometric. Homozygous ft cells grow faster than their twin cells in clones and generate larger territories, albeit delimited by normal clonal restrictions. Moreover, ft cells in clones tend to grow towards the wing proximal regions. These behaviors can be related with failures in cell adhesiveness and cell recognition (Garoia, 2000).

The cadherins Fat and Dachsous regulate dorsal/ventral signaling in the Drosophila eye

Planar polarity refers to the asymmetry of a cell within the plane of the epithelium; for example, cells may form hairs that point in a posterior direction, or cilia may beat uniformly. This property implies that cells have information about their orientation; it is of interested to discover the nature of this information. Relevant also is the body plan of insects, which, in the ectoderm and somatic mesoderm, consists of a chain of alternating anterior and posterior compartments -- basic units of development with independent cell lineage and subject to independent genetic control. Using the abdomen of adult Drosophila, genes required for normal polarity were either removed or constitutively expressed in small clones of cells and the effects on polarity were observed. Hitherto, all such studies of polarity genes have not found any difference in behavior between the different compartments. This study shows that the three genes, four-jointed, dachsous, and fat, cause opposite effects in anterior and posterior compartments. For example, in anterior compartments, clones ectopically expressing four-jointed reverse the polarity of cells in front of the clone, while, in posterior compartments, they reverse behind the clone. These three genes have been reported by others to be functionally linked. This discovery impacts on models of how cells read polarity. At the heart of one class of models is the hypothesis that cell polarity is determined by the vector of a morphogen gradient. Evidence is presented that cell polarity in the abdomen depends on at least two protein gradients (Fj and Ds), each of which is reflected at compartment borders. Consequently, these gradients have opposing slopes in the two compartments. Because all polarized structures made by abdominal cells point posteriorly, it is surmised that cells in each compartment are programmed to interpret these protein gradients with opposite signs, pointing up the gradient in one compartment and down the gradient in the other (Casal, 2002).

Two other genes resemble fj with regard to compartment-specific effects: dachsous (ds) and fat (ft). In both cases, UAS transgenes cannot be easily made, so only the effects of removing the genes have been studied. Dachsous is a giant integral membrane protein with many cadherin domains. ds gene expression has been monitored using a ds.lacZ transgene. In each segment of the tergites, ds.lacZ is expressed in one band per metamere with a peak near the A/P border that extends into both compartments. This single band is more clearly apparent in the pleura and appears to be centered in a more anterior location than in the tergite or sternite (Casal, 2002).

ds^- flies are lethal, but some hypomorphic mutants survive to adulthood with defective limbs -- the tarsi show polarity defects. In the abdomen of these flies, the anterior parts (a2) of the A compartments are fairly normal, but much of the rest of the A and P compartments is affected by whorls. Remarkably, hair orientation in the back half of the P compartments, both dorsal and ventral, is reversed (Casal, 2002).

In the tergites, ds^- clones are characterized by whorling hairs within the clone. They cause some swirly repolarization of the hairs in front of the clone in the A compartment, but not behind. These whorls could indicate that there has been a loss of overall polarity, even though some local coordination between adjacent cells remains. In the P compartment, ds^- clones induce clear reversal of hairs behind the clone without affecting the front. Just as with clones ectopically expressing fj, those situated at the back boundary of the A compartment reorient hairs outside the clone, both anterior to the clone (A cells) and posterior to it (P cells) — but hairs within the clone are more whorly than with fj-expressing clones (Casal, 2002).

Thus, apart from the whorls, ds^- clones are reminiscent of UAS.fj clones; both cause non-autonomous reversals in opposite ways in the A and the P compartment. Accordingly, Ds, like Fj, may form opposing gradients in A and P, each being interpreted with opposite signs. Because loss of Ds activity mimics gain of Fj activity, it is deduced that the gradients of Fj and Ds activity are reciprocal to each other, a conclusion that fits with the expression pattern of both genes in the pleura (Casal, 2002).

Like ds, ft encodes a huge molecule with many cadherin repeats, and as with ds, null mutant flies do not develop. The mutant imaginal discs grow excessively, and there are some effects on the polarity of bristles. Clones of ft^- cells in otherwise wild-type discs are abnormally large; in the abdomen, these clones tend to be creased, as if they were trying to grow beyond their normal compass (Casal, 2002).

In the A compartments of the tergites, ft^- clones tend to disturb and reverse polarity behind the clone, while, in the P compartments, they tend to reverse in front. Thus, ft^- clones, like ds^- and fj^- clones, have opposite effects on polarity in A and P. When the ft^- clones are near the A/P boundary, they behave as would be expected from the provenance of the cells neighboring the clone: clones at the back of the A compartment fail to reverse the P cells behind (P cells normally reverse in front of a ft^- clone), and P clones fail to reverse A cells in front of them (A cells normally reverse behind a ft^- clone) (Casal, 2002).

Thus, ft^- clones, like ds^- and fj^- clones, have opposite effects on polarity in A and P. Further, the effects of ft^- clones are similar to those of fj^- clones but are opposite those of UAS-fj and ds^- clones. For example, in the A compartment, hairs point toward ft^- clones but away from UAS-fj clones, whereas, in P, they point away from ft^- clones but toward UAS-fj clones. Using the logic deployed with fj and ds, it is inferred that Ft activity is reflected like that of Fj, forming a peak at the segment boundary and declining to a trough at the A/P boundary. But note that ft^- clones can cause polarity reversals anywhere within A, as well as in anterior P -- but fj^- clones do so only in anterior A. This difference argues for a model in which Fj is produced only by cells flanking the segment boundary, acting non-autonomously on cells further away, whereas Ft activity might be required autonomously in all cells, with any differential in Ft activity between neighboring cells determining their polarity (Casal, 2002).

The three genes ds, ft, and fj are functionally linked: mutations in all three damage the tarsi in a similar way; ds and ft encode similar cadherin molecules, and they and fj interact genetically. For the Drosophila eye, it has been proposed that the products of ds, ft, and fj work together in a linear pathway in the developing ommatidia. This pathway begins with a gradient of Wg and leads to the differential activation of Fz in the presumptive R3 and R4 cells. According to this model, graded Wg spreads into the eye from sources at the dorsal and ventral poles, induces Ds expression, represses Fj expression, and thereby generates reciprocal Ds and Fj gradients. Fj activity then represses Ds activity and reinforces this reciprocity. In turn, the Ds gradient then patterns the activity of Ft, which is ubiquitously expressed. Finally, the gradient of Ft activity promotes the activation of Fz in the more equatorial cell and directs it to become the R3 cell, while the more polar cell becomes the R4 cell (Casal, 2002).

The present results point to parallels between the action of Fj, Ds, and Ft in the eye and abdomen. In both cases, a morphogen (Wg in the eye, and Hh in the abdomen) appears to govern polarity through the induction of reciprocal gradients of Fj and Ds expression. Further, in the abdomen, Hh organizes polarity at least in part through the induction of Wg. Hence, as in the eye, peak Wg activity occurs where fj is repressed and where ds is expressed. Finally, the results suggest that the gradient of Ds in the abdomen is reciprocal to that of Ft activity, consistent with the model proposed for the eye. These parallels suggest that the three genes are part of a mechanism common to the eye and abdomen and presumably elsewhere (Casal, 2002).

These results argue that, in the abdomen, the compartmental provenance of responding cells is crucial. This is particularly clear for clones that either lack or overexpress fj. It is found that, in the A compartment, hairs point down gradients of Fj activity, while, in the P compartment, they point up. This discovery can help explain how all cells in the abdominal epidermis make hairs that have the same polarity, even though, in both compartments, the gradients of Fj and Ds decline in opposite directions. However, it presents other problems (Casal, 2002).

One problem is that it has been proposed that Hh drives polarity by inducing a gradient morphogen, X, whose slope specifies polarity. The model is that Hh enters the A compartment from the P compartment behind it and acts through wg and optomotor blind (omb) to induce X. For simplicity, it was conjectured that X might form a monotonic gradient, spreading forward from its peak at the back of the A compartment all the way to the front of the P compartment of the next segment. According to this conjecture, all cells in both A and P make structures that point posteriorly because all respond to the common vector of a monotonic gradient of X (Casal, 2002).

However, the present results argue for reflected gradients centered around the A/P compartment boundary and against a monotonic gradient for X. Thus, it is now surmised that Hh induces reflected gradients of Fj, Ds, and Ft activity. It is instructive to compare the imaginal discs with the abdomen. In the discs, unidirectional Hh signaling across the A/P boundary induces the morphogens Decapentaplegic (Dpp) and Wg, and these then spread both anteriorly and posteriorly and create reflected gradients that pattern both compartments. In the abdomen, Hh also induces Wg (in the tergites and sternites) and Dpp (in the pleura). At least in the tergites, Wg then spreads posteriorly from its source at the back of the A compartment to induce omb and specify cell pattern in the P compartment. Thus, the combined activities of Hh in A cells, and of Hh-induced Wg moving back into P cells, generates a zone of Omb expression spanning the A/P boundary. It is now suggested that this band of Omb organizes the reflected gradients of Ds and Fj, which in turn, through Ft, help polarize the cells. Thus, the combined actions of Fj, Ds, and Ft might constitute what was previously called X (Casal, 2002).

Another problem is raised by the finding that cells in the A and P compartments interpret the polarizing activities of Fj, Ds, and Ft with opposite response. In the wing, gene products such as Fz and Dsh accumulate transiently along the distal edge of each cell and forecast both the site and distal direction of hair outgrowth. Further, wing hairs invariably point away from UAS.fz clones and toward fz^- clones, and this suggests that these subcellular localizations could be a readout of differential Fz activity. It was found that UAS.fz and fz^- clones in the abdomen behave like their counterparts in the wing, whether in the A or P compartment -- in all cases, hairs point away from UAS.fz clones and toward fz^- clones. Thus, it is inferred that in the normal abdomen, Fz and Dsh accumulate along the posterior edge of both A and P cells, even though the controlling gradients of Fj, Ds, and Ft in the A compartment have the opposite slopes of those in the P compartment (Casal, 2002).

How might A and P cells be programmed so that bidirectional activity gradients of Fj, Ds, or Ft would lead to a unidirectional slope of Fz activity? It is suggested that a transcription factor, Engrailed, encoded by the selector gene that distinguishes P from A cells, also alters the response of P cells relative to A cells, so that in A cells, Fz might accumulate at the cell edge where Fj is lowest, while, in the P cells, it might accumulate where it is highest. The result would be a localized accumulation of Fz along the posterior edge in all cells, whether in A or P. A precedent comes from yeast, where haploid (a or a) cells bud axially near prior budding sites, while diploid (a/a) cells bud in a bipolar fashion at the site farthest from the previous bud. In yeast, this switch in polarity is also governed by transcription factors encoded by the mating-type locus (Casal, 2002).

In the abdomen, there are observations that do not fit with a simple linear pathway as proposed for the eye. For example, hair polarities are not randomized in fj^-, ds^-, or ft^- mutant tissues, and even entirely fz^- flies show relatively normal polarity in most regions. Nevertheless, consistent changes in polarity are generated by disparities in the activity of each of these polarity genes, usually across clone borders. Hence, cell polarity may depend on multiple signals of which the mutually reinforcing effects of Fj and Ds are but one example (Casal, 2002).

An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).

Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent to the Wg domains are non-overlapping cell populations that express the TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).

It is concluded that Drosophila optic lobe glia use axon fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless. When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).

Dachsous and planar polarity in the eye

Fat also plays an important role in planar polarity. This phenomenon is evidenced by the coordinated orientation of ommatidia in the Drosophila eye. Planar polarity requires that the R3 photoreceptor precursor of each ommatidium has a higher level of Frizzled signaling than its neighboring R4 precursor. Two cadherin superfamily members, Fat and Dachsous, and the transmembrane/secreted protein Four-jointed play important roles in this process. The data support a model in which the bias of Frizzled signaling between the R3/R4 precursors results from higher Fat function in the precursor cell closer to the equator -- the cell that becomes R3. Evidence is also provided that positional information regulating Fat action is provided by graded expression of Dachsous across the eye and the action of Four-jointed, which is expressed in an opposing expression gradient and appears to modulate Dachsous function. It is suggested that the presence of relatively higher Ds function in the polar cell could result in a difference in Ft function between the R3/R4 precursors by either inhibiting Ft function in a cell-autonomous fashion or by stimulating Ft function in the equatorial cell. The difference in Ft function between the precursor cells biases Fz signaling so that the equatorial cell has higher Fz activity (Yang, 2002).

The lack of a graded Ft expression suggested that Ft might be regulated by proteins that are themselves expressed in gradients in the eye disc. Two previous findings prompted a test of the role of dachsous (ds, which encodes a Fat-like transmembrane protein containing 27 cadherin repeats in its extracellular domain), as a Ft regulator: (1) loss of Ds function in the wing causes planar polarity defects; (2) removal of a single copy of ds suppresses defects caused by a dominant ft mutation, suggesting that Ds might act in conjunction with Ft. To determine whether Ds provides positional information regulating Ft function in the eye, ds expression was examined in the eye imaginal disc using a ds-lacZ enhancer trap (a ß-gal reporter gene inserted in the first intron of ds) that faithfully reproduces ds expression during third instar larval development. Interestingly, ds-lacZ is expressed in a graded pattern that is high at the two poles and low at the equator. In order to confirm that this gradient of ds transcription results in a gradient of Ds protein expression, antibodies were raised against the intracellular domain of Ds and used to stain eye discs. In the region near the morphogenetic furrow where the R3 and R4 fates are being specified, graded expression of Ds from the poles was readily apparent. In older ommatidia in the posterior region of the disc, Ds, like Ft, accumulates in a subset of cells surrounding each ommatidium (Yang, 2002).

If Ds is an important regulator of Ft, then the absence of Ds should lead to randomized d-v polarity. This prediction was examined using animals homozygous for the ds loss-of-function mutation ds^UA071. While Ds function is important for normal viability, a few homozygous ds^UA071 mutant animals survive for a few days after eclosion. Similar to ft ommatidia, the ds ommatidia randomly adopted either d or v polarity. Similar results were observed in animals transheterozygous for ds^UA071 and another strong ds allele (ds^38K) and in marked clones of homozygous ds^UA071 cells (~40% polarity reversals). The examination of ds mutant clones also showed the presence of nonautonomous effects on the polarity of neighboring wild-type tissue along the equatorial border of the clone (Yang, 2002).

When the pattern of R3/R4 specification and Fz signaling in ds eye discs was examined by staining for either the E(spl)mdelta0.5 marker or Fmi, the results were very similar to those described above for ft mutant clones. In both cases, the mutant ommatidia exhibited polarized Fmi localization and R4 reporter expression, but the direction of polarization was randomized. Thus, Ds, like Ft, plays an essential role in the establishment of properly biased Fz signaling during R3/R4 specification (Yang, 2002).

The requirement for Ds during the establishment of ommatidial polarity and the gradient of ds expression suggested that higher ds expression in the polar member of the R3/R4 precursor pair might contribute to the normal pattern of R3/R4 specification by modulating Ft function. For example, the presence of higher Ds in the polar precursor cell might either inhibit Ft function within the polar cell or else promote Ft function in the neighboring equatorial precursor cell. This proposal makes several predictions: (1) loss of Ds function from the polar cell, which would reverse the relative levels of Ds within an R3/R4 precursor pair, should lead to reversals in R3/R4 specification pattern and reversals of ommatidial polarity; (2) in contrast, loss of Ds from the equatorial cell, which does not change the direction of the ds gradient within an ommatidium, should have little effect on polarity; (3) loss of Ds from the polar cell should have no effect if that cell also lacks functional Ft (Yang, 2002).

To analyze the effects of a lack of Ds function in one member of an R3/R4 precursor pair, homozygous ds^UA071 clones were generated in heterozygous animals and R3/R4 mosaic ommatidia were examined. The loss of Ds function from the equatorial R3/R4 precursor cell had only a mild effect on ommatidial polarity (13% polarity reversals in 127 examples. In contrast, loss of Ds function from the polar cell led to polarity reversals in 43% of 98 examples. These data demonstrate that Ds acts primarily in the polar precursor cell and are consistent with the idea that the graded ds expression contributes to the normal pattern of R3/R4 specification by providing a higher Ds level to the polar cell of each R3/R4 precursor pair. However, the observation that differences in Ds function between the R3/R4 precursor cells are less effective than Ft differences at directing the pattern of R3/R4 specification suggests that, in addition to its primary role in the polar precursor cell, Ds may also play a role in regulating Fz signaling from the equatorial cell (Yang, 2002).

In order to test whether Ds regulates Fz signaling by modulating Ft function, the genetic interaction of ft and ds in specifying R3/R4 cell fates was examined. Since removal of Ds from the polar precursor cell frequently causes this cell to become R3 rather than R4 while the absence of Ft from an R3/R4 precursor cell leads to its specification as R4, the effects of removing both Ds and Ft from the polar cell were examined using marked clones of ds^UA071;ft^Gr-V double mutant cells. Polarity reversals occurred in only 2.5% (2 out of 80 examples) of the R3/R4 mosaic ommatidia in which the polar precursor cell lacked Ds and Ft. This result contrasts with the 43% polarity reversals observed when only Ds function was lost from the polar cell. Indeed, the effect of removing both Ds and Ft functions was remarkably similar to that of removing Ft alone. In each case, the mutant precursor cell was preferentially specified as R4 (80% and 98%, respectively). Thus, the absence of Ft function from one R3/R4 precursor cell determines the pattern of R3/R4 specification in a manner that is largely independent of input from Ds. These results suggest that Ds acts upstream of Ft and are consistent with the idea that the presence of higher Ds levels in the polar R3/R4 precursor directs correct ommatidial polarity by ensuring that Ft activity is higher in the equatorial precursor cell (Yang, 2002).

To further explore how Ds might act in regulating Ft, it was asked whether Ft protein level is altered in ds mutant clones. Staining for Ft was carried out in ds clones and it was found that the level of Ft is increased slightly within the clones. This result suggests that one mode of Ds regulation might be to antagonize Ft protein expression or stability (Yang, 2002).

This analysis supports the idea that positional information controlling Fz signaling during ommatidial development is provided by the opposing gradients of fj and ds expression. The question arises as to how these gradients are established. Previous work has shown that a major determinant of the fj expression gradient is Wg, a secreted Wnt class ligand that negatively regulates fj expression and that is expressed at high levels at the two poles of the eye disc. To test whether the Wg gradient also contributes to the regulation of ds expression, clones of cells in which Wg signaling was either ectopically activated or reduced were examined in animals carrying the ds-lacZ reporter. Ectopic activation was achieved by overexpressing a constitutively activated form of Armadillo (Arm) and resulted in a dramatic increase in ds-lacZ expression. The effects of attenuating Wg signaling were assayed in clones of cells homozygous for the hypomorphic arm^H8.6 mutation. ds-lacZ expression was severely reduced in these clones. Combined with previous studies of fj-lacZ expression, these data suggest that the ds and fj expression gradients result in large part from the presence of a gradient of Wg signaling that increasingly activates ds and inhibits fj expression near the poles. It is worth emphasizing that the receptor mediating the effects of Wg on fj and ds expression is likely to be another member of the Fz family, perhaps dFrizzled2 (dFz2), rather than Fz itself. This is evident from the observation that fj-lacZ expression is not affected by the loss of Fz function (Yang, 2002).

Another study suggests that fat is involved not in the establishment of R3 and R4 cell fate, but instead functions in establishing the dorsal-ventral midline (equator) during eye morphogenesis. The Drosophila eye is a polarized epithelium in which ommatidia of opposing chirality fall on opposite sides of the eye's midline, the equator. The equator is established in at least two steps: photoreceptors R3 and R4 adopt their fates, and then ommatidia rotate clockwise or counterclockwise in accordance with the identity of these photoreceptors. Two cadherins, Fat (Ft) and Dachsous (Ds), play a role in conveying the polarizing signal from the D/V midline in the Drosophila eye. In eyes lacking Ft, the midline is abolished. In ft and ds mutant clones, wild-type tissue rescues genetically mutant tissue at the clonal borders, giving rise to ectopic equators. These ectopic equators distort a mosaic analysis of these genes and has led to a possible misinterpretation that ft and ds are required to specify the R3 and R4 cell fates, respectively (Yang, 2002). Interpretation of these data supports a significantly different model in which ft and ds are not necessarily required for fate determination. Rather, they are involved in long-range signaling during the formation of the equator, as defined by the presence of an organized arrangement of dorsal and ventral chiral ommatidial forms (Rawls, 2002).

ft has long been known for its role in proliferation control. The identification of new ft alleles in a FLP/FRT screen has revealed a role for Ft in establishing epithelial polarity. In ft⁴²² null clones, approximately 52.5% of ommatidia—including mosaic and genetically mutant ommatidia—exhibit defects in polarity. Of these, 50.5% are inverted on their D/V axis. The remaining 2.0% of ommatidia are inverted on their A/P axis or on both their A/P and D/V axes. Furthermore, 98% of mosaic ommatidia that are phenotypically mutant are inverted on their D/V axis (Rawls, 2002).

Dorsoventrally inverted ommatidia are not randomly distributed within ft clones. Rather, they are preferentially localized toward the polar border such that the phenotypically mutant ommatidia are found in the polar region of the clone and phenotypically wild-type ommatidia are found along the equatorial border. The consequence of this biased distribution of ommatidia is an 'inverted equator' (originally called a pseudoequator) within the mutant clone, in which the points of opposing trapezoids face each other. Inverted equators in ft clones consistently arise approximately two rows from the equatorial border of the clone. These inverted equators were seen in 35/41 (85%) ft⁴²² clones. The 15% of ft clones with no apparent ectopic equator were either small or long and narrow and therefore not broad enough to detect this phenotype (Rawls, 2002).

While a small percentage of ft clones lie along the equator, of over 200 ft clones examined, none cross the equator. A closer examination of these clones has revealed that the position of the endogenous equator, which can be identified in neighboring wild-type tissue, gets shifted by one to two ommatidial rows along the mutant border to accommodate the ft mutant clone. These observations suggest that the juxtaposition of ommatidia with high versus low Ft activity influences the placement of the equator (Rawls, 2002).

ds, known for its role in morphogenesis, plays a role in setting up polarity in the eye. The bias of D/V:A/P errors is similar to that described for ft clones, although fewer ommatidia are disrupted. Approximately 29% of ommatidia (both mosaic and genetically mutant) in ds^38K clones (strong hypomorphic allele) display D/V inversions, while fewer than 1% of genetically mutant ommatidia display A/P inversions (517 ommatidia scored from 12 ds^38k clones). Inverted equators also occur in ds mutant clones at a similar frequency as is seen with ft: 15 out of 18 clones (83%) had inverted equators. However, it is interesting to note that ds inverted equators arise along the equatorial border of the clone rather than within the clone, as is seen with ft. Finally, although ectopic ds equators are rare, they do occur. This phenotype might be more penetrant in a null allele of Ds function; however, no such alleles have been reported (Rawls, 2002).

Inverted and/or ectopic equators have also been observed in mirr, fng, and four-jointed (fj). One significant difference between ft/ds and these genes is that, in these other examples, mutant tissue nonautonomously disrupts wild-type tissue, generating a contiguous patch of nonautonomous D/V inversions. In ft, nonautonomous effects are never observed in wild-type ommatidia. In ds, the nonautonomy can extend many rows beyond the clone and may be separated by up to eight rows of unaffected ommatidia. It is appealing to speculate that this 'extended' nonautonomy is an effect of the twin-spot clone, in that a difference in relative amounts of Ds activity reverses the polarity. However, the scattered occurrence of these inverted ommatidia and the fact that a range cannot be seen in the number of affected ommatidia make this hypothesis somewhat unsatisfying. In order to test this 'twin-spot' hypothesis unambiguously, again, a protein null allele of ds is necessary (Rawls, 2002).

The clonal phenotypes of ft and ds suggest that they are involved in establishing equators. Since the establishment of the equator is known to involve long-range signaling, the nonautonomous effects seen in the clonal phenotypes might be a consequence of a requirement for ft and ds in the transduction of a global patterning signal. To remove any effects of long-range signaling, mutant eyes were generated completely devoid of wild-type Ft using the EGUF system (Stowers, 1999). In contrast to ft clones, in EGUF-ft eyes, the D/V axis is so severely perturbed that the endogenous equator is abolished. The greater degree of disruption observed in EGUF-ft eyes compared to mosaic clones suggests that wild-type tissue communicates with mutant tissue, perhaps via cell-cell relay of the signal transduced by Ft. If this is the case, then the presence of the inverted equator within the mutant clones is established as a consequence of signaling from wild-type tissue (Rawls, 2002).

The EGUF-ds phenotype mimics the clonal ds phenotype: 31% of ommatidia display D/V inversions. In EGUF-ds eyes, the endogenous equator is evident. This may be due to the weaker nature of this allele. Alternatively, it may suggest that Ds plays a more modulating role in establishing the D/V midline than does Ft (Rawls, 2002).

Ft and Ds act nonautonomously in the eye. In ft clones, the majority of polarity defects occur in the polar region of the clone. In contrast, ommatidia in the equatorial region of the clone are phenotypically wild-type, suggesting that wild-type ommatidia outside the equatorial boundary of the clone rescue genetically mutant ommatidia within the equatorial region of the clone. Furthermore, wild-type tissue on the polar border of the clone does not rescue mutant ommatidia within the polar region of the clone, indicating rescue takes place only in an equatorial to polar direction and not from the poles to the equator. If this is the case, the Ft signal is propagated in a directional fashion from wild-type tissue at the equatorial border into the mutant clone. Finally, mutant tissue never nonautonomously affects wild-type tissue -- in over 200 clones analyzed, no inverted ommatidia were seen in which all eight photoreceptors were wild-type. In contrast to ft, rescue takes place in a polar to equatorial direction in ds clones (Rawls, 2002).

The tissue polarity genes fz and stbm are required to specify R3 and R4. These cells then regulate the direction of ommatidial rotation. Given the importance of these two cells in the establishment of polarity, ommatidia were examined that were mosaic for ft within the R3 and R4 pair. In the majority of cases, the Ft⁺ cell becomes R3. A similar analysis of the other developmental pairs of photoreceptors, R1/R6 and R2/R5, revealed that there is a strong tendency for the Ft⁺ cell to adopt the fate of the anterior (R1 and R2) rather than the posterior (R5 and R6) photoreceptor cell (Rawls, 2002).

There are two possible interpretations of these data. First, ft may be involved in specifying the anterior photoreceptor fates, as the data imply. However, if Ft is required to specify the R3 fate, at least a small fraction of ommatidia should be seen in which no R3 fate is specified in ft mutant tissue (i.e., ommatidia that have two R4s), as is the case in fz mutants, this phenotype is not seen in ft clones. Alternatively, this finding could reflect the link between how cells are recruited into the growing ommatidium, how ommatidia rotate to establish polarity, and how this process is disrupted in ft mutant ommatidia (Rawls, 2002).

In doing this mosaic analysis, it was essential to recognize that a property inherent to eye development is that ommatidia that arise at the polar border of a clone predominantly recruit their polar cells from wild-type tissue and their equatorial cells from mutant tissue. Phenotypically mutant ommatidia occur only in the polar region of ft clones. This phenotype complicates the analysis and makes it difficult to draw conclusions regarding the specific cell(s) in which Ft is required for cell fate (Rawls, 2002).

In wild-type clones, the cells that are recruited from the polar side of the clone (R4, R5, and R6) will face the posterior side of the clone at the end of rotation. In ft clones, ommatidia that are both phenotypically mutant and mosaic occur only at the polar boundary of the clone. Since these ommatidia are almost always D/V inversions, then they will have recruited their ft⁺ cells from the polar side of the clone, but rather than these polar-derived cells becoming posterior photoreceptors (R4, R5, and R6) as they would have in wild-type, they become anterior photoreceptors (R1, R2, and R3). Together, these factors create an artifactual bias in which the anterior photoreceptor cells are ft⁺ and the posterior cells are ft^-. Consequently, even though the data appear to indicate ft is required for the anterior cell fates (R1, R2, R3), a more detailed analysis reveals that the nature of the ft phenotype introduces a developmental bias that must be considered (Rawls, 2002).

The mosaic analysis of ds mutant clones revealed a trend the opposite of that described for ft. In ommatidia mosaic for ds in the pairs R1/R6, R2/R5, and R3/R4, the majority of photoreceptors are wild-type for the posterior fates (R4, R5, and R6). Since D/V inversions are found on the opposite side of the clone in ds compared to ft, this is the expected result if one applies the same logic as described above for ft. As with ft, no functional autonomy can be assigned to a single cell (Rawls, 2002).

A contrasting interpretation of a mosaic analysis of ft and ds, presented by Yang (2002), suggests ft and ds are required to specify the fates of photoreceptors R3 and R4, respectively. However, mosaic analyses of ft and ds are inherently biased due to the clonal phenotypes, as described above. This bias might mask a role for ft or ds in the R3/R4 fate decision, but currently there is no compelling evidence for such a functional requirement. The genetic data reported here and in Yang (2002) are insufficient to draw conclusions about the role of ft and ds in fate specification. Extensive experimentation and a better understanding of mechanism are necessary to discriminate between a role for ft in global D/V signaling versus a requirement for ft and ds in specification of the R3 and R4 cell fates (Rawls, 2002).

Early acting genes, for example, mirror (mirr) and fringe (fng), specify the dorsal and ventral halves of the eye, respectively, thereby setting up the D/V boundary. The ft clonal phenotype suggests ft might mediate D/V boundary formation. To address this possibility, the effect of ft on mirr expression was assessed. The expression pattern of the enhancer trap line 8A5, in which the mirr promoter drives expression of white, is unaffected in EGUF-ft eyes. In these eyes, mirr expression remains restricted to the dorsal half of the eye, indicating that ft is required either downstream of or in parallel to mirr (Rawls, 2002).

A proposed model for ft activity differs from that of Yang (2002) because this study (1) takes into account the observations that ectopic equators are generated in mutant clones and that the ft clonal phenotype differs significantly from the EGUF-ft phenotype and (2) considers fundamental properties of eye development in conjunction with the clonal phenotype. A model is proposed in which ft conveys D/V positional information to developing ommatidia to create the D/V midline (Rawls, 2002).

Ft functions to inhibit D/V signaling in the wing and haltere. The data are consistent with this proposal -- new equators are generated in ft mutant clones in the eye. It is proposed that a consistent level of Ft activity throughout the eye inhibits the D/V signaling required to form the equator. At the equator, Ft activity must be inhibited. The molecule that inhibits Ft could be expressed in several ommatidial rows encompassing the future midline. In a ft mutant clone, the phenotype is rescued for two rows in the equatorial region of the clone, suggesting the D/V signal propagated by Ft can be relayed for a distance of two ommatidial rows. Therefore, it is proposed that in the presence of the regulatory protein, the D/V signal can also be relayed an equivalent distance at the endogenous equator. When the D/V signal reaches its minimum, tissue with Ft activity apposes tissue without Ft activity, and it is at this point that the equator is established (Rawls, 2002).

Nonautonomous planar polarity patterning in Drosophila: Dishevelled-independent functions of Frizzled

The frizzled (fz) gene of Drosophila is required for planar polarity establishment in the adult cuticle, acting both cell autonomously and nonautonomously. These two activities of fz in planar polarity are temporally separable in both the eye and wing. The nonautonomous function is dishevelled (dsh) independent, and its loss results in polarity phenotypes that resemble those seen for mutations in dachsous (ds). Genetic interactions and epistasis analysis suggest that fz, ds, and fat (ft) act together in the long-range propagation of polarity signals in the eye and wing. Evidence has been found that polarity information may be propagated by modulation of the binding affinities of the cadherins encoded by the ds and ft loci (Strutt, 2002).

In the wild-type wing, each cell produces a single trichome at its distal vertex that points distally. Flies that lack fz function exhibit defects in trichome polarity. Most cells still produce a single trichome, but these are arranged in a distinctive swirling pattern known as the fz/in-like pattern. Furthermore, in the pupal wing, there is no asymmetric localization of the polarity proteins Fz, Dsh, Fmi, Pk, and Dgo and the majority of trichomes form in the cell center. An almost identical phenotype is caused by mutations that remove only the cell-autonomous functions of fz or mutations in the cell autonomously-acting polarity genes dsh and fmi. This indicates that this phenotype is likely to be solely the result of removing cell-autonomous polarity gene function (Strutt, 2002).

In contrast, ds is thought to be required only for the nonautonomous transmission of polarity information. Wings from ds individuals show a trichome swirling pattern, which is distinct from the fz/in pattern. Asymmetric polarity protein localization still occurs—albeit often in an aberrant pattern Similarly, cells adjacent to a fz clone, which have aberrant polarity because of the nonautonomous phenotype of fz, also asymmetrically localize polarity proteins (Strutt, 2002).

On the basis of these observations, it seems likely that the loss of fz function from the entire wing results in both cell-autonomous and -nonautonomous phenotypes, but that the former masks the latter. To study the phenotype caused by loss of the nonautonomous component only, a method of rescuing only the cell-autonomous phenotype must be devised. It was hypothesized that the nonautonomous function of fz might precede the cell-autonomous function. If this is correct, it should be possible to rescue only the cell-autonomous function of fz by adding back fz activity to a fz mutant fly at a stage of development after the nonautonomous requirement, but before the autonomous requirement. To test this, a transgene was used consisting of the Actin5C promoter separated from the fz coding sequence by an FRT-flanked transcription termination sequence. The fz coding sequence is fused to the coding sequence of GFP, to permit monitoring of fz expression, giving rise to the transgene Actin>stop>Fz-GFP. Expression of Fz-GFP from this transgene can be induced by heat-shocking flies that also carry a transgene containing yeast FLP recombinase under control of the hsp70 promoter (Strutt, 2002).

This expression system was used to rescue the phenotype of strong fz mutations that lack both cell-autonomous and -nonautonomous activity. Activation of Fz-GFP expression at time points up to about 6 hr after prepupa formation (APF) results in a wild-type trichome polarity pattern. However, activation of Fz-GFP expression at about 6 hr APF results in a weak polarity defect in some parts of the wing. Successively later activation results in an increasingly strong trichome polarity phenotype. Remarkably, this trichome polarity phenotype appears more ds-like than fz/in-like, in particular, showing complete inversions of trichome polarity in some proximal regions of the wing. Activation of Fz-GFP expression at 16 hr APF produces a pattern most similar to that seen in strong ds mutants. Activation at 24 hr APF gives a phenotype that is interpreted as a stronger form of the reported ds-like pattern (and that is stronger than the fz/in pattern). Notably, the characteristic wing shape and vein phenotypes seen in ds mutations are not observed, suggesting that the effects of ds on trichome polarity and wing morphology could be separable functions (Strutt, 2002).

Activation of Fz-GFP expression at later time points results in no increase in strength of the ds-like polarity phenotype. Instead, after 27 hr APF, progressively weaker phenotypes are seen that resemble a mixture of the ds-like and fz/in-like patterns, until about 31 hr APF, when the adult polarity pattern is typically fz/in-like and is identical to that produced in the absence of Fz-GFP expression (Strutt, 2002).

The effects of expression of Fz-GFP at different time points were also examined in the pupal wing at the time of trichome initiation (about 32 hr APF), monitoring Fz-GFP localization and trichome formation. Activation of Fz-GFP expression up to about 24 hr APF results in asymmetric Fz-GFP localization in an aberrant pattern and trichome formation at the corresponding cell edge. Activation at time points from 24 hr APF onward results in reduced asymmetric protein localization and an increase in the number of trichomes initiating away from the cell edge, until, by 30 hr APF, little Fz-GFP expression is seen at the time of trichome initiation, and trichomes are forming at the cell center (Strutt, 2002).

Thus, the temporal requirement for fz shows two phases. Activation of Fz-GFP expression prior to 6 hr APF elicits complete rescue of fz functions. Between 6 and 24 hr APF, lack of fz activity results in a ds-like polarity pattern (which gets progressively stronger, the longer fz activity is not present), but asymmetric polarity protein complexes still form, and trichomes emerge at the corresponding sites at the cell periphery. Between 24 and 31 hr APF, loss of fz activity results in a progressively more dominant fz/in-like trichome polarity pattern, failure to form asymmetric polarity protein complexes, and formation of trichomes in the cell center (Strutt, 2002).

Consistent with the hypothesis that the early phase of fz activity is required for nonautonomous polarity signaling, expression of Fz-GFP at 12 hr APF largely rescues the phenotype of an fz allele that is classified as lacking only cell-autonomous activity. At first sight, it is surprising that such an allele is not completely rescued. However, it has recently been reported that such alleles are also partially deficient in nonautonomous signaling activity (Strutt, 2002).

The results show that there are two temporally separable activities of fz. The later corresponds to the well-characterized fz cell-autonomous function. The early fz activity, the loss of which produces a more ds-like phenotype, is the fz nonautonomous pathway. Notably, this early activity is required over a significant period of time (16-18 hr in the eye and wing). In addition, this nonautonomous activity is dsh independent in the eye (Strutt, 2002).

A number of conclusions follow from these observations: (1) fz exhibits a similar dsh-independent nonautonomous activity in two different tissues (eye and wing), and, therefore, this is likely to be a conserved pathway; (2) the similarity of the fz nonautonomous phenotype to the ds phenotype and genetic interactions between these loci suggests that these molecules might cooperate in a common mechanism to propagate polarity signals; (3) since the nonautonomous activity of fz precedes the autonomous activity, the former is apparently not dependent on the latter. This supports models of polarity patterning, in which a long-range signal is propagated through the tissue prior to the cell-autonomous response to that signal (Strutt, 2002).

It is noted that, although these two activities of fz are temporally separable, during normal development it is likely that the period of nonautonomous activity nevertheless overlaps the beginning of autonomous polarity functions (Strutt, 2002).

In the eye, the clonal phenotypes of long-range patterning factors, such as the canonical Wnt pathway, JAK/STAT, and fj, have led to models in which they act to establish an activity gradient of a polarity signal (the 'secondary signal'). There are a number of reasons for supposing that these three pathways perform distinct functions that differ from that of the fz nonautonomous activity and that fz is likely to act downstream of these other factors: (1) gradients of components of all three of these pathways are apparent in the second instar stage of development, whereas fz nonautonomous activity in the eye is required over a period of up to 16 hr in the third instar; (2) fz nonautonomous activity is epistatic to (functions downstream of) dsh nonautonomous activity, and there are no genetic interactions between fz nonautonomous activity and canonical Wnt, JAK/STAT, or fj activities; (3) all three activities show similar nonautonomous clonal phenotypes with normal ommatidial polarities in the center of clones, but fz exhibits partial randomization of ommatidial polarities inside the clones (Strutt, 2002).

Conversely, there are a number of reasons for thinking that fz nonautonomous activity in the eye is closely related to ds and ft function. The phenotypes of clones lacking early fz function are similar to those of ds clones and ft clones. Furthermore, there are strong genetic interactions between these factors. Finally, an epistasis test between the clonal phenotypes of fz and ds gives an apparently additive (or possibly synergistic) phenotype. These results are consistent with fz acting jointly with ds and ft in the nonautonomous propagation of polarity information. A similar function for ds has been suggested on the basis of studies in the wing, it having been shown that ds nonautonomously affects trichome polarity and that it is likely to be involved in the maintenance or propagation of an fz-dependent nonautonomous polarity signal (Strutt, 2002).

Thus, overall data from both the eye and wing support fj acting upstream of ds and ft, which then act jointly with fz nonautonomous function in the long-range propagation of polarity information. Uncharacterized mechanisms of intercellular signaling then lead to autonomous activation of fz and assembly of asymmetric polarity protein complexes. Note is taken of the contrast with the recent suggestion that ds and ft act directly upstream of the autonomous function of fz (Strutt, 2002).

Other factors or mechanisms must also be involved in nonautonomous propagation of polarity information, in order to explain all of the observations. For instance, complete loss of fj function does not result in a loss of polarity patterning in the wing, indicating that there must be other upstream patterning factors. Furthermore, clones of fj and ft give stronger nonautononomous phenotypes in a central portion of the wing, whereas ds and fz seem to give rather similar phenotypes throughout. This suggests that there are other modulators of pathway activity that have region-specific effects (Strutt, 2002).

Groups of cells lacking fj function tend to round up into tight foci, appearing to have greater affinity for each other than for their fj-expressing neighbors. Furthermore, in mutant cells abutting fj-expressing neighbors, the cadherins Ds and Ft are preferentially found at the cell junctions touching fj⁺ cells. These observations support the notion that one role of fj in wing patterning is to alter the adhesive properties of cells and also of the cadherins Ft and Ds. It is also noteworthy that loss of ft activity results in Ds no longer being tightly localized in the apical junctional zone of cells and that, similarly, loss of ds seems to result in reduction of apical Ft localization (Strutt, 2002).

It is speculated that a gradient of fj activity in the wing might lead to graded Ds/Ft activity and, hence, cell adhesion. Such a gradient of cell adhesion constitutes a possible mechanism for the long-range transmission of polarity information, although direct evidence for this is lacking. It is noteworthy that fj, ft, and ds mutations also all result in truncations of the wing on the proximodistal axis, and it is possible that this phenotype is in some way due to effects on cell adhesion (Strutt, 2002).

Interestingly, the effect of fj clones on Ds/Ft is cell autonomous. It was suggested that, on the basis of its amino acid sequence and in vitro studies, fj encodes a secreted factor and that this property could explain its nonautonomous phenotypes. These results indicate that at least some functions of fj are cell autonomous (Strutt, 2002).

fourjointed interacts genetically with ft and ds in planar polarity and proximodistal patterning

four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localized to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. Cleavage and secretion of Fj is shown to not be necessary for activity, and Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). fj is shown to interact genetically with ft and ds in planar polarity and proximodistal patterning. It is proposed that Fj may act in the Golgi to regulate the activity of Ft and Ds (Stutt, 2004).

In Drosophila, the atypical cadherins Ft and Ds are good candidates for being the ultimate targets of fj activity. They are required for both planar polarity and PD patterning, and have similar mutant phenotypes to fj. In addition, fj interacts genetically with ds and ft in both planar polarity and PD patterning. Interestingly, ds fj double mutants have surprisingly strong phenotypes, which were qualitatively different to those of the single mutants, including duplications or transformations of limb structures. However, no such phenotypes are seen in any of the double mutant combinations, suggesting that the duplications/transformations may be specific to the combination of chromosomes used in classical experiments. The current results instead show that mutations in fj enhance the phenotypes of both ft and ds hypomorphic mutations, suggesting that these genes act in a common pathway (Stutt, 2004).

Epistasis experiments further demonstrate that ds is required to mediate fj function, and therefore ds acts downstream of fj; this is in agreement with data based on clonal analysis of ds and fj. Interestingly, recent experiments have also revealed a role for fj in regulating the intracellular distribution of Ds and Ft. In wild-type tissue, Ds and Ft colocalize at apicolateral membranes, and their localization is mutually dependent. Inside fj mutant clones, Ds and Ft localization is largely unaltered. However, in the row of mutant cells immediately adjacent to wild-type tissue, Ft and Ds preferentially accumulate on the boundary between fj⁺/fj^- cells. In addition, cells inside the fj clones appear to be 'rounded-up', suggesting that they prefer to adhere to each other rather than to non-mutant cells. Thus, it is thought that fj modulates the activity and intermolecular binding properties of Ft and Ds (Stutt, 2004).

An interesting point to note is that both ds and ft show planar polarity phenotypes as homozygotes, whereas fj only shows polarity phenotypes on the boundaries of mutant clones. The fj phenotypes have been explained by models in which fj acts redundantly to regulate the production of a gradient, the direction of which determines polarity. Thus, in homozygotes the direction of the gradient is unchanged, and animals show no major defects; but at clone boundaries there is a discontinuity in the direction of the gradient, leading to inversions of polarity. This model can be extended to suppose that Fj may modulate Ds/Ft activity, but that it does not act as a simple on-off switch; rather Ds/Ft retain some activity even when Fj is not present (Stutt, 2004).

In the absence of a known enzymatic function for Fj, the mechanism by which it might modulate Ft and Ds activity remains uncertain. It is speculated that since Fj acts intracellularly, it is possible that it promotes or mediates the post-translational modification of Ds and/or Ft proteins, and that these molecules mediate the non-autonomous signalling functions of Fj. However, the large size of the Ft and Ds gene products (5147 and 3380 amino acids, respectively) renders the analysis of their post-translational modification highly challenging (Stutt, 2004).

Interactions between Fat and Dachsous and the regulation of planar cell polarity in the Drosophila wing

It has been suggested that a proximal to distal gradient of the protocadherin Dachsous (Ds) acts as a cue for planar cell polarity (PCP) in the Drosophila wing, orienting cell-cell interactions by inhibiting the activity of the protocadherin Fat (Ft). This Ft-Ds signaling model is based on mutant loss-of-function phenotypes, leaving open the question of whether Ds is instructive or permissive for PCP. Tools have been developed for misexpressing ds and ft in vitro and in vivo, and these have been used to test aspects of the model. (1) This model predicts that Ds and Ft can bind. Ft and Ds are shown to mediate preferentially heterophilic cell adhesion in vitro, and each stabilizes the other on the cell surface. (2) The model predicts that artificial gradients of Ds are sufficient to reorient PCP in the wing; the data confirms this prediction. (3) Loss-of-function phenotypes suggest that the gradient of ds expression is necessary for correct PCP throughout the wing. Surprisingly, this is not the case. Uniform levels of ds drive normally oriented PCP and, in all but the most proximal regions of the wing, uniform ds rescues the ds mutant PCP phenotype. Nor are distal PCP defects increased by the loss of spatial information from the distally expressed four-jointed (fj) gene, which encodes putative modulator of Ft-Ds signaling. Thus, while the results support the existence of Ft-Ds binding and show that it is sufficient to alter PCP, ds expression is permissive or redundant with other PCP cues in much of the wing (Matakatsu, 2004).

Several gain-of-function findings are consistent with previous loss-of-function findings, and support the model that Ft-Ds signaling is sufficient to influence wing PCP. Ft and Ds preferentially bind in vitro. Patterned misexpression of ds is sufficient to alter wing PCP, consistent with its proposed role as a ligand. The effects of ft or ds misexpression on the direction of hair polarization are usually the opposite of those previously reported from ft or ds loss of function. The direction of hair polarity induced by ectopic ft is usually the opposite of that induced by ectopic ds, consistent with the proposal that Ds binding inhibits Ft activity. Finally, the effects of Ft misexpression are reduced in a ds mutant background, consistent with the proposed role of Ft as a receptor (Matakatsu, 2004),

Nonetheless, the data also show that the proximal to distal gradient of ds expression is not necessary for PCP throughout the wing, despite the distal defects observed in loss-of-function ds mutants. Instead, the experiments show that uniform ds misexpression can rescue the PCP defects caused by a ds mutation in all but the most proximal portions of the wing. Thus, ds is permissive for PCP in most of the wing, and there must be another polarity cue in the distal wing that is sufficient to orient PCP in the presence of uniformly transcribed ds. The experiments indicate that this distal cue is not provided by the distally expressed Fj protein: distal PCP is not disrupted either by uniform misexpression of both ds and fj, or by uniform misexpression of ds in a fj null mutant (Matakatsu, 2004),

It remains possible that the distal cue functions by regulating Ft-Ds signaling. These studies tested the PCP inputs from the patterns of ds and fj transcription, but unknown factors might post-transcriptionally regulate the forms of Ds or Ft protein produced, or their availability at the cell surface. It also is possible that Ft activity is spatially regulated by binding partners other than Ds. ft mutants have stronger PCP and disc overgrowth defects than do ds mutants, and misexpression of ft still causes PCP defects in a ds mutant lacking detectable cell surface protein (Matakatsu, 2004),

Alternatively, the cue may be provided by a mechanism that is completely independent of Ft or Ds. One often-proposed candidate is signaling via the Drosophila Wnts, especially given their patterned (distal or marginal) expression. However, although the misexpression of Drosophila wnt4 can disrupt wing PCP, PCP defects have not been reported in Drosophila Wnt mutants (Matakatsu, 2004),

Although a ds gradient is not required for PCP in most of the wing, it is possible that such a gradient is required locally in the portion of the wing near and proximal to the anterior cross vein. Proximal ds mutant PCP defects could not be rescued with uniform Ds expression, and the data suggest that this is not simply a failure caused by insufficient Ds levels. Thus, the view is favored that this sharp Ds gradient acts as a PCP cue in the proximal wing. If so, this indicates that the cues that orient PCP in the wing are not generally distributed; rather, the wing may be a patchwork of different regions that rely on different cues. This would provide a mechanism for locally altering PCP during evolution without globally affecting polarity in the wing (Matakatsu, 2004),

The hypothesis that Ft acts as a receptor and Ds acts as a ligand for PCP is based, not only on the uniform expression pattern Ft, but also on epistasis experiments in the eye, where the PCP activity of ds clones appears to depend on the presence of ft. Wing PCP can also be disrupted by the expression of a truncated form of Ds lacking its intracellular domain, which is consistent with Ds acting as a ligand (Matakatsu, 2004),

However, this study has shown that misexpressed Ft retains PCP activity in a ds mutant that eliminates detectable cell surface Ds. Thus, Ft activity is apparently not strictly dependent on patterned Ds expression. Again, this is consistent with the greater severity of ft mutant phenotypes compared with ds, and with the finding that uniform misexpression of ft but not ds can cause PCP defects. Since there is no evidence for homophilic Ft binding, the unbound Ft molecule may have basal PCP activity. Alternatively, low-level homophilic binding or heterophilic binding to some unknown ligand may activate Ft in the absence of Ds (Matakatsu, 2004),

It is not yet known how Ft-Ds interactions regulate the polarized redistribution of the core polarity proteins in the older pupal wing. The cytoplasmic domains of Ft and Ds contain potential regions for ß-catenin binding, and ft and ds mutants can enhance the effects of ß-catenin (Armadillo) misexpression. However, although expression of DE-cadherin in vitro results in a detectable concentration of Armadillo at the cell membrane, no similar effects were detected after expression of ft or ds. Moreover, clones homozygous for a strong armadillo mutation do not affect PCP. It has also been suggested that the cytoplasmic domain of Ft binds to and changes the activity of Grunge, the Drosophila homolog of the Atrophin transcriptional co-repressor, but it is not known whether this interaction is altered by Ft-Ds binding (Matakatsu, 2004),

The studies examining the timing of Ds activity suggest that its effects on the polarization of the core polarity proteins are likely to be indirect, since Ds acts before the polarized redistribution of the core polarity proteins within cells can be detected. Patterned misexpression of Ds at later stages, during the time of core protein polarization, has no effect on PCP. The period sensitive to ds misexpression is roughly congruent with the period of early Fz activity; if loss of Fz is limited to a period from 6 to 24 hours AP it leads to distinct, ds-like PCP defects. Thus, early Fz and Ds activity may be linked, or they may share a common target (Matakatsu, 2004),

The only known sign of cell polarization during the stages sensitive to Ds and early Fz activity is the redistribution of the Widerborst PP2A regulatory subunit from the anterior-proximal side to the distal side of wing cells at some time between 8 and 18 hours AP. Reductions in Widerborst activity can disrupt the polarized redistribution of Fmi and Dsh, suggesting an instructive role. However, Widerborst polarization is not affected by ectopic Fz expression, making it less likely that Widerborst polarization mediates early Fz activity (Matakatsu, 2004 and references therein),

A final interesting feature of the results is the preferentially heterophilic binding observed between Ft and Ds in vitro. This result is consistent with analyses of protein distribution within and adjacent to ft and ds mutant and overexpression clones. With the exception of the desmosomal cadherins, this kind of binding is unusual for cadherin-like proteins (Matakatsu, 2004),

A number of mammalian Fat-like (Fat1, Fat2, Fat3, XP_227060) and Ds-like (Protocadherin 16, Cdh23) proteins have been identified. Mutations and knockouts have been examined for a few of these; however, conjectures about the bases of the mutant phenotypes have largely assumed that these proteins mediate homophilic cell adhesion. It will be interesting to see whether the preferentially heterophilic interactions observed in Drosophila are preserved in similar mammalian proteins (Matakatsu, 2004),

The orientation of cell divisions determines the shape of Drosophila organs: Mutations in the Drosophila PCP genes dachsous (ds) and fat (ft) cause changes in the shape of different organs

Organ shape depends on the coordination between cell proliferation and the spatial arrangement of cells during development. Much is known about the mechanisms that regulate cell proliferation, but the processes by which the cells are distributed in an orderly manner remain unknown. This can be accomplished either by random division of cells that later migrate locally to new positions (cell allocation) or through polarized cell division (oriented cell division; OCD). Recent data suggest that the OCD is involved in some morphogenetic processes such as vertebrate gastrulation, neural tube closure, and growth of shoot apex in plants; however, little is known about the contribution of OCD during organogenesis. The orientation patterns of cell division was examined throughout the development of wild-type and mutant imaginal discs of Drosophila. The results show a causal relationship between the orientation of cell divisions in the imaginal disc and the adult morphology of the corresponding organs, indicating a key role of OCD in organ-shape definition. In addition, a subset of planar cell polarity genes was found to be required for the proper orientation of cell division during organ development (Baena-López, 2005).

Drosophila imaginal discs are a classical model system for studying general mechanisms involved in the control of organ growth and patterning. The imaginal discs are epithelial structures that originate from the embryonic ectoderm, and, after a period of cell proliferation during the larval stages, give rise to most adult organs. The wing disc is divided into lineage units known as compartments. The boundaries between compartments play a key role in the control of wing disc growth and patterning. Analysis of mitotic recombination clones in animals and plants allows for tracing the descendants of single marked cells during development. These experiments have shown a clear correlation between the shape of the clones and adult morphology of the organ where the clones are studied, i.e., clone growth defines organ shape. Most clones in the wing blade are very elongated and grow along the proximal-distal (P/D) axis of the wing, perpendicular to the D/V border. In contrast, clones within the wing margin grow along the D/V border. This study has considered the dorsal-ventral (D/V) boundary as a reference to measure the orientation of cell divisions during wing disc development. A striking relationship was observed between the shape of the clones and the orientation of cell divisions. Thus, the majority of cells divide along the proximal-distal (P/D) axis of the wing blade; 59.4% (n = 549) of mitoses form angles higher than 55° with respect to the D/V boundary, while only 13.8% are lower than 35°. In contrast, in the wing margin, most cells divide nearly parallel to the D/V boundary, forming angles lower than 35° (71.4%; n = 70). Furthermore, the characteristic shape of each intervein region is also reflected in the cell-orientation patterns. Thus, 65.8% (n = 116) of the mitotic figures studied in intervein regions C and D, where clones are very elongated and grow perpendicular to the wing margin, are nearly perpendicular to the D/V border, whereas in regions A and E, where clones are wider and grow parallel to the D/V border, only 50.3% of mitoses (n = 81) have this orientation (Baena-López, 2005).

Early-induced clones also show an elongated shape along the P/D axis of the wing. Accordingly, most of the cell divisions in the second instar wing discs appear with a P/D orientation. In everted wings in pupae, most cells also divide preferentially along the P/D axis, indicating that the correlation between the orientation of cell divisions and the shape of the clones is maintained throughout development (Baena-López, 2005).

Interestingly, the orientation of postmitotic daughter cells, analyzed in clones of two cells, conserves the positions determined by the angle of the OCD. The orientation of the first cell division tends to be maintained in subsequent divisions, since it is observed that 57% (n = 121) of clones of four cells form straight lines of one cell width. Although these results suggest that the cell relocation plays a minor role to define the clone shape, it cannot be ruled out that this process might refine clone shapes and therefore organ shape. Finally, it is suggested that the width of the clones mainly depends on the general probability of OCD (Baena-López, 2005).

To evaluate the general requirement of OCD in organogenesis, the patterns of mitotic orientation were examined in other wing disc regions. The thorax shows an isodiametric morphology, and mitotic recombination clones grow isodiametrically. Accordingly, no preferential orientation of the planar axes of cell divisions was found. In the peripodial epithelium, it was also observed that the orientation of cell divisions define the characteristic shape of the clones. As in the wing blade, postmitotic cells show the same orientation of previous cell divisions (Baena-López, 2005).

The general requirement of the OCD in the definition of wing disc morphology led to a study of the contribution of this process to the development of other organs. The pattern of cell-division orientations was examined during eye disc development. The dorsoventral midline of the Drosophila eye is known as the equator and defines a line of mirror-image symmetry, with ommatidia on each side having opposite chirality. This clonal boundary also plays an important role in the patterning and growth of the eye. Clones in the eye grow symmetrically oblique with respect to the equator. During eye development, an indentation known as the morphogenetic furrow (MF) marks the front of a wave of differentiation that sweeps from posterior to anterior across the disc. The orientation of cell divisions was monitored with respect to the MF in the region anterior to the furrow. The orientation of mitotic spindles in most mitotic figures in the ventral half of the eye discs form angles between 40° and 60°, whereas in the dorsal half, they form angles between 120° and 140°. In the eye disc, the postmitotic cell allocation again reflects the orientation of mitosis. Attempts were made to measure the orientation of cell divisions in a tube-shaped organ like the leg, but the highly folded epithelial organization of these discs prevented the reaching of any clear conclusion. However, clones in the adult legs appear narrow, running proximodistally over several joints. The results indicate that the OCD may be a general mechanism to generate shape during organogenesis (Baena-López, 2005).

One interesting group of genes involved in the orientation of cell divisions during Zebrafish gastrulation and growth of shoot apex in Arabidopsis is composed of planar cell polarity (PCP) genes. The function of these genes is evolutionary conserved to define the polarity and positional information of the cells within an epithelium. Mutations in the Drosophila PCP genes dachsous (ds) and fat (ft) cause changes in the shape of different organs: wings and eyes are rounder than the wild-type, whereas legs are wider and shorter. The activity of these genes is required upstream of other PCP genes that do not affect adult organ shape such as the core PCP genes strabismus (stbm), prickled (pk), and flamingo (fmi) or the effector PCP gene multiple wing hair (mwh). Whether the abnormal organ shapes, observed in ds and ft mutant backgrounds, are associated with changes in the clone shape and the orientation of cell division was analyzed (Baena-López, 2005).

Mitotic recombination clones in ds mutant wing discs show rounded shapes, losing the elongated shape of wild-type clones. Interestingly, loss-of-function clones for ds or ft fail to adopt their typical enlarged shape in the wing blade or in the eye, showing a rounder shape than wild-type clones. In contrast to control wing discs and wild-type clones, it was observed that the orientation of cell divisions is randomized in ds mutant cells (wing discs or ds mutant clones). Accordingly, postmitotic cells do not show any preferential orientation. The rounded shape and disturbed OCD is also observed in clones of ds- or ft-expressing cells and in adult wings where ds or ft is ectopically expressed using the UAS/G4 system. Similar results are observed in the eye imaginal discs. These results suggest that the polarization cues mediated by PCP genes are required for the control of the orientation of cell division and, therefore, are involved in organ shape definition. These findings show that some of the genes required for PCP are acting in early stages of the development, suggesting that the base for PCP may be established during larval development. The conserved function of PCP genes controlling OCD in different organisms suggests that this process might be an evolutionary mechanism in organ shape definition. Although the mechanistic details underlying the new functions of PCP genes are unknown, it is likely that the asymmetric cell distribution of a classical core of PCP proteins is not required. Finally, similar phenotypes were observed in both the lack and excess of function for ds and ft, suggesting that the default organ shape is circular when the cell polarization is disrupted (Baena-López, 2005).

To conclude, it is proposed that the shape of organs can be accounted for by the oriented pattern of cell divisions rather than postmitotic relocation of proliferating cells. However, how the preferential orientation is topologically determined is poorly understood. It is speculated that the orientation of cell divisions in different territories during development may result from the integration of signals coming from restriction borders and territorial local cell interactions. The existence of clusters of cells, synchronized in the same stage of the cell cycle, support the idea of a local control of cell proliferation. It has been found that the orientations of cell divisions in these clusters tend to be highly aligned, suggesting also a local control of OCD superimposed to more general and long-range signals. Although the signals exchanged between neighboring cells to determine local OCD are still unknown, it is likely that a subset of PCP genes could be involved in this process (Baena-López, 2005).

Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins

The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including controlling growth of imaginal discs, planar cell polarity (PCP) and the proximodistal patterning of appendages. Ft and Ds bind in a preferentially heterophilic fashion, and Ds is expressed in distinct patterns along the axes of polarity. It has thus been suggested that Ft and Ds serve not as adhesion molecules, but as receptor and ligand in a poorly understood signaling pathway. To test this hypothesis, a structure-function analysis of Ft and Ds was performed, separating their adhesive and signaling functions. It was found that the extracellular domain of Ft is not required for its activity in growth, PCP and proximodistal patterning. Thus, ligand binding is not necessary for Ft activity. By contrast, the extracellular domain of Ds is necessary and sufficient to mediate its effects on PCP, consistent with the model that Ds acts as a ligand during PCP. However, evidence is also provided that Ds can regulate growth independently of Ft, and that the intracellular domain of Ds can affect proximodistal patterning, both suggestive of functions independent of binding Ft. Finally, it is shown that ft mutants or a dominant-negative Ft construct can affect disc growth without changes in the expression of wingless and Wingless target genes (Matakatsu, 2006).

Chief amongst the findings of this study is that Ft activity is not simply a byproduct of changes in cell-cell adhesion. The FtΔECD construct lacks almost the entire extracellular domain and cannot bind or stabilize Ds in vitro or in vivo. Nonetheless, it can rescue the lethality, overgrowth and PCP defects of ft alleles that should be null for any adhesive or receptor function, and in a wild-type background can disrupt proximodistal patterning. This suggests that the intracellular domain of Ft can act in the absence of binding between endogenous Ft and Ds, or indeed between Ft and any other extracellular ligand, as long as sufficient levels are expressed (Matakatsu, 2006).

Conversely, it was found that a form of Ft lacking the intracellular domain (FtΔICD) failed to rescue overgrowth in ft mutants. In fact, this form acts as a strong dominant negative, inducing overgrowth of wild-type and ft mutant imaginal discs. This occurs despite the ability of FtΔICD to stabilize endogenous cell surface Ds and Ft, raising the possibility that FtΔICD binds to Ds and Ft is a way that blocks their activities. The possibility that FtΔICD alters the activity of some additional, unknown player cannot be ruled out. Although lethality prevents determining whether FtΔICD can rescue ft mutant PCP defects, expression of FtΔICD in wild-type wings also disrupts PCP. These PCP defects are weaker than those observed in ft mutants, suggesting that FtΔICD might have stronger effects on growth control than PCP (Matakatsu, 2006).

In contrast to Ft, the extracellular domain of Ds is sufficient for its effects on PCP. The DsΔICD construct lacks almost the entire intracellular domain, but nonetheless can rescue the PCP defects of strong ds mutants and disrupt PCP in wild-type wings. The DsΔECD construct, however, cannot bind or stabilize Ft and cannot rescue ds mutant PCP defects or influence PCP in wild-type wings. The results thus support the hypothesis that in PCP Ds acts chiefly as a ligand for Ft, modulating its activity (Matakatsu, 2006).

Nonetheless, the possibility that the intracellular domain of Ds has some PCP activity within the context of the whole protein cannot be ruled out, and the conservation of large regions of the Ds intracellular domain in its vertebrate homologs dachsous 1 and dachsous 2 suggests that Ds may have activity beyond that of a ligand. Thus, it is intriguing that expression of DsΔECD can disrupt another ds-sensitive phenotype, crossvein spacing in wild-type wings. Since crossvein spacing defects can result from either gains or losses in Ds or Ft function, it is possible that this defect is caused by disrupting the function of endogenous Ds, and thus the ability of that Ds to signal via Ft. However, DsΔECD did not cause any obvious change in the levels of endogenous Ds. Moreover, loss of Ds normally causes visible destabilization of cell surface Ft, and no changes were seen in Ft levels in cells misexpressing DsΔECD (Matakatsu, 2006). ds mutations can also enhance the overgrowth observed in mutants that lack the intracellular domain of Ft, indicating that in overgrowth, Ds activity is not completely dependent on regulating the activity of the intracellular domain of Ft. In this respect, overgrowth differs from PCP; ft mutants and ds ft double mutants produce identical PCP phenotypes. The result could be explained if Ds regulates growth via its intracellular domain. Alternatively, Ds may be acting as an extracellular ligand for a binding partner other than Ft (Matakatsu, 2006).

The results support the hypothesis the Ft signals via its intracellular domain in growth control, PCP and proximodistal patterning. Similarly, it is likely that the intracellular domain of Ds contributes to proximodistal patterning and perhaps growth control. The conservation of long stretches of the intracellular domain of Ft and Ds in the vertebrate homologs Fat4, dachsous 1 and dachsous 2 also suggests that there is conserved binding to intracellular factors (Matakatsu, 2006).

There are no known binding partners for the intracellular domain of Ds or dachsous-like proteins. The intracellular domain of Drosophila Ft also lacks the ENA-VASP binding sites that mediate at least some of the function of vertebrate Fat1 in vitro. The intracellular domain of Drosophila Ft can bind the atrophin Grunge, and genetic evidence suggests a link between Grunge and PCP. However, it is not yet clear if Grunge acts downstream of Ft, nor is it clear how atrophins, which act as transcriptional co-repressors, could polarize cells. grunge mutants also do not apparently reproduce the effects of ft mutants on disc growth or on wg expression in the prospective wing hinge (Matakatsu, 2006).

Some evidence suggests that Ds and Ft regulate growth and patterning by altering either the expression of wg in the prospective wing hinge or the response to Wg signaling. However, the current results make it unlikely that this can explain all but a small part of the overgrowth phenotype. The overgrowth induced by ft mutations or FtΔICD occurs without any consistent change in the expression of Wg target genes Dll or Vg, or in the expression of wg. Moreover, FtΔICD induced overgrowth in the entire wing disc, but whereas increased Wg signaling can induce overgrowth in the hinge, in the prospective wing blade Wg signaling reduces growth. The results are consistent with the failure of mutants in the Wg signaling pathway to modify the ft overgrowth phenotype (Matakatsu, 2006).

A recent study has suggested a possible link between overgrowth and Ras signaling; mild reductions in Ras function that have little effect on the growth of wild-type cells can block the overgrowth observed in ft mutant clones. It remains to be seen whether Ft can actually affect Ras signaling, or whether this represents the convergence of the two pathways on a shared target (Matakatsu, 2006).

Because Ds is expressed in an apparently graded fashion along the axes of polarity, it was suggested that Ds provides a global cue that orients PCP in the eye, wing and abdomen. But whereas patterned Ds misexpression is sufficient to reorient PCP, and patterned Ds expression does appear to be necessary for normal PCP in the eye, in the wing uniform Ds expression is able to rescue most of the ds mutant PCP defects. This suggests that most of the PCP defects in ds mutant wings are caused, not by a change in the spatial regulation of Ds-Ft signaling, but rather by the loss of a basal level of signaling required for the proper activity of some other polarizing cue. These results left open the possibility that Ft activity is being spatially regulated by an extracellular ligand other than Ds. However, this study shows that ft mutant PCP defects can be substantially rescued by uniform expression of FtΔECD, a form of Ft that cannot bind Ds, or probably any other ligand (Matakatsu, 2006).

There is, however, a region in the proximal wing where PCP defects cannot be rescued with uniform expression of either Ds, Ft, or FtΔECD. This is also the region of the wing where there is a boundary or sharp gradient between proximal regions with high and distal regions with low ds expression. Thus, it remains possible that Ds and Ft activities are permissive in much of the wing but, in the proximal wing, spatially instructive. The different sensitivities of different regions to changes in Ds and Ft may reflect localized differences in the strength of other partially redundant polarizing cues (Matakatsu, 2006).

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. This study reports that Ds and Fj regulate Hippo signaling to control growth. Interestingly, it was found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, it was found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size (Willecke, 2008).

These data show that Ds and Fj regulate the Hippo pathway in an unusual manner. Most interestingly, it was found that discontinuities or boundaries of Ds and Fj activity, rather than their absolute amounts, modulate the Hippo pathway. The effects of Ds and Fj on wg expression in the hinge region are also consistent with the proposed boundary model. Importantly, artificial Ds/Fj boundaries cause an up-regulation (de-repression) of Hippo pathway target genes and drive extra cell proliferation, whereas flattening of the endogenous Ds and Fj gradients reduced normal growth. Together, these data are consistent with a model in which Ds/Fj discontinuities suppress the activity of the Hippo pathway, thereby driving imaginal disc growth and thus contributing to the regulation of organ size (Willecke, 2008).

How much growth is controlled by Ds/Fj signaling? Flies with uniform Ds/Fj expression have significantly reduced wings, legs, and other body parts, but growth is not abolished. The Ds boundary effect thus accounts for some but not all growth control. Given that fat mutants have severely overgrown imaginal discs, how do these growth defects caused by flat Ds/Fj expression fit with a model that Ds and Fj act through Fat to regulate growth? The dachs mutant phenotype gives insights into that question. Dachs acts downstream of Fat and is required for the growth control function of Fat. Unlike Fat, however, Dachs is a positive regulator of growth. Fat thus suppresses growth by inhibiting Dachs, and the dachs mutant phenotype thus reflects the situation where Fat is fully (hyper) active. because Fat functions through the inactivation of Dachs, the growth defects caused by Fat hyperactivation cannot be stronger than the dachs mutant phenotype. The boundary model proposes that flattening the Ds and Fj gradients results in hyperactivation of Fat, thereby causing reduced growth. Remarkably, dachs mutants have small wings and short legs, and the strength of these growth defects are similar to those caused by uniform Ds and Fj expression. The phenotypes caused by uniform Ds and Fj expression are thus consistent with the model that discontinuities of Ds and Fj inactivate Fat signaling to promote growth (Willecke, 2008).

The observation that flies with uniform Ds/Fj expression as well as dachs mutants retain some growth indicates that other signaling mechanisms act in addition to the Ds boundary effect to control imaginal disc size. The Ds boundary effect is thus one of possibly several separate mechanisms that contribute to control the final size of imaginal discs. These other, currently unknown signals may act in parallel to the Hippo pathway to regulate tissue growth. In addition, other signals may regulate the Hippo pathway independently of the Ds boundary effect. For example, Mer acts in parallel to Fat, thus identifying another input into the Hippo pathway. It will be interesting to elucidate these additional signaling systems and to understand how they cooperate with Hippo signaling to control imaginal disc growth (Willecke, 2008).

Ds, Fj, and Fat regulate growth and planar cell polarity (PCP). Interestingly, discontinuities in Ds/Fj activity rather than their absolute amounts also regulate PCP. Ommatidial polarity reversals are associated with ds and fj mutant clone borders and ommatidia inside and outside of clones are affected. Boundary effects of Ds and Fj are also observed on hair polarity in the wing and abdomen. Thus, Ds/Fj discontinuities modulate Hippo signaling and PCP. However, the effects on the Hippo pathway are different from those on PCP. In contrast to the effects on Hippo target genes, which are induced all around clone borders, PCP effects are observed only on one side of clones. This difference can be explained because the Hippo readout is scalar (levels of target gene expression), whereas the PCP readout is vectorial (direction of polarity). Thus, the direction of the Ds/Fj gradients determines the direction of cell polarity, whereas the disparity in Ds/Fj activity (steepness of the gradients) modulates Hippo signaling (Willecke, 2008).

The effect of Ds/Fj boundaries appears to spread over several cells. Although β-Gal perdurance may contribute to this effect when assaying reporter gene expression, it was found that Ex degradation as well as up-regulation of DIAP1 protein, which has a short (30-min) half-life, is also observed over several cell diameters, indicating that the boundary signal is propagated over several cells. A similar propagation is also observed for the effects of Ds/Fj boundaries on PCP, and it has been suggested that Ds/Fj boundaries cause an asymmetric localization of Ds and Fat which may then propagate between cells (Willecke, 2008).

The boundary model proposes that cells respond to disparities in the levels of Ds/Fj between cells. How do cells sense Ds/Fj disparities to modulate downstream effectors? Ds forms heterodimers with Fat on neighboring cells and Fat cell autonomously regulates the activity of the Hippo pathway. This suggested that Fat and Ds may act as receptor and ligand, respectively. Surprisingly, however, Ds and Fat do not behave like a classical ligand-receptor pair. First, Ds does not regulate Fat in a dose-dependent manner, but rather acts through a boundary effect. Second, Ds is required in signal-sending cells as well as in responding cells, indicating that Ds has ligand- and receptor-like functions. This is true for the regulation of Hippo signaling as well as for PCP signaling in the abdomen. The finding that the intracellular domain of Ds is not required for the generation but for the sensing of the boundary signal further exposes this dual function of Ds. Two alternative models could explain how cells sense Ds discontinuities. In a first model, cells may compare the amount of Ds presented by neighboring cells on opposite sides. Cells may then sense a differential in the number of bound Fat molecules from one side of the cell to the other. In an alternative model, cells may compute the difference between the amount of Ds presented by neighboring cells (sensed by the amount of bound Fat molecules) with the amount of Ds expressed by a cell itself. This model may explain why Ds and its intracellular domain are required cell-autonomously to respond to the boundary signal. In both models, a differential in Ds activity between cells may regulate the activity of Fat, which then transduces the signal to downstream components. Fat is cell-autonomously required to regulate the Hippo pathway, and the intracellular domain of Fat is sufficient to promote the growth control and at least some PCP functions of Fat. The intracellular domain of Fat may thus transduce the boundary signal to downstream components regulating PCP and Hippo. Because Ex, Dachs, Hpo, Wts, and Yki do not or only slightly affect PCP, Fat may engage different downstream effectors to regulate PCP and the Hippo pathway. It will be fascinating to decipher the molecular mechanisms of how boundaries of Ds/Fj activity regulate the activity of Fat and how they are translated into a vector to control PCP and a scalar to modulate the Hippo pathway (Willecke, 2008).

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. This study used a cricket model (the two-spotted cricket, Gryllus bimaculatus) to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. Knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. A model is presented to explain these results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg (Bando, 2009).

Regeneration-dependent RNAi is a type of RNAi that occurs specifically after leg amputation in cricket nymphs that have been injected with double-stranded RNA (dsRNA) for a target gene (Nakamura, 2008a). In this system, when the metathoracic (T3) tibia of the third-instar nymph is amputated, it takes approximately 40 days (six ecdyses) to restore the adult leg. The process begins with the covering of the amputated region by newly formed cuticle. A ligand of Epidermal growth factor receptor (Gb'Egfr) is then induced by Decapentaplegic (Gb'Dpp) and Wingless (Gb'Wg) in a blastema composed of epithelial stem cells, which begins to undergo rapid proliferation to restore the lost portion in the fourth instar (Mito, 2002; Nakamura, 2008b). In the fifth instar, the tibiae, tibial spurs, tarsi and tarsal claws are restored in miniature. In the seventh instar, the amputated legs restore the missing portion to regain a nearly normal appearance. As no leg regeneration was observed after amputation in the case of rdRNAi against Gb'armadillo (Gb'arm), the canonical Wnt pathway should be involved in the initiation of the regeneration (Nakamura, 2007; Bando, 2009 and references therein).

Using an RNAi knockdown approach against 23 candidate genes, this study identified 15 components of the Ds/Ft signalling pathway that are involved in cricket leg regeneration. Based on additional data from Gryllus and Drosophila, a model signalling cascade was proposed for the regulation of leg regeneration by the Ds/Ft signalling pathway. As the main components of the Ds/Ft signalling pathways are conserved in vertebrates, this signalling cascade may also be involved in vertebrate leg regeneration (Bando, 2009).

The most typical phenotypes were the short and thick legs induced by rdRNAi against Gb'ft or Gb'ds. It is known that the size of each leg segment normally scales with overall body size, a phenomenon known as allometry. Surprisingly, the size of the regenerated legs in the phenotypes that were observed did not scale with overall body size. Furthermore, the size of the regenerated legs depended upon the site of tibial amputation. It is noteworthy that, although the expression patterns of Gb'ft and Gb'ds were different, their short and thick phenotypes were similar. This is consistent with the fact that Drosophila mutant phenotypes of both ft and ds in adult legs are short and thick, despite the fact that ft and ds have distinct expression patterns in Drosophila imaginal discs. Thus, it is concluded that the activity of Ds/Ft signalling regulates leg segment size and shape during regeneration. Furthermore, it was shown that the Ds/Ft signalling pathway may regulate leg size during regeneration through the Hpo signalling pathway. This is also supported by the fact that the Hpo signalling pathway is involved in an intrinsic mechanism that restricts organ size and that the Ds/Ft signalling system defines a cell-to-cell signalling mechanism that regulates the Hpo pathway, thereby contributing to the control of organ size (Bando, 2009).

Meinhardt pointed out that two processes operate during leg regeneration. One, which operates during the restoration of distal structures, is instructed by a morphogen epidermal growth factor (Egf), which is itself induced by two morphogens, Dpp and Wg, at the amputated surface (Meinhardt, 1982; Mito, 2002; Nakamura, 2008a). The other, operating in intercalary regeneration, is directly controlled by neighbouring cells at the junction between host and graft, but not by long-range morphogens (Meinhardt, 2007; Nakamura, 2008a). It is likely that the Ds/Ft signalling pathway participates in both mechanisms, because rdRNAi against Gb'ft or Gb'ds affected leg regeneration after either distal amputation or intercalary transplantation. In the case of distal amputation, as the Gryllus tarsi and claws were not restored after tibial amputation in the Gb'Egfr rdRNAi nymphs (Nakamura, 2008a), it has been speculated that Gb'Egf functions as a morphogen in the leg regeneration, as found in Drosophila leg imaginal discs. Recently, it has been demonstrated in the Drosophila wing disc that the Fat signalling pathway links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth. Thus, it is speculated that the Ds/Ft system links the Egf-mediated establishment of gradients of positional values across regenerating blastemal cells to the regulation of regenerate growth (Bando, 2009).

As Gb'd rdRNAi legs exhibited the short-leg phenotypes, but not thick ones, and Gb'd (decapentaplegic) is epistatic of Gb'ft and Gb'ds, Gb'D may mediate the components of Ds/Ft signalling controlling leg size. The enlarged phenotype of Gb'wts RNAi nymphs was suppressed by RNAi against Gb'd in Gryllus, indicating that Gb'd is downstream of Gb'wts. This result differs from Drosophila data. A genetic analysis is necessary to confirm the difference, because the epistatic allele is not null in RNAi experiments. As the phenotype of rdRNAi treatment against Gb'ds was weaker than that against Gb'ft, as reported in the corresponding Drosophila mutants, Gb'Ft may interact with factors that are as yet unidentified. Although the effect of rdRNAi against Gb'fj on leg size was very mild, the possible involvement of Gb'fj in allometric growth cannot be excluded. The short phenotypes were observed in the Gb'sd RNAi nymphal legs, so it remains a possibility that Gb'sd is involved in allometric leg growth. However, the apparent contribution of the Hpo-Sav-Mats complex is as yet uncertain (Bando, 2009).

Regenerated legs of Gb'ex and Gb'Mer RNAi adults become longer than normal control legs, and Gb'ex and Gb'Mer regulate cell proliferation induced by the presence of positional disparity. These results suggest that Gb'ex and Gb'Mer are also involved in allometric growth of the leg segment. In Drosophila, Ex and Mer negatively regulate cell growth and proliferation through the Hpo/Wts pathway. In mammalian cells, Nf2 (merlin) is known to be a crucial regulator of contact-dependent inhibition of proliferation (Curto, 2008). Thus, it is concluded that activities of Ex and Mer may regulate contact-dependent inhibition of proliferation via the Wts signalling pathway to restore the proper leg segment size during regeneration (Bando, 2009).

A widely accepted model for leg regeneration is the intercalation model, based on positional information. This model is based on the intercalation of new structures so as to re-establish continuity of positional values during regeneration. However, on the basis of this model, it is difficult to explain the changes in leg size that were observed in the present study. Thus, the model need to be extended to include the control of growth and tissue size during regeneration. Several models have been proposed to explain how organ size is regulated. Lawrence (2008) proposed a model, which is referred to here as the Ds/Ft steepness model, to explain the mechanisms underlying the determination of organ size and PCP, including the Warts/Hippo pathway as the mechanism for controlling growth. In this model, it was hypothesized that: (1) the morphogens responsible for the overall pattern of an organ establish and orient the Ds/Ft system, which then forms a linear Ds/Ft gradient. The nature of the Ds/Ft gradient is unknown, although the number of Ds/Ft trans heterodimers is the key variable; (2) the steepness of the Ds/Ft gradient regulates Hpo target expression and cell proliferation, and its direction provides information used to establish the correct cellular polarity; (3) growth would be expected to cease when the slope of the gradient declines below a certain threshold value; and (4) the maximum and minimum limits of the system are conserved, while recently divided cells take up intermediate scalar values from their neighbours (Bando, 2009).

Using this model, a modified Ds/Ft steepness model is proposed for leg regeneration acting as follows. The results indicate that nymphal leg regeneration depends on two major processes: (1) proliferation and differentiation of blastemal cells and (2) growth of the pre-existing stump. In each of these processes, new positional identities are specified in relation to new segment boundaries. According to the Ds/Ft steepness model, in normal regeneration, a very steep gradient should be formed in the regenerating blastema. The regenerate may grow so as to restore the normal pre-existing steepness. Reassignment of positional identities after amputation will correlate with a similar re-setting of the minimum Ds/Ft scalar value, and the results are consistent with the steepness hypothesis (Bando, 2009).

Growth of the pre-existing stump is a normal component of leg growth, in which the pre-existing stump cells proliferate according to some allometric signals, which may be related to the maximum scalar value and a slope of the gradient, keeping their original positional information. This was observed in the truncated leg of Gb'arm rdRNAi adults (Nakamura, 2007; Bando, 2009).

In the absence of the proliferation and differentiation of blastemal cells, as observed in the Gb'ft rdRNAi leg, the minimum scalar value, which is the most distal positional value, would be established at the site of amputation, and the Ds/Ft gradient would be expected, in turn, to shift down with the same slope as the pre-existing one. The Ds/Ft steepness model provides an explanation for the observation that the final leg size depends on the amputated position, if it is assumed that the gradient shifts down with the same slope as that where cells at an amputated position have the minimum scalar value. Thus, the observed re-specification of regeneration legs induced in the legs treated with rdRNAi against Gb'ft or Gb'ds is as would be predicted by the Ds/Ft steepness model. Thus, it is likely that the Ds/Ft gradient functions to link positional and allometric information to the regulation of leg segment growth. Furthermore, if it is assumed that the activity of Ex/Mer is related to a threshold value of the slope of the gradient that determines when growth ceases, all rdRNAi phenotypes in the present study can be interpreted consistently with the Ds/Ft steepness model for regeneration (Bando, 2009).

Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway

The regular array of distally pointing hairs on the mature Drosophila wing is evidence for the fine control of Planar Cell Polarity (PCP) during wing development. Normal wing PCP requires both the Frizzled (Fz) PCP pathway and the Fat/Dachsous (Ft/Ds) pathway, although the functional relationship between these pathways remains under debate. There is strong evidence that the Fz PCP pathway signals twice during wing development, and a Bidirectional-Biphasic Fz PCP signaling model has been presented which proposes that the Early and Late Fz PCP signals are in different directions and employ different isoforms of the Prickle protein. The goal of this study was to investigate the role of the Ft/Ds pathway in the context of the Fz PCP signaling model. The results lead to the following conclusions: (1) The Early Fz PCP signals are in opposing directions in the anterior and posterior wing and converge precisely at the site of the L3 wing vein. (2) Increased or decreased expression of Ft/Ds pathway genes can alter the direction of the Early Fz PCP signal without affecting the Late Fz PCP signal. (3) Lowfat (Lft), a Ft/Ds pathway regulator, is required for the normal orientation of the Early Fz PCP signal but not the Late Fz PCP signal. (4) At the time of the Early Fz PCP signal there are symmetric gradients of dachsous (ds) expression centered on the L3 wing vein, suggesting Ds activity gradients may orient the Fz signal. (5) Localized knockdown or over-expression of Ft/Ds pathway genes shows that boundaries/gradients of Ft/Ds pathway gene expression can redirect the Early Fz PCP signal specifically. (6) Altering the timing of ds knockdown during wing development can separate the role of the Ft/Ds pathway in wing morphogenesis from its role in Early Fz PCP signaling (Hogan, 2011).

The data presented in this report allow refinement Bidirectional-Biphasic (Bid-Bip) Fz PCP signaling model, particularly the nature of the proposed Early Fz(Sple) signal (Sple is an isoform of Prickle). The Early Fz(Sple) signal is in opposing directions in the anterior and posterior wing and converges precisely at the site of the L3 vein. The site of the L3 vein, therefore, represents a discontinuity in Early Fz(Sple) signaling that is called the PCP-D (see A model for PCP specification in the Drosophila wing). However, it is clear that physical differentiation of the L3 vein is not required for the formation of the PCP discontinuity (PCP-D). The correspondence of the PCP-D with the site of the L3 vein is perhaps surprising as the compartment boundary (a barrier to clonal growth that runs a few cells anterior to the L4 vein) appears a more obvious boundary between the anterior and posterior wing. However, the L3 vein has been defined as a specific region of low Hedgehog signaling within the wing, suggesting this region has the molecular autonomy needed to function as a signaling centre. In addition, recently published work from the Eaton lab (Aigouy, 2010) has also identified the L3 vein as the boundary between oppositely polarized cells in the anterior and posterior of early pupal wings (Hogan, 2011).

Both reduced activity and uniform over-expression of Ft/Ds pathway genes have similar effects on the direction of the Fz(Sple) signal, which becomes more distal in both the anterior wing and distal regions of the posterior wing. Significantly, the Eaton lab has shown that the subcellular localization of Vang/Stbm protein in the early (15 hours a.p.f.) pupal wing of a ds mutant is more distal than wild-type in both the anterior and distal posterior wing (Aigouy, 2010). The current results are consistent with the idea that the normal direction of the Fz(Sple) signal is controlled by gradients of Ft/Ds pathway activity that can be flattened through either reduced or uniform expression of individual pathway components. An observation made by Matakatsu (2004) that ds is expressed transiently in a P-D stripe within the pupal wing blade at around the time of Early Fz PCP signaling and the peak of Ds expression has been localized to the site of the L3 vein, the same location as the wing PCP-D. This implies that there are symmetric gradients of ds expression in the anterior and posterior wing and that the Early Fz(Sple) signal points up a ds expression gradient. This conclusion is supported by the finding that the Fz(Sple) signal reorients to point away from localized ds knockdown, but not from localized ds over-expression. The Early Fz(Sple) signal also points away from over-expressed ft or fj, which suggests that Ft or Fj activity has the opposite effect to Ds activity on direction of the Fz(Sple) signal. This is the same relationship between Ft, Ds and Fj activity that has been established in the Drosophila eye. Recent molecular studies have shown that Fj, a golgi kinase, can phosphorylate cadherin domains within both Ft and Ds proteins. It has been proposed that this modification increases Ft activity, but decreases Ds activity (Hogan, 2011).

Reducing ds expression (or increasing ft or fj expression) under the control of the sal-Gal4 driver redirects the Early Fz(Sple) signal for a significant distance (ten or more cell diameters) beyond the sal-Gal4 expression domain. In principle, reducing ds expression within the sal-Gal4 domain should generate a local reversal of the ds expression gradient at the boundary of sal-Gal4 expression (e.g. the L2 vein). This short reversed ds gradient should generate a correspondingly short region of reversed Fz(Sple) signal which should be visible (on a pk^pk mutant wing) as a short region of reversed hair polarity adjacent to the L2 vein. Therefore, the propagation of reversed hair polarity significantly anterior to the L2 vein is surprising. However, a similar propagation of reversed polarity is seen adjacent to loss-of-function and over-expression clones of ds, ft or fj in the Drosophila abdomen. The model proposed for the propagation of altered polarity in the abdomen may, therefore, also apply to the Early Fz(Sple) signal in the wing (Hogan, 2011).

Since it has been established that wing hair polarity points down a gradient of Fz activity and it is proposed that the direction of the Early Fz(Sple) signal (i.e. the hair polarity that would be specified by the signal) points up a Ds expression gradient, it appears that there are opposing gradients of Ds and Fz activity during Early Fz(Sple) signaling. This relationship between Ds and Fz gradients is consistent with that described in the Drosophila eye, although it is opposite to that previously proposed in the wing. These findings, therefore, may help resolve this discrepancy between the proposed relationships of Fz and Ds activity in the eye and wing that has been highlighted by others (Hogan, 2011).

From this work it is concluded that for substantial regions of the wing (including most of the anterior wing and distal regions of the posterior wing), Ft/Ds pathway activity can be altered such that the Early Fz(Sple) signal is redirected, but the Late Fz(Pk) signal remains unaffected. For any specific experiment, this result might be explained by the specific properties of the mutant allele used or by the specific spatial or temporal activity of the Gal4 driver used to drive gene knockdown or over-expression. However, this study has shown that numerous alleles, as well as both knockdown and over-expression, of Ft/Ds pathway genes, can redirect the Fz(Sple) signal in a similar way, without affecting the Fz(Pk) signal in the same region. This suggests that across most of the wing there is a different requirement for the Ft/Ds pathway in the Early Fz(Sple) and Late Fz(Pk) signals. Moreover, it was found that loss of the Ft/Ds pathway regulator Lft affects the Early Fz(Sple) signal, but not the Late Fz(Pk) signal. This suggests that the mechanism used by the Ft/Ds pathway to direct the Early Fz(Sple) signal differs from that used to organize the Late Fz(Pk) signal (Hogan, 2011).

What, then, is the role of the Ft/Ds pathway in the Late Fz(Pk) signal? Since the Late Fz(Pk) signal organizes hair polarity, characterizing the loss of Ft/Ds pathway activity on hair polarity should be informative. It was found that driving ft or ds RNAi uniformly in the wing results in altered wing morphology, but only localized proximal hair polarity changes. This might be due to incomplete gene knockdown, coupled with different requirements for Ft/Ds activity for Late Fz PCP signaling in different regions of the wing. However, it is suggestive that wings homozygous for a fj amorphic allele show only a localized hair polarity phenotype in this same proximal region, implying that Fj is only required for hair polarity in the proximal wing. These results raise the possibility the Ft/Ds pathway is normally only required for hair polarity in the proximal wing (Hogan, 2011).

Since neither ft nor ds null flies are adult viable, previous studies have inferred the role of Ft and Ds in wing hair polarity from analyzing phenotypes of viable hypomorphic alleles, clones of amorphic alleles and localized over-expression. Some hypomorphic ds allele combinations display extensive wing hair polarity disruptions, although the residual activity of these specific alleles has not been well characterized. Wing clones homozygous for amorphic ft or ds alleles can show hair phenotypes, although this is dependent upon the position and/or size of the clone. However, mutant clones generate ectopic Ft or Ds activity boundaries/gradients in the wing and it is known that localized mis-expression of Ft/Ds pathway genes can generate hair phenotypes in wing regions not affected by uniform over-expression. Most telling, clones of fj affect hair polarity in regions of the wing that are not affected in amorphic fj wings. These results clearly show that mis-regulated Ft/Ds activity can change wing hair polarity. However, they do not definitively establish a role for Ft/Ds pathway in the normal organization of hair polarity outside of the proximal wing. Therefore, it remains possible that Ft/Ds pathway activity is only required for hair polarity in the proximal wing, but mis-regulated Ft/Ds pathway activity can induce changes in hair polarity in other wing regions. This may restrict the normal role of the Ft/Ds pathway to organizing the Late Fz(Pk) signal in the proximal wing alone (Hogan, 2011).

According to the Bid-Bip model, the two Fz PCP signaling events aligned with different axes of the developing wing allow membrane ridges to be organized in different directions in the anterior and posterior. The ability of the insect wing to deform specifically is vital for insect flight and it has been proposed that wing membrane structure helps provide the appropriate wing rigidity and flexibility. In the case of membrane ridges, the membrane should be flexible parallel to the ridges, but be resistant to folding perpendicular to the ridges. The A-P ridges in the anterior wing are perpendicular to longitudinal wing veins which suggests a rigid anterior wing structure, whereas the posterior ridges are almost parallel with longitudinal wing veins suggesting a more flexible posterior wing structure. This organization is typical for Dipteran wings which usually have a well-supported leading edge and a flexible trailing edge. Indeed, similar ridge organization have been seen in wings of other Drosophila species. Therefore, the different orientation of ridges in the anterior and posterior wing may have a functional basis. The reason for the uniform distal hair polarity across the Drosophila wing is not well understood, but is conserved in a wide range of Dipteran species suggesting a functional constraint. Therefore, the two Fz PCP signals in different directions during Drosophila wing development may provide a mechanism that allows hairs and ridges to be organized appropriately using a single signaling pathway (Hogan, 2011).

Are multiple Fz PCP signaling events active in other Drosophila tissues besides the developing wing? Intriguingly, the Prickle isoforms, Pk and Sple, play different roles in PCP in numerous Drosophila tissues, including the wing, eye, abdomen and leg. This raises the possibility that there are multiple Fz PCP signals involving differential use of Pk and Sple isoforms in each of these tissues. However, the specific phenotypes associated with loss of either or both isoforms within the different tissues suggest that the details of the Bid-Bip model are unlikely to hold true for all tissues. How can multiple Fz PCP signals occur in different directions in the same developing tissue? One possibility is that changes in the molecular makeup of the Fz PCP pathway allow it to respond to different global signals within the tissue, or to respond in different ways to the same global signal. In the Drosophila wing, this might result from the differential use of the Pk and Sple isoforms. Alternatively, the individual Fz PCP signals may respond to different global signals present at different times during tissue development or to a single dynamic global cue. The significance of Prickle isoform switching and the possibility of dynamic global PCP signals are ongoing topics of interest (Hogan, 2011).

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components Ras^V12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by Ras^V12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when Ras^V12 was expressed. In this model, Ras^V12 drives NB differentiation in Ras^V12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (ds^ΔICD) or ft lacking its extracellular domain (ft^ΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway

During animal development, several planar cell polarity (PCP) pathways control tissue shape by coordinating collective cell behavior. This study characterizes, by means of multiscale imaging, epithelium morphogenesis in the Drosophila dorsal thorax and shows how the Fat/Dachsous/Four-jointed PCP pathway controls morphogenesis. The proto-cadherin Dachsous is polarized within a domain of its tissue-wide expression gradient. Furthermore, Dachsous polarizes the myosin Dachs, which in turn promotes anisotropy of junction tension. By combining physical modeling with quantitative image analyses, it was determined that this tension anisotropy defines the pattern of local tissue contraction that contributes to shaping the epithelium mainly via oriented cell rearrangements. The results establish how tissue planar polarization coordinates the local changes of cell mechanical properties to control tissue morphogenesis (Bosveld, 2012).

Altogether this study found that Ds polarization promotes Dachs polarization within a domain of the opposing tissue-wide ds and fj gradients. Their local polarization produces an anisotropic distribution of junction tensions, which increases the contraction rates along the lines of Ds and Dachs planar polarization to shape the epithelial tissue mainly through oriented cell rearrangements. The Dachs myosin has the necessary domains to be an actin binding motor and, in complex with Dachsous, it may directly contribute to junction contractility, thereby favoring cell rearrangements. Since MyosinII also contributes to junction tension and cell rearrangements, future work should dissect the respective roles of Dachs and MyosinII in these processes. Morphogenesis is accomplished by the concerted activity of multiple signaling pathways. The subtractive method of tissue deformation rates is general enough to isolate the contribution of a given pathway to morphogenesis without making assumptions on its magnitude and its spatial dependence. Finally, given the multitude of cell shapes, cell sizes, and division patterns occurring in the thor ax epithelium,future work on this tissue should reveal how multiple signaling pathways are integrated to regulate proliferation, planar polarization, and morphogenesis (Bosveld, 2012).

dachsous and frizzled contribute separately to planar polarity in the Drosophila ventral epidermis.

Cells that comprise tissues often need to coordinate cytoskeletal events to execute morphogenesis properly. For epithelial tissues, some of that coordination is accomplished by polarization of the cells within the plane of the epithelium. Two groups of genes--the Dachsous (Ds) and Frizzled (Fz) systems--play key roles in the establishment and maintenance of such polarity. There has been great progress in uncovering the how these genes work together to produce planar polarity, yet fundamental questions remain unanswered. The Drosophila larval ventral epidermis has been studied to begin to address several of these questions. ds and fz are shown to contribute independently to polarity, and they do so over spatially distinct domains. Furthermore, it was found that the requirement for the Ds system changes as field size increases. Lastly, it was found that Ds and its putative receptor Fat (Ft) are enriched in distinct patterns in the epithelium during embryonic development (Donoughe, 2011).

In early embryos, the body axis is subdivided into parasegments, each of which is further subdivided into two domains. One half of the epithelial cells will secrete smooth cuticle and the other half will form cuticular protrusions called denticles (the denticle field). The denticle field pattern is the product of a series of distinct polarized events. First, cells align into columns as a consequence of the reorganization of select cell interfaces. Second, one to three F-actin bundles protrude from the posterior edge of each cell. Third, the F-actin bundles guide the secretion of extracellular matrix (cuticle) such that denticles take on their final tapered orientation and hooked shapes. The result is that each column of denticles corresponds to a single column of underlying cells. This study has taken advantage of this polarized pattern to investigate the roles of ds, ft and fz in establishing this planar polarity (Donoughe, 2011).

With each molt, a growing larva secretes a new cuticle that is patterned on the underlying epidermis. Since there are no major cell rearrangements nor any increase in cell number during larval growth, cells of this epithelium maintain their specific fates and relative positions. Thus, the denticle pattern is resynthesized for each successive cuticle, where the columns of protruding denticles remain intact until the next molt, enabling the crawling larvae to grip the substrate during locomotion (Donoughe, 2011).

This study addresses long-standing questions in the planar cell polarity (PCP) field: (1) how do Fz and the members of the Ds system each contribute to planar polarity in an epithelium and (2) how do Ds and Ft influence the polarized placement of F-actin protrusions (Donoughe, 2011)?

This study has elucidated the contributions of several key polarity genes in the larval ventral epidermis. The genes in the Ds system are essential for proper polarity in this tissue. Notably, the Ds extracellular domain is able to reorient adjacent cells even when they are null for ds. The Fz protein operates largely redundantly and in parallel to the Ds system, and appears to contribute more in some columns than others. As field size increases, it is likely that Fz is less able to polarize the tissue on its own. By contrast, the Ds system is able to polarize the tissue equally well at small and large field sizes. Finally, it was found that in embryos, Ds and Ft are enriched in the posterior half of each denticle field. This correlates with the domain of the embryonic denticle field where actin protrusion placement defects appear in ds M^-Z^- embryos (Donoughe, 2011).

Several observations are in line with what is understood from other tissues. First, the polarity disruptions in ds^- or ft^- single mutants are comparable in severity to those observed in ds^- ft^- double mutants. This confirms that Ds and Ft act within the same process to polarize tissues. Second, in the adult abdomen, an experimentally induced high point of Ds extracellular domain expression causes an adjacent cell to reorient its polarity toward that high point (see Ds extracellular domain can reorient neighboring denticle columns). Likewise, overexpression of the Ds extracellular domain in one cell column of an otherwise wild-type larva causes the flanking cell columns to reorient toward this (presumed) enhanced source of Ds. By repeating this experiment in the ds^- mutant, any potentially confounding contributions from the superimposed distribution of endogenous Ds were avoided. Therefore, it can be concluded that cells polarize toward high levels of Ds. Whether this is the case during normal patterning is more difficult to address (Donoughe, 2011).

Finally, it was found that gain-of-function effects are propagated farther than just the adjacent cell. Thus, in a wild-type background, excess Ds in column 1 caused reorientation in columns 2 and 3. This implies that the signal was received in column 2 (resulting in altered polarity there), and then a polarizing effect was propagated to column 3. When such overexpression was repeated in a ds^- background, however, column 2 reoriented whereas column 3 largely did not. This demonstrates that Ds is not required for a cell to respond to a Ds polarity signal, but it is important in propagating that signal onward. Altogether, these findings support the hypothesis that Ds and Ft work together to send, implement and propagate a polarity signal (Donoughe, 2011).

A central focus of ongoing research is to determine how the Fz and Ds systems each contribute to the establishment and maintenance of planar polarity. In both the Drosophila eye and wing it appears that the Ds system provides a directional cue that is amplified and implemented by the Fz system. In the abdomen, by contrast, the Ds system can polarize in the absence of Fz and Stan, both of which are essential for the non-cell-autonomous effects of the Fz system. The current findings make it clear that for the larval denticle field, the Fz protein acts in a way that is inconsistent with its proposed role downstream of the Ds system. However, this observation still leaves room for the possibility that Ds-Ft engages downstream components within the Fz system (Donoughe, 2011).

The larval epidermis is unique in that the relative requirements for the Ds and Fz systems differ in different domains. The most obvious example of this is that when the Ds system is removed, polarity is completely removed in some columns (e.g., columns 0 and 4) but at least some polarity is still present in others (e.g., columns 1, 2, 3, 5, 6). Thus, it appears that the Fz system (still intact) is acting in those columns to impart polarity, suggesting that the two systems have independent and redundant inputs to polarity (Donoughe, 2011).

It was also demonstrated that Ds extracellular domain overexpression is able to reorient adjacent columns in an fz null background, and this signal is propagated onward. This shows that the Ds system can send, receive and propagate polarity information without contribution from the Fz protein. It remains possible that even when Fz-dependent intercellular signaling is absent, intracellular components of the Fz-system, such as Dsh, act in implementing the Ds signal. This function of Dsh would have to be unaffected in dsh[1] MZ mutants, as the polarity of dsh[1] MZ and dsh[1] MZ ds^- larvae appear similar to that of fz^- and ds^- fz^- larvae, respectively. Testing for Ds-mediated polarity in dsh null cells would be the true test of this hypothesis, but is precluded by the essential role of dsh in canonical Wnt signaling (Donoughe, 2011).

If, however, the Ds system operates independently of the Fz system, this would have significant ramifications for understanding of the molecular mechanisms that must be engaged downstream of each polarity system. The two systems must eventually converge at the point when cells create the oriented read-out (in this case, denticle formation). It is possible that the common polarity effectors might be far downstream of the initial effects in signaled cells. Given that the Fz and Ft receptors are so dissimilar from a molecular standpoint, their immediate effectors are likely to be distinct. Only by identifying the proteins that interact with Ft to implement Ds system polarity will it be possible to determine whether these effectors intersect downstream components of the Fz system or act independently on the polarity read-out (Donoughe, 2011).

Another observation that requires explanation is that the Ds and Fz systems seem to operate serially in some contexts (e.g. in the eye or wing) but in parallel in others (e.g. in the abdomen or the larval epidermis). Ds system-mediated microtubule (MT) orientation has been suggested as one mechanism by which the Ds system could feed into the Fz system. When MTs are oriented along the axis of polarity of wing cells, MT-mediated polarized transport brings Fz to the cell membrane, and it was recently shown that the maintenance of the correct MT orientation is Ds dependent. In the embryonic ventral epidermis, however, MTs are oriented perpendicular to the axis of planar polarity, at least at steady state. Therefore, unless careful imaging uncovers a minor, posteriorly polarized and Ds-dependent MT track, it seems unlikely that the Ds system is operating in the ventral epidermis in the manner proposed for wing polarity. This could explain why the Ds system only functions independently of Fz protein in the denticle field (Donoughe, 2011).

In ds^- fz^- larvae, all columns were largely disordered, but the flanking cell columns exhibited a slight, yet statistically significant, tendency toward reversed polarity. It is difficult to explain why there is residual polarity rather than randomization. Although the ds and fz alleles that were used are nulls, fz2 cannot be additionally removed owing to its essential role in canonical Wnt signaling. Thus, it is possible that Fz2-dependent polarization makes some contribution in the larval epidermis, although Fz2 has not as yet been implicated in PCP in any tissue. Alternatively, even if fz were the only Fz system receptor active for PCP, some latent activation of downstream components of the Fz system could, in principle, be responsible for imparting this subtle but polarized output (Donoughe, 2011).

An alternative explanation for the residual polarization in ds^- fz^- mutants is that there is an underlying bias in the tissue that is ordinarily masked in the presence of Ds or Fz proteins, but uncovered when both are removed. Since the residual orientation in double mutants tends to be directed away from the smooth field, perhaps that domain is somehow responsible for the latent polarity. Alternatively, the orientation signal might derive from within the denticle field. For instance, the 4-5 column interface is a boundary for Notch and EGFR signaling. Perhaps a low-level orientation signal emanates from that position (Donoughe, 2011).

This work also suggests that the Ds and Fz systems have different capacities to adjust to changes in field size. Current models for creating planar polarity begin with a gradient across the field of unpolarized tissue. A subtle bias is presumably then established within each cell across the field, as cells compare the level of the polarizing gradient they detect with that detected by their neighbors. This bias is then reinforced in each cell through a feedback mechanism, converting it into a sharp intracellular gradient of effector protein distribution (Axelrod, 2009). At those initial stages, when a given cell compares the level it perceives with that of adjacent cells, the magnitude of the difference under comparison should be influenced by the size of the field: as field size increases, the contrast perceived by adjacent cells decreases. Correspondingly, any comparison mechanism will be challenged as field size increases (Donoughe, 2011).

The larval epidermis presents such a challenge to the polarizing systems as tremendous growth occurs across the field between each larval molt. This study succeeded in analyzing the effects on the Fz system as field size increased by examining ds null animals at each molt. At small field size (i.e. first instar), polarity defects are rare; however, at large field size (i.e., third instar, five times larger), the disruption to polarity is dramatic. This suggests that the Fz system loses potency as field size increases. By contrast, the Ds system did not appear to be affected, as there are only rare defects in fz null animals at first or third instar. Since the change in field size through the larval instars occurs in the absence of cell division, it will be of interest to explore what other parameters of cell growth affect the Fz system in this tissue (Donoughe, 2011).

Note also that this work demonstrates that denticle field polarity can change over the course of larval growth. This supports the recent finding that third instar polarity is not determined at the embryonic stage. Together, these findings strongly imply that planar polarity in the larval epidermis is not permanently set, but rather requires input throughout larval growth (Donoughe, 2011).

The ventral epidermis also provides the opportunity to study how the two polarity systems influence distinct polarized outputs from the same tissue. Cell alignment and denticle orientation were largely unaffected in ds M^-Z^- embryos/first instar larvae, but there were F-actin protrusion placement defects in cell columns 3 through 5. This result is compelling, as the domain affected matched the region of peak Ds and Ft accumulation. In fz M^-Z^- and dsh[1] MZ backgrounds, there are subtle column 1 and 2 defects in F-actin protrusion placement. It is intriguing that the embryonic protrusion placement defects appear in complementary patterns for the Fz system as compared with the Ds system; this suggests that in embryos, as in larvae, the two systems function mainly in spatially distinct domains (Donoughe, 2011).

In several tissues, protein distributions have provided a window into the mechanism of polarization. However, in the embryonic epidermis, this analysis so far has not been suggestive. As neither Ds nor Ft showed an obvious bias toward particular interfaces around a given cell, it is not immediately apparent how these accumulation patterns might be related to proposed Ds-Ft dimer distributions or to the polarity of the tissue. It is of course possible that the protein accumulations would be more suggestive if one could analyze them during the larval molts, but this cannot presently be done (Donoughe, 2011).

In this context, it is worth noting that the endogenous distributions of Fz system components have not yet been determined in the ventral epidermis. Staining for Fz-GFP and Dsh-GFP, however, reveals a difference in their enrichments as compared with Ds and Ft: both Fz system members are strongly enriched along cell interfaces that separate cell columns and are depleted from interfaces between cells within the same column. Whether these putative enrichments are necessary for polarity in this tissue remains to be tested (Donoughe, 2011).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size

The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. This study has modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. This model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which were confirmed experimentally (Aegerter-Wilmsen, 2012).

This paper presents a new model for the regulation of wing disc size. The model contains a rather complex regulatory network, which consists of a considerable number of interactions, receives nonuniform input of protein activities, and interacts with a mechanical stress pattern that emerges over time and space. It is assumed that the regulatory network represents protein activities and interactions that regulate these activities. The model does not distinguish between interactions at the transcriptional and protein activity level, but considers effects on net activities. All protein activities emerge from the network, except for those of Dpp, Wg and N, which are implemented in the model. In the regulatory network, differences in Ds and Fj concentrations between neighboring cells lead to activation of Dichate (D) by changing its intracellular localization. In addition, it is assumed that a weighted average of the area of a cell and its neighbors is a good readout for mechanical stress, that cells do not rearrange when exposed to mechanical tension, and that the planar polarization of D imposes a bias on the direction of the division plane. The interactions are hypothetical and form the main untested assumptions underlying the model. The regulation of ds by mechanical compression is not essential for the principle behind size regulation in the model, but improves the fit of simulation results with experimental data (Aegerter-Wilmsen, 2012).

A qualitative understanding can be gained by considering it in terms of a compression gradient model. During growth, compression increases in the center of the disc. Growth ceases when compression in the center reaches a certain threshold and the gradient of the compression gradient drops below a certain threshold in the rest of the disc. Read-out of the compression gradient is accomplished by a mechanism that involves Vg and the Hippo pathway. Numerical simulations were used to show that the model can account for growth termination and that it reproduces a large range of additional data on growth regulation, including some emergent properties of the system. Based upon the principle underlying the model, predictions can be made with respect to cell shape patterns. In order to take into account the curved surface of the wing pouch, an open source image analysis program was developed. The results showed that the general dynamics of the formation of cell shape patterns is indeed similar to the one predicted by the model. This analysis is, however, based on images from different discs and, especially during the early stages, there is variation among discs. It would therefore be interesting to assess whether the predicted dynamics is also present in the temporal evolution of single discs. However, this first requires the development of experimental methods with which single discs can be followed over time (Aegerter-Wilmsen, 2012).

Even though the development of cell shape patterns constitutes a fundamental prediction of the model, it would be an interesting future experimental challenge to test the model's basic assumptions directly, i.e., the regulation of Yki, Arm and ds by mechanical forces. The regulation of Yki by mechanical compression is most relevant for the model's behavior and appears necessary to obtain growth termination in combination with roughly uniform growth. The regulation of Arm by compression seems to be involved in stabilizing the Vg gradient, which could be relatively unstable if it would be regulated by Vg autoregulation alone. In addition, this interaction smoothens the compression gradient, which might have implications for the 3D structure of the wing disc. Last, the regulation of ds by mechanical forces is not essential for the principle behind size regulation, but improves the modeling results and also contributes to smoothening of the compression gradient. While developing the model, focus was placed on its ability to reproduce specific features of growth dynamics, as well as a number of key experiments that are used to argue in favor and against current models. One of the latter results, the decrease of medial growth upon induction of uniform Dpp signaling, could not be reproduced. In the simulations, these discs grow very fast. It is conceivable that such growth rates cannot be sustained in vivo because of a limited availability of nutrients and oxygen. When imposing a maximum total growth rate on disc growth, it is indeed possible to obtain growth rates in the medial part that are lower than those in wildtype discs, whereas lateral growth rates are higher, in agreement with experiments. Thus, with this additional assumption, the model can reproduce the results it was aimed to reproduce (Aegerter-Wilmsen, 2012).

There are currently no experimental data available on the parameters underlying the model and therefore they were fitted manually. As has become clear from the parameter analysis, there are only a few parameter combinations that can reproduce all results. However, it is not known whether this set is reproduced robustly in vivo and there is no natural selection on reproducing experimental manipulations robustly. Nevertheless, it is entirely possible that a larger set of parameter values should reproduce the results. In addition, even though the model can reproduce the selected set of experimentally observed features, there are related observations it cannot reproduce. For example, the final size reached in the model is too small, the experimentally observed nonautonomous growth induction by clones overexpressing brk is nearly absent in the model, and growth induction along the boundary of ds overexpressing clones extends further inside the clone than measured experimentally. It would be interesting to study whether there are factors missing in the model, which would make the parameter space less strict. For example, the parameter space was strongly restricted by the stipulation to reproduce the absence of Vg-BE activity in ap0 mutants upon ectopic wg expression. If it could be assumed that smaller discs have a different geometry in vivo than larger ones, the number of possible parameter combinations would increase. It will be interesting to assess the geometrical properties of discs in young larvae and evaluate whether the model should be adjusted in this respect (Aegerter-Wilmsen, 2012).

Very recently, another model has been formulated for growth regulation that assumes that growth is regulated by increases of Dpp signaling levels over time. However, growth is increased in wing discs in which Brk and Dpp signaling are removed. This either contradicts this model or the current understanding of Dpp signaling needs to be revised. The current model reproduces increased growth in such mutants, including its non-uniformity (Aegerter-Wilmsen, 2012).

The adult wing is covered by bristles, which point towards the distal part of the wing. This orientation is regulated by planar polarity genes. Regulation of planar polarity seems to be related to growth regulation. For example, Ds and Fj are not only important for growth regulation, but are also required for the development of a proximodistal polarity pattern. It is currently not clear whether Ds and Fj are directly involved in regulating planar polarity. If this were the case, then the model would suggest that planar polarity may, at least in part, arise from an interplay between morphogens and mechanical forces. The model presented in this study was developed for the wing imaginal disc of Drosophila. It would be interesting to see whether a similar model could also reproduce size regulation and additional experimental results in other systems. For other imaginal discs, it has been shown that their centers are also compressed at the end of growth. The precise regulatory networks involved in growth and size regulation are different for the different discs, but it would be interesting to see whether certain principles are conserved. In mammals, mechanical forces regulate growth in many tissues. However, the situation is often very different from that in the wing disc in that most mammalian tissues reach their final size while they perform a biological function. Thus, it would be interesting to study whether principles similar to those described here apply for mammalian organs early during development (Aegerter-Wilmsen, 2012).

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

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

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

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

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

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

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

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

Local cell death changes the orientation of cell division in the developing Drosophila wing imaginal disc without using Fat or Dachsous as orienting signals

Drosophila imaginal disc cells exhibit preferred cell division orientations according to location within the disc. These orientations are altered if cell death occurs within the epithelium, such as is caused by cell competition or by genotypes affecting cell survival. Both normal cell division orientations, and their orientations after cell death, depend on the Fat-Dachsous pathway of planar cell polarity (PCP). The hypothesis that cell death initiates a planar polarity signal was investigated. When clones homozygous for the pineapple eye (pie) mutation were made to initiate cell death, neither Dachsous nor Fat was required in pie cells for the re-orientation of nearby cells, indicating a distinct signal for this PCP pathway. Dpp and Wg were also not needed for pie clones to re-orient cell division. Cell shapes were evaluated in wild type and mosaic wing discs to assess mechanical consequences of cell loss. Although proximal wing disc cells and cells close to the dorso-ventral boundary were elongated in their preferred cell division axes in wild type discs, cell shapes in much of the wing pouch were symmetrical on average and did not predict their preferred division axis. Cells in pie mutant clones were slightly larger than their normal counterparts, consistent with mechanical stretching following cell loss, but no bias in cell shape was detected in the surrounding cells. These findings indicate that an unidentified signal influences PCP-dependent cell division orientation in imaginal discs (Kale, 2016).

This paper made use of the observation that clones of imaginal disc cells mutant for pie, which exhibit an elevated rate of apoptosis, bias the cell division orientation of other cells nearby in a search for a signal responsible for cell division orientation. It is hypothesized that dying pie cells may be the source of a polarizing signal that is detected by other cells, and the roles of candidate signals were evaluated by removing them genetically from pie mutant cells. It is further hypothesized that the result may also be relevant to the orientation of cell divisions in normal development (Kale, 2016).

Since cell division orientation requires the PCP receptor Fat, this study tested whether its PCP ligand Dachsous was required, but this model was excluded. Since cell division orientation also requires Dachsous in the dividing cells, tests were performed to see whether Fat itself was a signal required in the apoptotic clones, but this was also excluded. In fact both Fat and Dacshous could be eliminated together from the dying cell population without preventing the orientation of nearby cells. The possibility was considered that rather than expressing Fat or Dachsous, apoptotic cells might down-regulate one or both proteins and that this might affect nearby cells, but it was found that eliminating one or both genes was not sufficient to orient nearby cell divisions. The possible contribution of Four-jointed, a kinase that phosphorylates Fat and Dachsous proteins in the Golgi, was not tested because Four-jointed should be unable to signal in cells already mutated for both ft and ds. Altogether, the experiments eliminated known ligands for the Fat/Dachsous PCP pathway, suggesting that the pathway must be required to orient cell division in response to some other signal (Kale, 2016).

It has been suggested that apoptotic imaginal disc cells secrete the morphogens Dpp and Wg in the process of stimulating compensatory proliferation. Since Dpp and Wg pattern many aspects of imaginal disc development, including the expression of some PCP genes, they were candidates to orient the division of imaginal disc cells. Contrary to this prediction, clones of apoptotic cells lacking Dpp and Wg continued to orient nearby cell divisions. It cannot be excluded that there may be other biochemical signals from dying cells that orient cell division. For example, there are other Wnt proteins in Drosophila that might affect cell polarity (Kale, 2016).

One other model consistent with these results is that cell division is oriented by physical constraints rather than biochemical signals. It is thought that in the wild type wing disc, the characteristic circumferential division pattern of the peripheral cells is a result of their being stretched around the growing wing pouch. Consistent with this conclusion, it has been reported that when a clone of cells grows more rapidly than the surrounding epithelium, cells around the clone are stretched circumferentially to accommodate the hyperplastic region, and this change in shape tends to orient cell divisions in a circumferential pattern around the hyperplastic clones. By analogy to these findings concerning enhanced growth, it might be expected that clones of cells experiencing high rates of cell death would expand more slowly than surrounding cells, and that this would stretch the cells around the clone inwards towards the slow growing region, leading to a reorientation of cell divisions towards the slow growing clone, opposite to the case of more rapidly growing clones. As expected given their persistent cell death, clones of pie homozygous cells grow more slowly than control clones, and exhibit a small increase in apical cell size, consistent with local tension in the epithelium. The changed orientation of cell division near to pie clones has been reported previously. This study was unable, however, to measure a consistent change in shape of the wing cells adjacent to pie homozygous clones, the population of cells where the altered division orientation is measured. This lack of correlation between cell shape and cell division orientation is also seen for wing pouch cells in the wild type wing disc, which show a proximo-distal division preference but no obvious proximo-distal polarization. The shapes of mitotic cells were not measured separately, and so the possibility cannot be excluded that only the mitotic cells exhibit altered shapes in the wing pouch. Recently, it has been reported that the orientation of epithelial cell division is determined by microtubule interactions with cell junction vertices, and that cell shape is a poor predictor of cell division in rounded cells, where the disposition of cell junction vertices varies. This may explain why both the normal cell division orientation and the response to cell death do not correlate with cell shape within the wing pouch region, where cells are more rounded than in peripheral regions of the wing disc (Kale, 2016).

Oriented cell divisions are suggested to contribute to organogenesis. It was suggested that oriented cell divisions are responsible for the shape of cell clones in the wing disc, which ultimately determines the shape of the whole tissue (which is a collection of clones). Oriented cell divisions may have other functions, for example they may represent a homeostatic mechanism that ameliorates growth-induced mechanical stress (Kale, 2016).

The shape of cell clones becomes less regular during cell competition, and the interfaces between wild type and Minute cell populations become more convoluted and interdigitated. Previously, it was suggested that oriented cell division could be responsible for the intermingling of wild type and Minute cells. Recently, Levayer described very similar intermingling between cells in the pupal notum that are induced to compete by expression of different levels of Myc protein (Levayer, 2015). Very little cell division occurs in pupal notum, and Levayer describe cell neighbor exchanges that are responsible for intermingling the cell populations. They propose these exchanges are promoted by mechanical effects of differential growth rates. Wild type and Minute cells also grow at different rates, but the apoptotic protein baculovirus p35 reduces the degree of intermixing between wild type and Minute cells. There is now evidence that p35 also stimulates Minute growth rate, while having less effect on wild type cells. Although the precise mechanism is unclear, Minute cell growth is possibly stimulated by signals from the undead Rp/Rp cells that are preserved when p35 is expressed. Together these data raise the possibility that p35 may affect both cell division orientation and intermingling of wild type and Minute cells by equalizing their relative growth rates. In the case of pie clones that expand slowly, differential growth might result in local mechanical stretching which influences nearby cell divisions, although it cannot be excluded that the pie mutant clones have other differences from wild type (Kale, 2016).

Fat has a role as an upstream regulator of the Salvador-Warts-Hippo (SWH) pathway of tumor suppressors. There is substantial evidence that the SWH pathway responds to mechanical cues. Inputs are reported from actin polymerization status and from adhesion junctions via α-catenin and Juba proteins. Recent studies indicate that the SWH pathway itself promotes epithelial junctional tension, which is reduced in clones of ft or wts mutants. Cell division orientation also depends on atro, however, which has been thought not to affect SWH activity, since it does not affect growth. Recent studies suggest that mutations in the Fat-Dachsous pathway may affect PCP through a disruption of the Spiny Leg protein by de-repressed Dachs that is not a reflection of normal Dachs function. This does not explain how cell division orientation is affected by Fat or Dachsous but it does raise the possibility that Fat and Dachsous mutations might affect processes that depend little on their normal alleles. What this study reports is that the model developed for planar cell polarity, in which ligand-receptor interactions between Fat and gradients of Dachsous control cell polarity, do not seem applicable to the orientation of cell division in the wing disc, where mechanical factors may be important (Kale, 2016).

EVOLUTIONARY HOMOLOGS

FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrate full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization reveals FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression are observed in fetal tissue (as opposed to adult tissue), suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. It is proposed that this family of molecules is likely to be important in mammalian developmental processes and cell communication (Dunne, 1995).

The expression during rat embryogenesis of the protocadherin fat, the murine homolog of a Drosophila tumour suppressor gene, has been defined. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the beta-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. At E18 fat expression is seen in the brain throughout the entire neuraxis in the cells next to the ventricles, with more extensive labelling within the regions of the cortex containing the germinal or ventricular zones adjacent to the lateral ventricles. No labelling is seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat (Ponassi, 1999).

Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity

Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration. This study investigated the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. Fat4 and Dchs1 were found to be expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. The results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes (Zakaria, 2014).

REFERENCES

Search PubMed for articles about Drosophila dachsous

Abu-Shaar, M. and Mann, R. S. (1998). Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125(19): 3821-3830. PubMed Citation: 9729490

Adler P.N., Krasnow, R. E. and Liu, J. (1997). Tissue polarity points from cells that have higher Frizzled levels towards cells that have lower Frizzled levels. Curr. Biol. 7(12): 940-949. PubMed Citation: 9382848

Adler, P., Charlton, J. and Liu, J. (1998). Mutations in the cadherin superfamily member gene dachsous cause a tissue polarity phenotype by altering frizzled signaling. Development 125(5): 959-968. PubMed Citation: 9449678

Aegerter-Wilmsen, T., Heimlicher, M. B., Smith, A. C., de Reuille, P. B., Smith, R. S., Aegerter, C. M. and Basler, K. (2012). Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size. Development 139: 3221-3231. Pubmed: 22833127

Aigouy, B., et al. (2010). Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila. Cell 142: 773-786. PubMed Citation: 20813263

Ambegaonkar, A. A., Pan, G., Mani, M., Feng, Y. and Irvine, K. D. (2012). Propagation of Dachsous-Fat planar cell polarity. Curr Biol 22: 1302-1308. PubMed ID: 22727698

Axelrod, J. (2009). Progress and challenges in understanding planar cell polarity signaling. Semin. Cell Dev. Biol. 20: 964-971. PubMed Citation: 19665570

Ayukawa, T., Akiyama, M., Mummery-Widmer, J. L., Stoeger, T., Sasaki, J., Knoblich, J. A., Senoo, H., Sasaki, T., Yamazaki, M. (2014) Dachsous-dependent asymmetric localization of Spiny-legs determines planar cell polarity orientation in Drosophila. Cell Rep. 8(2):610-21. PubMed ID: 24998533

Baena-López, L. A., Baonza, A. and García-Bellido, A. (2005). The orientation of cell divisions determines the shape of Drosophila organs. Curr. Bio. 15: 1640-1644. 16169485

Baena-Lopez, L. A., Rodriguez, I. and Baonza, A. (2008). The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like. Proc Natl Acad Sci U S A 105: 9645-9650. PubMed ID: 18621676

Bando, T., Mito, T., Maeda, Y., Nakamura, T., Ito, F., Watanabe, T., Ohuchi, H. and Noji, S. (2009). Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136(13): 2235-45. PubMed Citation: 19474149

Bosveld, F.,et al. (2012). Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway. Science 336(6082): 724-7. PubMed Citation: 22499807

Brittle, A., Thomas, C. and Strutt, D. (2012). Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous. Curr. Biol. 22(10): 907-14. PubMed Citation: 22503504

Buratovich, M. A. and Bryant, P. J. (1997). Enhancement of overgrowth by gene interactions in lethal(2)giant discs imaginal discs from Drosophila melanogaster. Genetics 147(2): 657-670. PubMed Citation: 9335602

Casal, J., Struhl. G. and Lawrence, P. A. (2002). Developmental compartments and planar polarity in Drosophila. Curr. Biol. 12: 1189-1198. 12176328

Cho, E. and Irvine, K. D. (2004). Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development 131: 4489-4500. 15342474

Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and Irvine, K. D. (2006). Delineation of a Fat tumor suppressor pathway. Nat. Genet. 38(10): 1142-50. 16980976

Clark, H. F., et al. (1995). Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev. 9(12): 1530-1542. PubMed Citation: 7601355

Curto, M. and McClatchey, A. I. (2008). Nf2/Merlin: a coordinator of receptor signalling and intercellular contact. Br. J. Cancer 98(2): 256-62. PubMed Citation: 17971776

Dearborn, R. and Kunes, S. (2004). An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe. Development 131: 2291-2303. 15102705

Donoughe, S. and DiNardo, S. (2011). dachsous and frizzled contribute separately to planar polarity in the Drosophila ventral epidermis. Development 138(13): 2751-9. PubMed Citation: 21613320

Dunne, J., et al. (1995). Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30(2): 207-23. PubMed Citation: 8586420

Feng, Y. and Irvine, K. D. (2009). Processing and phosphorylation of the Fat receptor. Proc. Natl. Acad. Sci. 106: 11989-11994. PubMed Citation: 19574458

Garoia, F., et al. (2000). Cell behaviour of Drosophila fat cadherin mutations in wing development. Mech. Dev. 94: 95-109. 10842062

Gonzalez-Morales, N., Geminard, C., Lebreton, G., Cerezo, D., Coutelis, J.B. and Noselli, S. (2015). The atypical cadherin Dachsous controls left-right asymmetry in Drosophila. Dev Cell 33(6):675-89. PubMed ID: 26073018

Greaves, S., et al. (1999). A screen for identifying genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Genetics 153: 1753-1766. PubMed Citation: 10581282

Held, L. I., Duarte, C. M. and Derakhshanian, K. (1986). Extra tarsal joints and abnormal cuticular polarities in various mutants of Drosophila melanogaster. Roux Arch. 195: 145-157

Hogan, J., Valentine, M., Cox, C., Doyle, K. and Collier, S. (2011). Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway. PLoS Genet. 7(2): e1001305. PubMed Citation: 21379328

Ishikawa, H. O., Takeuchi, H., Haltiwanger, R. S. and Irvine, K. D. (2008). Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains. Science 321(5887): 401-4. PubMed citation: 18635802

Kale, A., Rimesso, G. and Baker, N. E. (2016). Local cell death changes the orientation of cell division in the developing Drosophila wing imaginal disc without using Fat or Dachsous as orienting signals. PLoS One 11(12): e0167637. PubMed ID: 28030539

Kawamori, H., Tai, M., Sato, M., Yasugi, T. and Tabata, T. (2011). Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe. Dev. Growth Differ. 53(5): 653-67. PubMed Citation: 21671914

Levayer, R., Hauert, B. and Moreno, E. (2015). Cell mixing induced by myc is required for competitive tissue invasion and destruction. Nature 524(7566): 476-480. PubMed ID: 26287461

Lawlor, K. T., Ly, D. C. and Dinardo, S. (2013). Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field. Dev Biol. 383(2): 285-94. PubMed ID: 24051227

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

Lawrence, P. A., Struhl, G. and Casal, J. (2008). Do the protocadherins Fat and Dachsous link up to determine both planar cell polarity and the dimensions of organs? Nat. Cell Biol. 10: 1379-1382. PubMed Citation: 19043429

Mangione, F. and Martin-Blanco, E. (2018). The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen. Cell Rep 25(10): 2836-2850. PubMed ID: 30517870

Mani, M., Goyal, S., Irvine, K. D. and Shraiman, B. I. (2013). Collective polarization model for gradient sensing via Dachsous-Fat intercellular signaling. Proc Natl Acad Sci U S A 110: 20420-20425. PubMed ID: 24282293

Mao, Y., et al. (2006). Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila. Development 133(13): 2539-51. PubMed Citation: 16735478

Mao, Y., Kucuk, B. and Irvine, K. D. (2009). Drosophila lowfat, a novel modulator of Fat signaling. Development 136(19): 3223-33. PubMed Citation: 19710173

Marcinkevicius, E. and Zallen, J. A. (2013). Regulation of cytoskeletal organization and junctional remodeling by the atypical cadherin Fat. Development 140: 433-443. PubMed ID: 23250217

Matakatsu, H. and Blair, B. S. (2004). Interactions between Fat and Dachsous and the regulation of planar cell polarity in the Drosophila wing. Development 131: 3785-3794. 15240556

Matakatsu, H. and Blair, S. S. (2006). Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins. Development 133(12): 2315-24. 16687445

Meinhardt, H. (1982). Models of Biological Pattern Formation. London: Academic Press.

Meinhardt, H. (2007). Computational modelling of epithelial patterning. Curr. Opin. Genet. Dev. 17: 272-280. PubMed Citation: 17627806

Misra, J.R. and Irvine, K.D. (2016). Vamana couples fat signaling to the Hippo pathway. Dev Cell 39(2):254-266. PubMed ID: 27746048

Mito, T., Inoue, Y., Kimura, S., Miyawaki, K., Niwa, N., Shinmyo, Y., Ohuchi, H. and Noji, S. (2002). Involvement of hedgehog, wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs, a verification of the modified boundary model. Mech. Dev. 114: 27-35. PubMed Citation: 12175487

Nakamura, T., Mito, T., Tanaka, Y., Bando, T., Ohuchi, H. and Noji, S. (2007). Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus. Dev. Growth Differ. 49: 79-88. PubMed Citation: 17335429

Nakamura, T., Mito, T., Bando, T., Ohuchi, H. and Noji, S. (2008a). Dissecting insect leg regeneration through RNA interference. Cell Mol. Life Sci. 65: 64-72. PubMed Citation: 18030418

Nakamura, T., Mito, T., Miyawaki, K., Ohuchi, H. and Noji, S. (2008b). EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph. Dev. Biol. 319: 46-55. PubMed Citation: 18486122

Neumann, C. J. and Cohen, S. M. (1996). Distinct mitogenic and cell fate specification functions of Wingless in different regions of the wing. Development 122: 1781-1789. 8674417

Olofsson, J., Sharp, K. A., Matis, M., Cho, B., Axelrod, J. D. (2014) Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity. Development 141: 2866-2874. PubMed ID: 25005476

Ponassi, M., et al. (1999). Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80(2): 207-12. PubMed Citation: 10072790

Rawls, A. S., Guinto, J. B. and Wolff, T. (2002). The cadherins Fat and Dachsous regulate dorsal/ventral signaling in the Drosophila eye. Curr. Biol. 12: 1021-1026. 12123577

Reddy, B. V. and Irvine, K. D. (2008). The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation. Development 135: 2827-2838. PubMed Citation: 18697904

Reddy, B. V., Rauskolb, C. and Irvine, K. D. (2010). Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia. Development 137(14): 2397-408. PubMed Citation: 20570939

Rodríguez, I. (2004). The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc. Development 131: 3195-3206. 15175250

Rodrigues-Campos, M. and Thompson, B. J. (2014). The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila. Development 141: 4098-4103. PubMed ID: 25256343

Rogulja, D., Rauskolb, C. and Irvine, K. D. (2008). Morphogen control of wing growth through the fat signaling pathway. Dev. Cell 15: 309-321. PubMed Citation: 18694569

Sagner, A., Merkel, M., Aigouy, B., Gaebel, J., Brankatschk, M., Jülicher, F. and Eaton, S. (2012). Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium. Curr. Biol. 22(14): 1296-301. PubMed Citation: 22727699

Simon, M. A. (2004). Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression. Development 131(24): 6175-84. 15548581

Simone, R. P. and DiNardo, S. (2010). Actomyosin contractility and Discs large contribute to junctional conversion in guiding cell alignment within the Drosophila embryonic epithelium. Development 137: 1385-1394. PubMed ID: 20332153

Sopko, R., Silva, E., Clayton, L., Gardano, L., Barrios-Rodiles, M., Wrana, J., Varelas, X., Arbouzova, N. I., Shaw, S., Saburi, S. et al. (2009). Phosphorylation of the tumor suppressor fat is regulated by its ligand Dachsous and the kinase discs overgrown. Curr. Biol. 19: 1112-1117. PubMed Citation: 19540118

Stowers, R. S. and Schwarz T. L. (1999). A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152: 1631-1639. 10430588

Strutt, H. and Strutt, D. (2002). Nonautonomous planar polarity patterning in Drosophila: Dishevelled-independent functions of Frizzled. Dev. Cell 3: 851-863. 12479810

Strutt, H., Mundy, J., Hofstra, K. and Strutt, D. (2004). Cleavage and secretion is not required for Four-jointed function in Drosophila patterning. Development 131: 881-890. 14757640

Willecke, M., Hamaratoglu, F., Sansores-Garcia, L., Tao, C. and Halder, G. (2008). Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation. Proc. Natl. Acad. Sci. 105: 14897-14902. PubMed Citation: 18809931

Yang, C.-h., Axelrod, J. D. and Simon, M. A. (2002). Regulation of Frizzled by Fat-like cadherins during planar polarity signaling in the Drosophila compound eye. Cell 108: 675-688. 11893338

Zakaria, S., Mao, Y., Kuta, A., Ferreira de Sousa, C., Gaufo, G. O., McNeill, H., Hindges, R., Guthrie, S., Irvine, K. D. and Francis-West, P. H. (2014). Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity. Curr Biol 24(14): 1620-7. PubMed ID: 24998526

Zecca, M. and Struhl, G. (2010). A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth. PLoS Biol. 8(6): e1000386. PubMed Citation: 20532238