Table of contents

Wingless targets at the wing margin

Two lines of evidence suggest that Distal-less and vestigial expression at the wing margin, as well as bristle specification, are organized by WG. First, using a temperature-sensitive mutation of wg, it is possible to remove wg activity at chosen times during wing development. Dll expression is abolished within 48 hours following a shift to the nonpermissive temperature, and vg expression is eliminated except for a thin stripe of cells straddling the D/V boundary compartment boundary. Second, ectopic expression of wg as well as ectopic activation of the Wg-signal transduction pathway, caused by eliminating the Shaggy/Zeste-white 3 activity, up-regulates the expression of Dll and Vg within the wing-blade primordium. Expression of a tethered WG protein, genetically engineered to be attached to the cell surface and thus unable to diffuse like normal WG protein, drastically alters the up-regulation of vg and Dll in sorrounding cells. Normal WG upregulates Dll in wild-type cells up to 10 or more cell diameters away from the WG source, while tethered WG up-regulates these genes only in their immediate wild-type neighbors. Thus normal WG can act as a long range morphogen, exerting a graded influence on vg and Dll in surrounding cells, while tethered WG exerts only a short-range, all-or-non influence on surrounding cells (Zecca, 1996).

Armadillo is required autonomously and continuously to mediate the response of wing cells to WG-Secreting cells located at a distance. Clones of arm mutant cells were generated in wing discs. These cells stop dividing and either die or are actively eliminated from the disc epithelium. When stained for either VG or DLL expression 36 hours after mitotic recombination is induced, none of the cells within such clones express either protein (Zecca, 1996).

Wingless targets neuralized at the wing disc margin. wingless-expressing cells induce only their immediate neighbors to express neur whereas wg-expressing cells exert a long range influence on Dll and vg expression. Thus WG appears to have the capacity to define multiple distinct outputs regulating the expression of target genes at different threshold concentrations. neur is induced in isolated cells close to the D/V boundary rather than in a swath of cells, all of which appear to be responding in a uniform fashion to WG. neur expressing cells are neuroblasts that arise from a population of proneural cells by a process of lateral specification. In this case, WG appears to define the population of proneural cells, and these cells then send signals other than WG, which are transduced by the Notch receptor, leading to the segregation of the neur expressing cells (Zecca, 1996).

Short-range interaction between dorsal and ventral (D and V) cells establishes an organizing center at the DV compartment boundary that controls growth and specifies cell fate along the dorsal-ventral axis of the Drosophila wing. The secreted signaling molecule Wingless (WG) is expressed by cells at the DV compartment boundary and has been implicated in mediating its long-range patterning activities. Does WG acts directly at a long-range to specify cell fates in the wing? To investigate this question, mutant clones of two components of the WG transduction pathway, dishevelled and armadillo were examined. Cells mutant for dsh show reduced levels of Dll and vg expression. Cells mutant for a temperature sensitive hypomorphic allele of arm, likewise show loss of expression of Dll and vg when larvae are shifted to non-permissive temperatures. Reducing WG levels at the margin reduces both the maximum level of Dll expression near the DV boundary and the distance from the DV boundary at which Dll can be activated. An intermediate level of WG activity is not sufficient to support the specification of wing margin bristles, suggesting that WG has fallen below a critical threshold for activation of AS-C gene expression, while remaining above the respective thresholds of activation for both Dll and vg. Thus, WG acts directly, at long range, to define the expression domains of its target genes, Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and vestigial at different distances from the DV boundary is controlled by WG in a concentration-dependent manner, with AS-C requiring the highest levels of WG. Dll, expressed in a wider range, requires the next highest level, and vg, which is expressed across the entire wing pouch, requires the lowest levels. It is proposed that WG acts as a morphogen in patterning the D/V axis of the wing (Neumann, 1997).

The expression of vestigial during wing development is regulated through two enhancers: the second intron or boundary enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect the patterns of expression directed by these regulatory regions: vgBE produces a thin stripe over the prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to the vgBE and which fill in the developing wing blade. Both, vgBE and vgQE, act as integrators of signaling systems that drive wing development and, in this manner, these regulatory regions determine the tempo and the mode of wing development (Klein, 1999 and references).

In addition to the activity of Notch, the activity of wg is required for the initiation of vg expression because in the second instar larval discs of wg mutants, the activity of the vgBE is absent over the region of the wing anlage. The possibility that Wingless acts on the vgBE is supported by the observation that a dominant negative version of DFrizzled2, a receptor for Wingless, reduces the activity of the vgBE. Altogether these results raise the possibility that Wingless has a direct input on the vgBE. Consistent with this the first 80 bp of the vgBE, which are required for the full activity of the enhancer, contain two putative TCF-1-binding sites associated with Wingless signaling. In contrast with ectopic expression of wg, which never leads to ectopic expression of the vgBE, ectopic expression of Delta leads to ectopic expression of the vgBE, but only where the levels of ectopic expression of Delta are high: within the developing wing blade and near the AP compartment boundary. An effect of Wingless on this enhancer can be detected when Wingless is coexpressed ectopically with Delta. Although Wingless alone has no effect on this enhancer, coexpression of Delta with Wingless extends the realm of activity of the vgBE into regions where expression of Delta on its own has no detectable effect. These results suggest that the major effect of Wingless on the vgBE is to collaborate with Notch/Su(H) signaling during the early stages of wing development (Klein, 1999).

In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted morphogen Wingless (Wg) at the dorsoventral compartment boundary. Late in wing development, Wg also induces a zone of non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin. How Wg coordinates the cell cycle with patterning has been investigated by studying the regulation of this growth arrest. Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the zone of non-proliferating cells (ZNC). The ZNC is composed of three subdomains, each about four cells wide. Cells in the central domain express wg. This domain is flanked by dorsal and ventral domains, which, in the anterior compartment, express Achaete and Scute. Cells in the ZNC stop proliferating 30 h before most of the other cells in the disc but re-enter the cell cycle for two or three divisions after pupariation. This arrest is seen by an absence of cells in the S phase of mitosis. The domain architecture of the ZNC is suggested by the expression of string and the G2 cyclins A and B. In the anterior compartment, cells in the dorsal and ventral domains do not express STG messenger RNA but accumulate high leves of G2 cyclins in the cytoplasm. Since Stg is required for mitosis and Stg and the G2 cyclins are degraded at cell division, these patterns are indicative of arrest in G2. In contrast, in the central domain CycA and CycB proteins are undetectable, but STG mRNA is expressed. This indicates that these cells may be arrested in G1. G1 arrest may be due to inactivation of dE2F, a factor required to activate the transcription of genes needed for DNA replication (Johnston, 1998).

Loss of wingless function during disc development abolishes both the G1 and G2 arrests and allows string expression in the anterior dorsal and ventral domains. Four observations suggest that the proneural genes achaete and scute regulate the G2 arrest of the ZNC:

  1. ac and sc are expressed in the stg-negative, G-2 arrested cells, but not in G1-arrested cells.
  2. Loss of Wg activity results in loss of expression of ac and sc.
  3. Ectopic Wg or activated Arm expands the domain of stg-negative, Ac-positive cells.
  4. Expression of ac and sc in the ZNC is extinguished just before re-entry into the cell cycle, after pupariation.

Together, these results indicate that Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute, which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing arrest at G2 in wg-expressing cells, resulting in their arrest in G1. To test whether Notch directly regulates the G1 arrest, discs were constructed lacking Wg activity, but expressing activated Notch in the ZNC. These discs do not form a ZNC at all. Thus, in the absence of Wg activity, Notch is not sufficient to induce a G1 arrest. It is noted that the string promoter contains putative Ac/Sc-binding sites, indicating that these basic helix-loop-helix proteins can repress string expression directly (Johnston, 1998).

cut and achaete are targets of shaggy signaling in the wing margin region reflecting the activity of wg and probably mediating its function. The functional relationship between these genes and wg is the same as that which exists during the patterning of the larval epidermis (Couso, 1994).

The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression, providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).

In wing development, decapentaplegic is expressed along the anterior-posterior compartment boundary. Early wingless expression is involved in setting up the dorsoventral boundary. Interaction between dpp- and wg-expressing cells promotes appendage outgrowth. optomotor-blind expression is required for distal wing development and is controlled by both dpp and wg. Ectopic omb expression can lead to the growth of additional wings. Thus, omb is essential for wing development and is controlled by two signaling pathways (Grimm, 1996).

The Wingless protein, in a role distinct from its embryonic segment polarity function, appears to be the earliest-acting member of the hierarchy and crucial for distinguishing the notum/wing subfields, and for the compartmentalization of the dorsal and ventral wing surfaces. The Wingless product is required to restrict the expression of the apterous gene to dorsal cells and to promote the expression of the vestigial and scalloped genes that demarcate the wing primordia and act in concert to promote morphogenesis (Williams, 1993).

araucan and caupolican, two members of the iroquois gene complex, are highly related proteins belonging to a new family of homeoproteins. ARA and CAUP regulate the pattern elements (sensory organs and veins) in wing imaginal discs by spatially restricting domains of expression of the proneural genes achaete and scute and the provein gene rhomboid. ara-caup expression is restricted to two symmetrical patches located one at each side of the dorsoventral compartment border. ara-caup expression in these patches is necessary for the specification of the prospective vein L3 and associated sensory organs. Here, ara-caup expression is mediated by the Hedgehog signal through its induction of high levels of Cubitus interruptus in anterior cells near the the AP compartment border. wingless is expressed in a narrow stripe of cells that stradles the DV compartment boundary of the wing disc corresponding to the prospective wing margin. The dorsal and ventral ara-caup L3 patches are separated by a gap that corresponds to the cells that detectably accumulate WG. Clones of mutant wg expressing cells spanning the gap between the L3 patches extend these patches toward the DV border and a narrow gap of only one or two cell diameters remains. Thus WG represses ara-caup expression at the prospective wing margin domain. Likewise repression by Engrailed is most likely to be responsible for the posterior border of ara-caup expression in the L3 patches (Gómez-Skarmeta, 1996).

Wingless is required for the expression of the proneural achaete and scute and subsequent formation of sensory bristles along the margin of the wing. In the developing wing margin of Drosophila, wingless is normally expressed in a narrow strip of cells adjacent to the proneural cells that form the sensory bristles of the margin. Wingless appears to be downstream of Notch, however. Loss of Notch from proneural cells produced cell-autonomous neurogenic phenotypes and precocious differentiation of sensory cells, as would be expected if Notch had a role in lateral inhibition within the proneural regions. Loss of scute expression and of sensory precursors is observed if clones mutant for Notch are in the normal region of wingless expression. These anti-proneural phenotypes are associated with the loss of wingless expression; indeed, this loss may be partially or wholly responsible for the anti-proneural phenotype. The role of Notch in the regulation of wingless expression precedes the requirement for lateral inhibition in proneural cells. Furthermore, overexpression of wingless with a heatshock-wingless construct rescues the loss of sensory precursors associated with the early loss of Notch (Rulifson, 1995).

In Drosophila wing imaginal discs, the Wingless (Wg) protein acts as a morphogen, emanating from the dorsal/ventral (D/V) boundary of the disc to directly define cell identities along the D/V axis at short and long range. High levels of a Wg receptor, Drosophila frizzled 2 (Dfz2), stabilize Wg, allowing it to reach cells far from its site of synthesis. Wg signaling represses Dfz2 expression, creating a gradient of decreasing Wg stability moving toward the D/V boundary. This repression of Dfz2 is crucial for the normal shape of Wg morphogen gradient as well as the response of cells to the Wg signal. In contrast to other ligand-receptor relationships where the receptor limits diffusion of the ligand, Dfz2 broadens the range of Wg action by protecting it from degradation (Cadigan, 1998).

Dfz2 binds and transduces the Wg signal in cell culture. To examine Dfz2's role in vivo, its expression pattern was examined in the developing wing. In the wing pouch, the region of the disc destined to become wing blade, Dfz2 is expressed in an inverse pattern to that of Wg, with the lowest levels found at the D/V boundary. This pattern is Wg-dependent, since Dfz2 expression near the D/V stripe is derepressed when Wg activity is blocked for 24 hr in wgts discs , as compared to wgts discs at the permissive temperature. To extend these findings, the Gal4/UAS system was used to express deleted versions of two Wg signaling components, Armadillo and dTCF (Pangolin), which constitutively activate (armact) or inhibit (dTCFDN) Wg signaling. Expression of armact throughout the wing pouch represses Dfz2 expression, while expression of dTCFDN in a Patched (Ptc) pattern (i.e., a stripe that runs perpendicular to the D/V wg stripe at the anterior/posterior boundary) leads to derepression of Dfz2 within the Ptc domain. Thus, Wg signaling is responsible for the lower expression of Dfz2 near the D/V boundary (Cadigan, 1998).

To test whether wg-dependent repression of Dfz2 expression is important for normal wing development, Dfz2 was misexpressed using UAS-Dfz2 lines crossed to various Gal4 drivers. This Gal4 driver is expressed in every cell in the wing pouch except those at the D/V boundary, and 1J3-Gal4/UAS-Dfz2 (1J3/Dfz2) expression overwhelms the endogenous graded Dfz2 pattern. Ectopic expression of Dfz2 throughout the wing disc causes an expansion of Wg target gene expression, resulting in "Hairy" wings. All surviving animals have ectopic bristles on their wing blades. These sensory organs are normally only found at the wing margin, the adult structure corresponding to the D/V boundary (bristle formation depends on Wg activity). The ectopic bristles in the anterior compartment are almost always of the slender or chemosensory type, though an occasional stout bristle is also observed. In the posterior compartment, the extra bristles are similar to the noninnervated ones found at the posterior margin (Cadigan, 1998).

The slender and chemosensory bristle cell fates are determined during the third larval instar by proneural genes such as ac, whose expression is wg-dependent. ac is initially expressed at mid-third instar in the anterior compartment in a stripe on each side of the D/V boundary. The cells destined to become bristle precursors gradually accumulate Ac to higher levels than their neighbors. Consistent with the hairy wing phenotype, 1J3/Dfz2 discs have a dramatic increase in cells expressing high levels of Ac. These cells are found at a greater distance from the D/V stripe than in controls and presumably cause the ectopic bristles seen in adult wings (Cadigan, 1998).

In the morphogen model of Wg action in the wing blade, ac is an example of a short-range target, requiring high levels of Wg signaling to be expressed. As was found for ac, the expression domain of another short-range target, Delta (Dl), normally expressed in a narrow stripe on either side of the wg stripe, is much broader in 1J3/Dfz2 wing discs. The model also states that Wg acts directly on long-range targets, such as Dll, which require less Wg signaling for activation and thus are normally expressed in wider domains centered on the D/V boundary. Dll is expressed at highest levels close to the Wg stripe and then at progressively lower levels at further distances. In 1J3/Dfz2 discs, the higher expression levels of Dll are seen much further from the stripe. Thus, misexpression of Dfz2 at high levels throughout the wing pouch expands the domains of both short- and long-range Wg targets (Cadigan, 1998).

The increased activation of Wg targets by misexpression of Dfz2 could be due to a heightened response of the cells to the Wg signal, or a constitutive activation of the signaling pathway. To address this, the effect of Dfz2 misexpression was examined in discs from wgts mutants reared at the restrictive temperature. Both Ac and Dll expression are dramatically reduced in these discs to levels seen in wgts discs grown under the same conditions. This indicates that the primary effect of Dfz2 misexpression is to potentiate the ability of Wg to signal to target cells (Cadigan, 1998).

The 1J3/Dfz2 experiments suggest that Dfz2 can transduce the Wg signal in the wing. Presumably, as has been shown in cell culture, this occurs through direct binding. To examine this in more detail, an altered Dfz2 cDNA was expressed in flies. The cDNA encodes the extracellular domain anchored to the cell surface via a glycerol-phosphatidyl inositol linkage. This is a truncated protein and consequently should not be able to transduce the signal to intracellular targets, since it lacks the seven transmembrane and intracellular domains. Therefore, if Dfz2 and Wg can interact in vivo, GPI-Dfz2 should block Wg signaling by binding the protein nonproductively. Expression of GPI-Dfz2 in the wing pouch does abolish the expression of the Wg targets ac and Dll and causes severe notching of the wings in adults. Experiments in the embryo and eye indicate that GPI-Dfz2 efficiently blocks Wg signaling in these tissues as well. These data are consistent with the hypothesis that Dfz2 is a physiologically relevant Wg receptor (Cadigan, 1998).

Misexpression of Dfz2 alters Wg distribution by increasing its stability. Wg is normally found at high levels in the cells expressing wg RNA but drops off sharply moving away from the stripe. Previously, it has been reported that Wg is undetectable more than 10 cell diameters from the D/V boundary. Using an affinity-purified Wg antibody, it is found that low levels of Wg are still detected up to 25 cell diameters away from the site of secretion. This Wg signal is punctate and favors the apical portion of the epithelium. It is not seen in wgts discs grown at the restrictive temperature, indicating that it is due to Wg and not a cross-reaction artifact. Thus, the physical distribution of Wg is consistent with the genetic evidence that it can directly affect gene expression over long distances (Cadigan, 1998).

Misexpression of Dfz2 or GPI-Dfz2 causes a dramatic posttranscriptional spread of Wg, with 1J3/Dfz2 discs, which overexpress Dfz2, having high levels of Wg several cell diameters away from the RNA stripe. A greater accumulation of Wg is seen with GPI-Dfz2, when compared to Dfz2. This could simply be due to a higher amount of truncated receptor present or caused by the inability of GPI-Dfz2 to internalize Wg after binding. While Dfz2 does not appear to facilitate the diffusion of Wg directly, its ability to protect Wg from degradation can indirectly promote the movement of Wg. Endogenous Dfz2 levels are modified by activating or inhibiting Wg signaling and determined the effect on Wg levels. Expression of dTCFDN in the posterior compartment of wing discs (which blocks Wg signaling) results in derepressed levels of Dfz2 transcripts and accumulation of Wg outside the RNA expression domain. Clones mutant for dishevelled (dsh) activity, which lack Wg signaling, also have a similar accumulation of Wg. Conversely, clones lacking zeste white 3 (zw3), also known as shaggy, which constitutively activate Wg signaling and are predicted to have repressed Dfz2 levels, have less Wg inside them, when compared to surrounding tissue. These results show that Wg signaling has a negative effect on the accumulation of Wg, which can be explained by the ability of Wg to inhibit Dfz2 expression (Cadigan, 1998).

It is proposed that the ability of Dfz2 to stabilize Wg, combined with the Dfz2 expression pattern, plays a major role in shaping the Wg morphogen gradient. Wg concentration initially decreases rapidly moving away from the D/V boundary but then plateaus at a low level. The data support a model where Wg is normally able to travel up to 25 cell diameters away from its source, consistent with genetic data on the range of action of Wg. This diffusion/transport of Wg does not appear to be enhanced by increased Dfz2 levels. However, the distribution of Dfz2 creates a situation where Wg is unstable near the D/V boundary and more stable at further distances. High levels of Dfz2 near the boundary, through expression of a transgene or derepression of the endogenous Dfz2 genes, stabilizes Wg so that elevated levels are observed. Repression of Dfz2 expression away from the boundary, via activation of the Wg signaling pathway, destabilizes Wg, resulting in lower levels found in these cells. Thus, Dfz2-mediated stabilization of Wg can, in large part, explain the biphasic nature of the Wg morphogen gradient. This work raises additional questions regarding Wg function. What is the biochemical basis for the enhanced Wg instability near the site of its production, and what is the basis for the enhanced Wg stability from its site of secretion? (Cadigan, 1998).

Dfrizzled3 is a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signaling, but in contrast to Dfz2, it is activated rather than repressed by Wg signaling in imaginal discs. Dfz3 is not required for viability but is necessary for optimal Wg signaling at the wing margin. Dfz3 was identified by characterizing a P-element line from a large scale Gal4 enhancer trap screen that allows direct visualization of gene expression patterns in living flies. A Gal4 insert found in the cytological position 1C exhibits an adult expression pattern resembling that of wg. The Gal4 expression pattern of this line has been visualized by a UAS-lacZ reporter gene. Depending on the tissue analysed, Dfz3-Gal4 is expressed in a broad domain centered over, or in a domain coinciding with, the wg expression domain. Dfz3 is expressed throughout the wing pouch but appears to be upregulated by Wg signaling at the presumptive wing margin and in a ring around the pouch. In the notum the expression is similar to the thoracic expression of wg. In the leg disc Dfz3 is expressed in a broad ventral wedge centered on the wg domain. Dfz3 expression in the eye disc is also coincident with wg expression and can be detected at the dorsal and ventral margins, which give rise to the head capsule (Sivasankaran, 2000).

Since the expression pattern in the wing disc strongly suggests that Dfz3 is a Wg target gene, an examination was carried out to see whether its expression is controlled by Wg signaling. When the Wg pathway is ectopically activated in clones of cells expressing a constitutively activated form of Armadillo, Dfz3 expression is upregulated in these cells in a cell-autonomous fashion. This supports the view that Dfz3 is a target of Wg signaling, and indicates that Wg acts directly and at long range in the wing pouch to control the expression of Dfz3 (Sivasankaran, 2000).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Short-range Hh signaling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3-L4 intervein tissue. N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue. These signals overlap spatially and temporally and lead to opposite fates. It is proposed that in and close to the DV boundary, N, Wg, and Hh signaling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin (Glise, 2002).

wingless regulates stripe expression to specify flight muscle attachment sites

In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

The Wg gradient in the presumptive notum controls sr transcription differentially and keeps different sr domains distinct. The actual regulation of sr by wg appears to be very complex. Lateral domain c appears more sensitive to perturbations in wg signaling as compared to b. This is interesting since all the cells of domain b receive uniform levels of Wg whereas cells at the posterior border of c, and those bordering the posterior sr domain receive high Wg. One possibility is that the latter cells block progress of the Wg gradient and thus determine responses of cells further away. This may be brought about by targeting Wg to lysosomes and degrading it, as in the embryo. Another possibility, not exclusive of the first, could be that the domain and levels of wg transcription determine the range and gradient of Wg. The precise definition of the domain of wg transcription could be by a mechanism similar to that used in the wing margin. While the data suggest that the posterior and lateral-most domain do not receive Wg and may lie outside its purview, the formal possibility still exists that wg affects these domains in some other unknown way (Ghazi, 2003).

wg controls sr expression by in segment border cells of the Drosophila embryo. Wg signaling restricts sr activation to a single row of cells. In the presumptive notum on the wing disc, hh expression is restricted to a very narrow region, which forms the posterior compartment. Its effects in the disc are mediated by dpp, which serves multiple functions. Dpp is required for induction of wg expression, as it positively regulates pnr, which in turn activates wg. However, once wg is induced, Dpp tightly restricts its domain. This antagonism is required for correct positioning of the DC bristles. This antagonism also defines domains of sr. It is unclear if dpp directly regulates sr, or its effect is by control of other genes. The similarity between sr phenotypes observed on expansion of wg expression, and in dpp mutants, is suggestive of its effects being mediated by wg only, but it is also possible that it influences sr expression directly (Ghazi, 2003).

Pnr, a GATA-binding protein normally functions as a transcriptional activator and is antagonized by Ush in its function. Loss of function pnr mutants show no sr expression in the domain covered by pnr. This, along with sr expansion in mutants of ush, would suggest that pnr activates sr in the notum, and is inhibited by ush. However, there is also loss of sr expression in pnr `gain of function' mutants. The reason for this is not completely clear. One possibility is that since the mutation causes an increase in wg activity in the region this may cause a down-regulation of sr. This is supported by a similar effect seen on misexpression of activated armadillo in the pnr domain. Results with both pnr and ush have been taken into account to suggest that pnr positively regulates sr and is antagonized by ush (Ghazi, 2003).

These results indicate that each sr domain is regulated by a combination of prepattern genes and signaling molecules. But, a precise description of the 'combinatorial code' for regulation of each sr domain is beyond the scope of this work and can be achieved by generation of domain specific markers for sr. Based on expression pattern data, and existing literature, it is suggested that high levels of Pnr, low (or absence of) Ush and moderate levels of Wg determine the initial induction of domain a. The distinction between medial (a) and lateral (b-d) domains is established by the presence of very high levels of Wg (the cells where the Wg gradient originates). Lateral expression domains are probably induced in domains controlled by the lateral prepattern gene iro. The differences between different lateral domains arise as a result of expression of different genes in the region. For instance, the lateral-most domain d appears to be regulated by ush and does not encounter Wg at all. Whereas, all cells of b receive uniformly moderate levels of Wg, only cells at the borders of c receive high Wg levels, and these differences result in the distinct identities of the two domains. Dpp, either through its effects on these regulatory genes and/or through direct effects on sr influences the process (Ghazi, 2003).

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G1 arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G1 arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G1-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G1-S progression. Thus, G1 arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G1 progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).

This investigation examines the mechanism by which Wg signaling promotes G1 arrest in the presumptive Drosophila wing margin. It was postulated that Rbf might mediate the ability of Wg to induce G1 arrest, since loss of Wg signaling promotes expression of dE2f1 target genes. Overexpression of Rbf can block this induction of dE2f1 target gene expression. Strikingly, loss of Rbf in the zone of nonproliferating cells (ZNC) prevents G1 arrest, as evidenced by ectopic BrdUrd incorporation in Rbf mutant clones. This requirement for Rbf in the ZNC is notable. Surprisingly few developing fly tissues display such an absolute requirement for Rbf to promote G1 arrest. To date, Rbf has been shown to be required to limit DNA replication in the embryo and in the ovary. However, in many tissues, loss of Rbf does not result in ectopic S phase; a likely explanation for this finding is that in other developing tissues, Rbf may function as one of several redundant mechanisms that function to promote G1 arrest. Such redundancy would help to ensure that the cell cycle is regulated tightly during development (Duman-Scheel, 2004).

In an attempt to better understand the mechanism by which Wg promotes Rbf function, this investigation uncovered interactions between dMyc and components of the Rbf/E2f pathway. Wg signaling normally inhibits dMyc expression in the ZNC. Ectopic expression of dMyc in the ZNC can induce expression of dE2f1 target genes, which can be blocked by the addition of Rbf-280 (a constitutively active form of Rbf). Thus, overexpression of dMyc, which results from loss of Wg signaling in the ZNC, must somehow inactivate Rbf. These data indicate that inhibition of dMyc expression in the ZNC is critical for Rbf function (Duman-Scheel, 2004).

The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G1-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G1-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G1-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G1 arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G1 arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G1 progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G1 progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G1 arrest in the ZNC. Still, it is possible that Wg promotes G1 arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).

It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).

Recent studies indicate that both dMyc and Rbf can regulate cellular growth in the Drosophila wing. dMyc induces cellular growth, whereas Rbf inhibits cellular growth and proliferation. dMyc can promote cellular growth in the presence of constitutively active Rbf, suggesting that dMyc can induce growth independently of the Rbf/E2f pathway. Such results are consistent with previous studies that indicate that Ras, which can induce growth by increasing levels of dMyc protein, also is capable of inducing growth in the presence of Rbf. It is likely that dMyc regulates growth through induction of genes encoding regulators of protein synthesis, such as ribosomal proteins and the DEAD-box helicase Pitchoune, as well as other proteins that regulate cellular metabolism (Duman-Scheel, 2004).

Wnt signaling is generally associated with the stimulation of cell proliferation during development and in tumor cells. However, in the ZNC, Wnt/Wg signaling actually promotes cell-cycle arrest. Ironically, in the ZNC, Wg signaling suppresses expression of dmyc; however, a cMyc reporter was found to be directly up-regulated by Tcf4 in a colon carcinoma cell line. Thus, Wg appears to be able to up-regulate Myc expression in some tissues and to repress it in others (Duman-Scheel, 2004).

The ability of Wg signaling to either activate or repress the same target gene in different situations has been observed in other cases. For example, in the developing Drosophila midgut, low levels of Wg signaling, in conjunction with Dpp, stimulate expression of Ubx and lab; high levels of Wg signaling result in the repression of Ubx and lab by means of the transcriptional repressor Teashirt. Thus, expression of Wnt target genes can be turned on or off in response to the modulation of Wg levels as well as by the presence or absence of the various proteins that can regulate transcription in conjunction with, or in response to, Wg signaling. Such flexibility is advantageous to a developing organism (Duman-Scheel, 2004).

Wg signaling can be modulated to affect expression of the same target gene differently in various situations. Moreover, Wg signaling can be modulated to promote or inhibit the different, somewhat conflicting cellular processes of patterning, growth, proliferation, and differentiation. The same is true for Hh signaling, which also regulates all of these cellular processes. Thus, it seems, at least in the case of Hh and Wg, that one signaling molecule can regulate many different types of cellular and developmental events. In order for various cellular programs to be implemented and coordinated during development, the way that a particular cell type responds to Wg or Hh signaling at any given time must be tightly regulated. The delicate balance between various processes that can occur in response to Hh or Wg signaling is likely maintained through tight control of the temporal and spatial expression patterns of Hh and Wnt targets and the molecules that regulate them (Duman-Scheel, 2004).

A regulatory receptor network directs the range and output of the Wingless signal

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg [Arrow (Arr) and Frizzled 2 (Fz2)] are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. This study analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. The experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by an in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, a 200 bp regulatory element in the arr locus was identified that can account for the Arr gradient, and it was shown that this is indirectly negatively controlled by Wg activity. Finally, the role of Frizzled 3 (Fz3) in was analyzed this system, and its expression, which is induced by Wg, was found to contribute to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, these results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system (Schilling, 2014).

During the development of a metazoan organism, signaling events are precisely regulated. One frequently employed mode of regulation is feedback loops. This study analyzed a network of feedback loops in the Drosophila wing pouch that regulate receptor abundance, and thus the range of distribution and signaling output of Wg (Schilling, 2014).

Receptors sequester their ligands and, thereby, impact upon the range of the signal. A transcriptional regulatory link between receptor expression and signaling activity, causing up- or downregulation of receptor levels in cells in response to the signal, can thus restrict or extend the signaling range. For example, the Hedghog (Hh) signal induces the expression of its receptor Patched (Ptc), a regulatory link which severely narrows the Hh activity. In the case of Wg, this study observed the opposite. Wg signaling appeared to extend the range of Wg distribution by transcriptionally downregulating expression of arr and fz2; downregulation of the receptors hinders the formation of Wg-Arr-Fz2 complexes. This allows superfluous Wg to diffuse further away from the source without being immobilized by its receptors. In agreement with this notion, a slightly narrower distribution was observed of extracellular Wg in discs that expressed Fz2 or Arr under the tubulinα1 promoter. Quantifying these observations in discs that compartmentally overexpressed the receptor, a subtle reduction of the decay length (corresponding to a slightly steeper Wg distribution) was observed in compartments that overexpressed the receptor compared with that in wild-type compartments (Schilling, 2014).

In apparent contradiction, a previous study has shown that high levels of Fz2 can stabilize Wg and promote Wg spreading; accordingly, this study observed an accumulation of Wg when repeating this experiment by overexpressing Fz2 using the GAL4-UAS system. These contradictory findings can be reconciled by taking into account the different strength of Fz2 upregulation in the two experimental setups -- Fz2 expression that is driven by the tubulinα1 promoter leads to a relatively mild upregulation of the receptor by, approximately, a factor of 2, whereas overexpression by using the GAL4-UAS system causes a much stronger overexpression. Presumably, Arr becomes the limiting factor in UAS-Fz2-overexpressing cells, a situation that might prevent the surplus Wg-Fz2 complexes from being internalized, thus causing an extracellular accumulation of Wg. If Fz2 is only moderately overexpressed, sufficient Arr protein might be available to allow this extra Fz2 to form Wg-Arr-Fz2 complexes, which are subsequently internalized, leading to a slight narrowing of the gradient because there is less free and diffusible Wg. Consistent with this notion, simultaneous strong overexpression of both of the receptors Fz2 and Arr, by means of Gal4, leads to a reduction of extracellular Wg levels (Schilling, 2014).

Although Wg signaling transcriptionally represses both Arr and Fz2, ubiquitous overexpression of Arr, or Fz2, had no phenotypic consequences. Unexpectedly, however, severe phenotypes arose upon mosaic expression of the tub>arr or tub>fz2 transgenes. Theoretical modeling and reporter gene analysis indicated that cells that had elevated receptor levels ectopically activated the pathway when situated close to wild-type cells. Apparently, the 'high-receptor-level state' allows tub-fz2 or tub-arr cells to engage in ligand-receptor interactions that depend on the 'low-receptor-level state' of their neighbors. One plausible explanation might be that tub-fz2, or tub-arr, cells bind to Wg that diffuses in from neighboring wild-type cells (Schilling, 2014).

The different outcome of clonal versus uniform alteration of the Wg pathway is reminiscent of observations that have been reported by Piddini and Vincent (2009), where loss of Wg signaling in the entire P compartment had no impact on the expression of low-threshold target genes but resulted in their repression, and in patterning defects, when Wg signaling was only clonally abolished. Piddini and Vincent also used different patterns of Wg receptor expression for their experiments, and they explained their findings by postulating that there is a Wg-induced, still to be identified, inhibitory signal that negatively regulates target gene expression in surrounding cells (Schilling, 2014).

In an additional layer of negative-feedback regulation in the wing pouch, Wg signaling activates the expression of the Frizzled family member Fz3. Fz3 seems to act as a negative regulator of Wg signaling by repressing Wg signaling readouts and downregulating Wg receptor levels. Various models could be envisaged of how Fz3 acts as an inhibitor of Wg signaling. As it has been demonstrated that Fz3 is able to bind Wg, Fz3 could work as a decoy receptor that acts as a molecular trap by binding to Wg without eliciting a signal. Decoy receptors are often part of negative-feedback mechanisms. In the Drosophila epidermal growth factor (EGF) system, the pathway inhibitor Argos is a target of EGF signaling and functions as a decoy receptor. In vertebrates, decoy Frizzled receptors have been identified that modulate Wnt signaling - secreted Frizzled-related molecules (sFRPs) have strong homology to the Frizzled extracellular domains. sFRPs inhibit signaling by directly binding to the Wnt ligands. No sFRP gene has been identified in the Drosophila genome (Schilling, 2014).

In another scenario, Fz3 could work as a negative regulator of Wg receptors. Its function could be analogous to that of ZNRF3 and RNF43 in crypt base columnar intestinal stem cells. These related E3 ubiquitin ligases have been shown to regulate the stability and levels of cell-surface Fz and LRP5/6, through internalization and lysosomal degradation of the receptor components in the presence of Wnt signaling. Several of the current experimental findings indicate that Fz3 might work as an inhibitor of Wg feedback at the receptor level - firstly, decreased Arr and Fz2 levels were observed in compartments that overexpressed Fz3, and secondly, Arr and Fz2 levels were increased in fz3 mutant wing discs. Most probably, Fz3 acts by more than one mechanism - cells that overexpressed Fz3 in the Wg stripe lead to Arr downregulation, whereas cells that overexpressed Fz3 outside of the Wg stripe lead to Arr upregulation. Furthermore, extracellular Wg was stabilized upon Fz3 overexpression. In a wild-type situation, this stabilization of Wg might contribute to a broader Wg gradient and promote signaling in the outskirts of the wing pouch. Taken together, these findings suggest that only Wg-bound Fz3 causes inhibition of the pathway (Schilling, 2014).

The post-translational regulation of Wg receptor levels was not the focus of this study, but substantial efforts were undertaken to further characterize the transcriptional regulation of the receptor genes. In particular, attempts were mead to identify the regulatory elements of these genes that mediated the feedback loops. The isolation of a 200 bp fragment of the arr locus and a 300 bp fragment of the fz3 gene (each of which was responsive to Wg signaling and drove reporter gene expression in a pattern that was reminiscent of the endogenous expression pattern) allowing an investigation of whether the Wg pathway controls these genes directly or indirectly; fz3 appeared to be a direct target of canonical Wg signaling, whereas arr did not. Pan-binding sites were dispensable in the minimal arr enhancer, indicating that either Arm regulates the transcriptional activity of arr through another DNA-binding protein, or that Arm and/or Pan transcriptionally induce one (or more) negative regulators that, in turn, regulate arr expression. Hence, although the Wg pathway has been reported to possess the capacity to directly negatively regulate transcription, it apparently does not use this mechanism to attenuate arr expression (Schilling, 2014).

Including transcriptional Wg receptor downregulation in the model led to a broader distribution of Wg - receptor downregulation by ligand-induced endocytosis consumes the ligand, this was not the case for transcriptional repression. The broadening of the Wg distribution area under a mechanism of transcriptional receptor repression might facilitate a robust signaling readout for high-threshold Wg target genes (Schilling, 2014).

A recent study indicates that the long-range Wg gradient might be less important for imaginal disc patterning than assumed previously. Hence, it is also conceivable that the receptor gradients are not essential, a notion supported by the finding that uniform misexpression of Arr or Fz2 in the wing imaginal disc had no phenotypic consequences. Nevertheless, it remains to be determined whether the Arr and Fz2 gradients are dispensable; the tubArr transgene is not able to rescue arr loss-of-function mutants (Schilling, 2014).

So far, most quantitative models of the Wnt-Wg pathway have focused on intracellular events, and only a few models have taken into account the spatial aspects of this signaling system. The model in this paper is the first to systematically study the roles of Wg-receptor complexes -- Wg-ArrFz2 and Wg-Fz3 -- in the spatial profile of Wg signaling, as well as being the first to be challenged experimentally by manipulations of the receptor levels. The cell-based modeling approach of ligand receptor interactions allowed varying of all parameters in a cell-autonomous manner, which has not been done in previous studies. This technique is, thus, an ideal tool to predict the impact of clonal conditions with cellular precision, which have historically formed the basis of experimental approaches in Drosophila but have also become increasingly available in vertebrates (Schilling, 2014)

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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