wingless

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The Wingless Pathway and Miscellaneous Wingless Targets

armadillo, disheveled and shaggy encode elements of a unique wingless signaling pathway used several times throughout development. WG signaling generates a hyperphosphorylated form of DSH, that is associated with a membrane fraction. Overexpressed dsh becomes hyperphosphorylated in the absence of extracellular WG and increases levels of the Armadillo protein, thereby mimicking the WG signal (Yanagawa, 1995).

Wingless acts through inactivation of the Shaggy/Zeste white 3 protein kinase to specify ventral cell fate in the leg. Ectopic expression of wg outside its normal ventral domain has been shown to reorganize the dorsal-ventral axis of the leg in a non-autonomous manner. Cells that lack Shaggy/Zeste-white 3 activity can influence the fate of neighboring cells to reorganize dorsal-ventral pattern in the leg, in the same manner as wg-expressing cells. Therefore, clones of cells that lack Shaggy/Zeste white 3 activity exhibit all of the organizer activity previously attributed to wg-expressing cells, but do so without expressing wg. The organizing activity of ventral cells depends upon the location of the clone along the dorsal-ventral axis (Diaz-Benjumea, 1994).

One enhancer element in the BXD region of Ultrabithorax, called 22186R is activated after the blastoderm stage, in contrast to other early enhancers. 22186R is not detectable until germ band extension is well under way. Ectodermal expression becomes visible in stage 9 embryos as a series of thin bands in PS6, 10, 11, 12, and 13, and shortly afterward staining appears in PS 7, 8 and 9, and then in the thoracic parasegments PS5, 4 and 3. The 2218R6 enhancer is unusual in directing expression in the central nervous system (CNS) at later embryonic stages. In wingless mutants expression from 22186R is absent (Poux, 1996).

The maintenance of gooseberry distal is controlled by the wg signal. A control element responsible for wg-dependent maintenance of gsb expression, gsb-late element, is separable from gsb-early element, which is required for the initial activation of gsb by pair-rule transcription factors (Li, 1993a).

In the dorsal epidermis and the terminal regions of the body, expression of wingless is independent of gooseberry but requires a wingless-lady bird regulatory feedback loop. Loss of lady bird function reduces the number of wingless-expressing cells in dorsal epidermis and leads to complete inactivation of wingless in the anal plate. Consequently, mutant lady bird embryos fail to develop anal plates and ubiquitous embryonic expression of either one or both lady bird genes leads to severe defects of the dorsal cuticle. Lack of late wingless expression and anal plate formation can be rescued with the use of a heat-shock-lady bird transgene (Jagla, 1997a).

Analysis of the expression of 18 wheeler in different mutant backgrounds shows that it is under control of segment polarity and homeotic genes. Initial accumulation of 18w is normal in wingless mutants. However, by full germband extension, the ventrolateral expression of 18wis narrower than in wild type. These changes appear well before cell death is seen in wg mutants. In patched mutants, the domains of wg and of 18w expand to include the expression domains of wingless and engrailed. These results suggest that wg and en positively regulate 18w expression within the ventromedial stripes (Eldon, 1994).

Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development. Hh signal transduction leads to increased stability of the transcription factor Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF. A new gene, slimb (for supernumerary limbs), is described which negatively regulates both of these signal transduction pathways. Loss of slimb function results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes. Clones of slimb1 cells in the leg or wing disc ectopically express dpp or wg when they arise in the anterior (but not the posterior) compartments of these discs. Anterior clones reorganize normal limb pattern, creating supernumerary 'double-anterior' limbs. Slimb, like PKA, is a negative regulator that normally prevents activity of the Hh signal transduction pathway in the absence of ligand. slimb mutant cells that arise in the presumptive wing blade ectopically express Scute and differentiate ectopic sensory bristles instead of epidermal hairs on the surface of the wing blade. Both phenotypes are strictly autonomous to the mutant cells, as is the case when the Wg signal transduction pathway is constitutively activated, but not when Wg is ectopically expressed. The slimb gene encodes a conserved F-box/WD40-repeat protein related to Cdc4p, a protein in budding yeast that targets cell-cycle regulators for degradation by the ubiquitin/proteasome pathway. It is proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis (J. Jiang, 1998).

The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary (Wessells, 1999).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. Nearly all the pair rule and segment polarity genes affect morphology of the gnathal segments and to varying degrees perturb pb accumulation. In general, mutations in the pair rule genes eliminate either the maxillary or labial lobe, as well as reduce the width of the respective segment. Despite these effects on morphology, pb expression can often be seen in the affected segments. Mutations in the segment polarity genes affect the morphology of both the maxillary and labial lobes. The overall effect is a reduction in the size of these lobes, resulting in a correspondingly reduced number of pb-expressing cells. Of the segment polarity genes tested, wingless has the strongest effect on pb expression. At early stages in wg^CX4 mutants, no pb expression is apparent in the presumptive labial lobe, though later, as head involution commences, some of these cells do begin to express pb (Rusch, 2000).

Chibby regulates Wingless targets

Inappropriate activation of downstream target genes by the oncoprotein ß-catenin is implicated in development of numerous human cancers. ß-catenin and its fruitfly counterpart Armadillo act as coactivators in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. A conserved nuclear protein, named Chibby, has been identified a screen for proteins that directly interact with the C-terminal region of ß-catenin. In mammalian cultured cells Chibby inhibits ß-catenin-mediated transcriptional activation by competing with Lef-1 to bind to ß-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo (Takemaru, 2003).

To investigate the role of Cby in Wnt signalling during development, Drosophila was examined because the fruitfly expresses a Cby ortholog that, like the human Cby described above, inhibits ß-catenin-dependent activation of reporter in mammalian cultured cells. cby double-stranded RNA was injected into wild-type embryos; this resulted in transformation of ventral denticles into naked cuticle. This phenotype is similar to embryos that overexpress wingless (wg) in all epidermal cells. In contrast, losing wg pathway activity results in transformation of naked cuticle into denticles. In addition, the head structures of cby(RNAi) embryos were missing, and the embryos were small. These phenotypes were also observed in embryos that express intron-spliced snapback RNA corresponding to cby. To confirm the specificity of the RNAi phenotype, a short interfering RNA was injected whose sequence did not overlap with either the dsRNA or snapback RNA used earlier. The siRNA duplex produced a highly similar phenotype, indicating that the phenotype is caused by specific reduction of cby activity. The cuticle phenotype suggests that cby functions as an antagonist of the wg pathway. To test this possibility further, expression of the gene engrailed (en), which is transcriptionally activated by wg signalling in the cuticle-secreting embryonic epidermis, was examined (Takemaru, 2003).

En is normally expressed in segmental stripes two cells wide, immediately behind cells expressing wg. In cby(RNAi) embryos, the En stripes expanded by another row of cells, a phenotype similar to one observed when embryos overexpress wg in the en domain. This indicates that cby normally represses expression of en. Because en is in turn required to maintain wg expression, wg activates its own expression through a paracrine feedback loop. RNAi depletion of cby results in expansion of wg messenger RNA and protein expression, further indicating that loss of cby function leads to wg pathway activation. The expanded width and intensity of the Wg stripes is greater than in embryos where wg signalling is maximally stimulated. This suggests that in addition to wg signalling, cby acts in a regulatory process at present unknown (Takemaru, 2003).

Experiments with mammalian Cby suggest that it inhibits Wnt signalling by binding to ß-catenin in the nucleus and blocking its interaction with Tcf/Lef transcription factors. If Drosophila Cby functions in a similar manner, then loss of cby should not affect the abundance or localization of Arm (ß-catenin). Arm localizes to nuclei in stripes of Wg-responding cells at stage 9 of embryogenesis (Takemaru, 2003).

Arm protein was examined in cby(RNAi) embryos; Arm abundance and localization were not detectably affected. If Cby were involved in transducing the Wg signal, then Wg would be predicted to lie upstream of Cby in an epistasis genetic pathway. To test for epistasis, the expression of a wg-responsive UbxB-lacZ reporter gene was examined in the embryonic midgut (Takemaru, 2003).

Expression is controlled by an enhancer that interacts with Drosophila Tcf and is activated in a manner dependent upon Wg signalling. Wg is expressed in parasegment (ps) 8, where it controls UbxB-lacZ expression in visceral mesoderm throughout ps7-9, as well as development of the second midgut constriction. In a wg null embryo, UbxB-lacZ expression is greatly reduced and the midgut constriction fails to form, indicating a strong dependence on wg function. In cby(RNAi) embryos, lacZ expression expanded into anterior and posterior parasegments, and the second but not the first midgut constriction formed; these phenotypes are reminiscent of moderate wg misexpression throughout the midgut. Thus, consistent with the observations of cby function in the epidermis, cby represses wg-dependent gene expression and development in visceral mesoderm. When cby was depleted by RNAi in wg null embryos, UbxB-lacZ expression was not blocked, and development of the second midgut constriction occurred. The wg;cby(RNAi) embryos resembled wild-type more than cby(RNAi) embryos; they sometimes exhibited more restricted lacZ expression and a first midgut constriction. Since embryonic RNAi usually leads to reduced levels of gene product, this implies that the residual cby product represses visceral mesoderm more effectively in the absence of wg. These results establish that wg function is mediated, at least in part, through cby (Takemaru, 2003).

It is concluded that Cby is a nuclear protein that is conserved throughout evolution. It antagonizes Wnt/Wg signalling by inhibiting ß-catenin/Arm function in mammalian cells and in Drosophila, raising the possibility that it may be a tumor suppressor gene. In this regard, Cby expression was found to be significantly downregulated in thyroid and metastatic uterine tumors, and nuclear Cby staining is missing or considerably weaker in thyroid tumors. Dysregulation of ß-catenin signalling has been reported in these types of cancers, so the decreased levels of Cby expression might be relevant to tumor formation (Takemaru, 2003).

Wingless in the formation of imaginal discs

wingless and dpp are required for allocation of cells to the thoracic imaginal primordia in the germ band extended embryo (corresponding to phase three of dpp expression). Narrow horiontal stripes of DPP intercept vertical stripes of WG secreting cells to form a ladder-like pattern in the ectoderm. It is at the points where WG and DPP stripes intersect that wing and leg imaginal discs are specified and Distal-less is induced (Cohen, 1993).

A third signaling molecule, Hedgehog, is also required for Distal-less induction. HH is secreted from the posterior compartments of imagial discs. Taken together, these three secreted signaling molecules, HH, WG and DPP specify the distal-axis of imaginal discs; each is required for distal-less induction (Diaz-Benjumea, 1994).

Wingless function in imaginal discs

Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. Wingless, but not Decapentaplegic, is responsible for the initial distal identity specification of the limb primordia. Proximal limb precursors expressing escargot encircle the Distal-less expressing distal primordium. Dll expressing cells show a dynamic cell migration in the early stage of limb formation, migrating basally during stage 12. Cells that have just started to express Dll also express thickveins. This suggests a requirement for regulated Dpp signaling at the level of receptor expression. Limb formation is restricted to the lateral position of the embryo through exertion of negative control by Decapentaplegic and the EGF receptor, both of which determine the global dorsoventral pattern. dpp specifies proximal cell identities. In the absence of dpp Escargot and Snail are lost. A late function of Decapentaplegic locally determines additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, in contrast to the deletion of distal structures when decapentaplegic mutations affect the imaginal disc. The limb pattern elements appear to be added in a distal to proximal direction in the embryo, which is just the opposite of what is happening in the growing imaginal disc. It is proposed that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis. This model is contrary to that of Cohen (1993) who argues that Dpp and Wingless are both required to induce the limb. Since Dll expression persists and expands dorsally in the absence of Dpp, it is clear that Dpp plays no role in inducing initial Dll expression but that the dorsoventral limit of Dll expression is defined by repression as a result of Dpp expression. Similarly, EGF-R is required to repress Dll expression in the ventral ectoderm (Goto, 1997).

Limb development requires the formation of a proximal-distal axis perpendicular to the main anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg is required to form the mature pattern (Lecuit, 1997).

aristaless is involved in the allocation of cells to the most distal elements of appendages. Like distal-less, aristaless is expressed at the intersection of Wingless and DPP stripes. Ectopic expression of aristaless is induced by ectopic wingless in regions expressing dpp. One or two cells expressing aristaless then invaginate with the formation of imaginal disc primordia, and are allocated to the distal-most element of appendages (Campbell, 1993).

In the leg disc, HH is secreted by posterior cells and acts at short range to induce dorsal anterior cells to secrete DPP and ventral anterior cells to secrete WG. Complementary patterns of decapentaplegic and wg expression are maintained by mutual repression. DPP signaling blocks wg transcription and WG signaling attenuates dpp transcription. This repression is essential for normal axial patterning because it ensures that the dorsalizing and ventralizing activities of DPP and WG are restricted to opposite sides of the leg primordium and meet only at the center of the primordium to distalize the appendage. A similar dorsoventral bias in the choice of dpp or wg expression is revealed by eliminating the activity of protein kinase A, an experimental intervention that mimics the reception of the HH signal. Constitutive activation of the WG signal transduction pathway by loss of Zeste white (Shaggy) kinase mimics the reception of WG signal, and is sufficient to bias dorsal cells to express wg rather than dpp (Jiang, 1996).

Different thresholds of Wg activity in the wing imaginal disc elicit different outcomes, which are mediated by regulation of decapentaplegic expression and result in alterations in the expression of homeotic genes. A high level of Wg activity leads to loss of all dorsal pattern elements and the formation of a complete complement of ventral pattern elements on the dorsal side of legs, and is correlated with repression of dpp expression. wg expression in dorsal cells of each disc also leads to dose-dependent transdetermination in those cells in homologous discs such as the labial, antennal and leg, but not in cells of dorsally located discs. When dpp expression is repressed by high levels of Wg, transdetermination does not occur, confirming that dpp participates with wg to induce transdetermination. These and other experiments suggest that dorsal expression of wg alters disc patterning and disc cell determination by modulating the expression of dpp. The dose-dependent effects of wg on dpp expression, ventralization of dorsal cells and transdetermination support a model in which wg functions as a morphogen in imaginal discs (Johnston, 1996).

Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both Eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).

During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).

The Bar homeobox genes function as latitudinal prepattern genes in the developing Drosophila notum. In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. The two Bar locus genes may belong to a different class of prepattern genes expressed latitudinally: it is suggested that the developing notum consists of checker-square- like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar genes is determined by wingless, whose expression is under the control of Decapentaplegic signaling (Sato, 1999).

The Drosophila notum is considered genetically divided into several longitudinal, side by side, domains whose boundaries are determined by pannier, wingless and iroquois expression (listed respectively from medial to lateral). To further clarify relative locations of pnr, wg and iro expression areas, third-instar larval and pupal future notum were stained with various combinations of molecular markers. In larval and pupal future notum, pnr-Gal4 is expressed medially and iro-lacZ laterally. pnr-Gal4 and iro-lacZ domains partially overlap one another, and wg-lacZ (or Wg) expression is noted in the pnr-iro overlapping region and its immediate neighbors. Bar homeobox genes may belong to an additional class of notal subdivision genes. Staining for BarH1 indicates that BarH1 is expressed latitudinally (anterior vs. posterior) in the anterior-most region of future notum and postnotum. BarH1 expression begins at early to mid third instar. Anti-Ac antibody staining and neur-lacZ expression indicates PS macrochaetae are situated in the vicinity of posterior-ventral corners of the anterior BarH1 expression domain. BarH1 and BarH2 are referred to as Bar collectively and the anterior portion of the prescutum or its precursor expressing Bar is referred to as Bar prescutum. The Bar expression domain overlaps that of pnr, wg and iro. Bar expression similar to that in wing discs is observed in haltere discs (Sato, 1999).

It is concluded that a checker-board-like subdivision of future notum is regulated by putative prepattern gene expression. Future notum may be divided into square subdomains in a checker-board-like manner, each with its own unique combinations of prepattern gene expression. Putative prepattern genes, iro and pnr, form longitudinal domains. Bar homeobox genes form the anterior-most domain. This is the first demonstration of the presence of latitudinal, front to back, prepattern genes in the notum. Bristle formation in each subdomain may be positively regulated by a region-specific combination of prepattern genes. Consistent with this, microchaetae formation in the anterolateral prescutum (the lateral Bar prescutum), where Bar and iro are coexpressed, requires the concerted action of Bar and iro (Sato, 1999).

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