Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).
Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).
In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).
Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, ArmS10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).
Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of Nts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).
Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or ArmS10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (ArmS2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing ArmS2 display 'wiggly' clone borders, unlike those expressing Dsh or ArmS10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).
To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing ArmS10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).
Somatic clones lacking Ft (ft-/ft-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft+/ft+) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).
The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).
Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).
In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).
The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).
One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).
Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).
Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).
Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin (Grunge) is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. Atrophin is shown to bind to the cytoplasmic domain of Fat and Atrophin mutants show strong genetic interactions with fat. Both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data suggest a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination (Fanto, 2003).
Mosaic analysis of ft mutant clones demonstrates a strong bias for the cell that retains ft function to become the R3 cell. This has been interpreted to indicate that Fat directly biases the cell to become an R3 cell. Atro clones also show a bias for the R3 cell to retain Atro function, supporting the model that Atro, like Fat, works in R3 fate determination (Fanto, 2003).
However, an extensive mosaic analysis found that all anterior cells (R1, R2 and R3) tend to be ft+, and all posterior cells (R4, R5 and R6) tend to be ft-. It has been suggested that this bias is due to spatial considerations, cells that are polar in the precluster, undergo a 90° rotation, leaving them in a posterior position in the adult. At the polar border of ft clones, ommatidia rotate in the opposite direction to wild type, therefore the bias is reversed, leading to an increase in ft- anterior cells. Therefore, it as been concluded that additional data are required to show that ft function is specifically needed in R3. To determine if ft and Atro are specifically required in the R3 photoreceptor, mosaic analysis of Atro, ft and wild-type clones has been undertaken. In wild-type clones, marked only by white, ommatidia at the polar border of the clone show a weak preference for posterior photoreceptors to be wild-type. This bias is strictly due to the spatial constraints of recruitment in clones. In ft clones, it has been found that at the equatorial border (where polarity is unaltered) there is no discernable difference between anterior photoreceptors subclass types; 85% of all photoreceptors that retain ft are anterior class. The increase from ~61% to 85% is probably due to the adhesive properties of ft, which result in smooth edged clones (Fanto, 2003).
By contrast, at the polar border, which is where planar polarity is altered, there is a marked tendency for ft function to be retained specifically in the R3 photoreceptor; 100% of R3 cells retained ft, whereas only 83% of all anterior photoreceptors retained ft. Mosaic analysis of Atro clones shows that the bias introduced by planar polarity (PP) alterations at the polar border of clones (similar to ft) introduces a general bias for anterior photoreceptors. However, again, the bias is stronger in the R3 cell than for other anterior photoreceptors. This increased bias in the R3 photoreceptor over the other anterior class member suggests that ft and Atro are important in R3 fate (Fanto, 2003).
The conclusion that Atro function is important for the R3 cell fate is also strongly supported by the observation that loss of Atro often results in symmetric ommatidia with two R4 cells. This is reflected by the increase in R4/R4 ommatidia seen in Atro clones in the eye disc marked by expression of the R4 marker, md-lacZ. In addition the overexpression of Atro in R3 and R4 generates symmetric ommatidia with two R3 cells. Together, these data support the proposal that Atro is needed for the R3 fate (Fanto, 2003).
The non-autonomous nature of the PP defects associated with ft and Atro mutant clones could have presented some problems to mosaic analysis. It might be expected that non-autonomous alterations in polarity would equally affect all photoreceptors, yet the data clearly show enhanced requirements for ft and Atro function in the R3 photoreceptor over other photoreceptors. In addition, the proposal that Atro is needed for the R3 cell fate is supported by analysis of the R4 marker in eye discs. Interestingly, the tendency to lose the R3 cell fate in Atro clones is seen throughout the clone, and does not appear to participate in the phenomena of equatorial rescue or polar nonautonomy (Fanto, 2003).
Because Frizzled (Fz) is also needed for R3 fate decisions, it has been suggested that Fat positively affects Fz signaling. The observation that Atro acts with Fat and also biases towards the R3 fate suggests that the regulation of Fz by Fat may not be direct. It is proposed instead that Atro is necessary for the ft-dependent bias to an R3 cell fate and for the production of a diffusible PP molecule that controls Fz activity (Fanto, 2003).
The proposal that Fat increases Fz activity, and thereby biases a cell towards the R3 fate, does not explain the non-autonomous disruptions of wild-type tissue on the polar side of ft and Atro clones, or the rescue of ft and Atro mutant tissue from wild-type tissue on the equatorial side of the clone. There are several models that could explain the non-autonomous disruptions of planar polarity. One model suggests that planar polarity is established through a 'domino effect'. This model is suggested by the striking accumulation of planar polarity components, such as Fz and Dsh on the distal edge of every cell in the wing. This observation, coupled with genetic data that suggest that high Fz activity on one side of the cell forces low Fz activity on the other side, leads to a model in which accumulation or loss of polarity in a cell leads to templating of that state onto the next cell, non-autonomously propogating PP defects. However in the eye, Fz and Dsh show differential distribution on only a subset of ommatidial precursor cells, and, importantly, intervening cells show no altered accumulation. These data argue against a simple templating model for PP in the eye (Fanto, 2003).
An alternative model suggests that the juxtapositioning of ft+ and ft- tissue contributes to midline determination and emphasizes the role of Fat in inhibiting DV signaling away from the equator. This inhibition would be relieved at the equator by an unidentified molecule that would inhibit Fat function. If this model were correct, a small ft clone should mimic the situation at the equator, where Fat function is predicted to be locally inhibited. One would therefore be expected to find an ectopic equator in the middle of the clone. Instead, however, the opposite phenotype is found, since the ommatidia on the two sides of the clone point toward the middle of the clone, rather than away from it (Fanto, 2003).
The model that best explains both the equatorial rescue and polar nonautonomy of ft and Atro clones is that Fat and Atro together control expression of a planar polarity morphogen, here called 'factor X'. It is imagined that factor X is in a gradient with high levels at the equator and low levels at the poles, thus all ommatidia will appear to 'point' down this gradient. If Fat and Atro are essential to the production of factor X, then the ft/Atro mutant tissue will be void of factor X, producing a sink in the gradient. The gradient will still be pointing in the same direction initially, explaining the wild-type polarity of ommatidia at the equatorial side of the clone and 'equatorial rescue' seen in ft and Atro clones. For ommatidia at the polar edge of the clone, the gradient will be reversed, and ommatidia will point in the opposite direction. The gradient will also be disrupted outside of the clone, leading to inversions of the polarity of wild-type tissue on the polar side of the clone and 'polar nonautonomy' seen in these mutant clones. In large clones, there will be a region in the center of the clone where there is no detectable factor X, and as a result polarity will be randomized. All of these predictions are met in ft and Atro clones. Loss of Ds, which inhibits Fat function, should increase factor X. As predicted by this model, ds clones show disruptions in wild-type tissue on the equatorial side of the clone, and rescue of mutant tissue on the polar side of the clone. Without ft or ds function there would be no gradient and, consistent with this prediction, complete loss of planar polarity is seen in eyes that are homozygous for strong alleles of ft or ds (Fanto, 2003).
A gradient of Wg protein (which is high at the poles and low at the equator) initially establishes a gradient of Ds protein over the eye field. This gradient of Ds protein in turn produces a gradient of Fat activity, which, it is believed, creates a gradient of Atro activity. It is proposed that each cell will produce factor X at a level that is proportionate to the level of Atro activity, which varies according to the position of that cell in the ds and ft activity gradients. This model assumes that Factor X is a short-range diffusible molecule, which provides polarity information to ommatidial preclusters to direct their rotation. Since Fat has been shown to be upstream of Fz, it is speculated that the Atro-dependant Factor X is a ligand for Fz (Fanto, 2003).
Both ft and Atro also act in other, apparently unrelated, pathways. One of the prominent features of ft mutant larvae is the loss of growth control, which leads to dramatically overgrown discs and mutant clones that are markedly larger than their sister twin spots. However, Atro- clones do not display overgrowth in the eye, suggesting that ft restricts growth via an Atro-independent pathway. In addition, in the adult eye Atro clones (unlike ft clones) show severe defects in photoreceptor number and type, suggesting Atro has additional roles in photoreceptor specification and/or survival that are not shared by Fat. One particularly surprising result was the finding that Atro- clones are markedly smooth before the furrow, and that this smoothness is lost after the furrow passes. This suggests that Atro may function in a cell adhesion process that is lost upon cell differentiation (Fanto, 2003).
Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neuronal degenerative disease characterized by the variable combination of ataxia, choreoathetosis, myoclonus, epilepsy and dementia. This disease is caused by the expansion of a polyglutamine tract within the Atrophin 1 protein. Atro is the sole fly homolog of human atrophins. Atro has been shown to act as a transcriptional co-repressor in vivo in Drosophila. Atro interacts genetically with even skipped, a transcriptional repressor, and is required for the in vivo repressive activity of even skipped. The transcriptional repressor activity of Atro has been localized to the highly conserved C-terminal region of Atro. This C-terminal region can bind to Even skipped in vitro and interacts with the minimal repression domain of Even skipped (Fanto, 2003).
This study has shown that the intracellular domain of Fat binds the C-terminal domain of Atro. The cytoplasmic expression of Atro and its interaction with Fat raises the possibility that instead of acting as a simple co-repressor, Atro functions in a more complex manner. Other transcriptional co-repressors are known to be converted to transcriptional activators upon cell signaling, and future work will determine if the interaction of Fat with Ds alters the transcriptional activity of Atro (Fanto, 2003).
Owing to the fact that Atro binds the cytoplasmic domain of Fat, a model is favored in which Atro acts downstream of Fat, possibly relaying a Fat-dependant signal to the nucleus. However, the similarity of the ft and atro loss-of-function phenotypes makes classical epistasis experiments difficult, therefore a model in which Atro acts upstream of ft cannot be excluded. Examination of the amount or subcellular distributions of Fat and Atro, suggest that Atro does not control Fat expression or localization, nor does ft control the levels or subcellular localization of Atro (Fanto, 2003).
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 (FjGGG; G, glycine). The expression levels and Golgi localization of FjGGG appear normal, but FjGGG 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 FjGGG. 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, Ds2-3S236A (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 (fig. S2) (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 Ds2-3S236A 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 sFjGGG was used as the enzyme. Moreover, Ds2-3S236A 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:FjGGG, 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 (fig. S5) (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 fjGGG 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 FjGGG: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 FjGGG: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).
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).
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 (dcole88) 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, DcoDbt-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).
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 pkpk 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).
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