The Wingless pathway In embryos mutant for
armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in
wg mutant embryos, suggesting that their gene products act in the wingless pathway (van den Heuvel,1993). dsh and porc act upstream of zw3, and arm acts downstream of zw3 (Siegfried, 1994).
A wg transgene, controlled by a heat-shock promoter,has been used for genetic epistasis experiments. It shows that wg acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed and cuticle differentiation (Noordermeer, 1994).
Loss of dishevelled function
in clones, in double heterozygotes with wg mutants, and in flies bearing a weak dishevelled
transgene leads to patterning defects which phenocopy defects observed in wg mutants alone.
Further, polarized cells in all body segments require dishevelled function to establish planar cell
polarity. The requirement for dishevelled in
establishing polarity is cell autonomous (Theisen, 1994).
A screening was carried out to identify components of the wingless signal transduction pathway; the screen sought dominant suppressors of the rough eye phenotype, caused by a transgene that drives ectopic expression of wingless during eye development. Essentially such a screen would identify cellular components that when mutated would fail to transduce signals from ectopically produced Wingless. Four strong suppressors were found that fall into two complementation groups. Two are recessive alleles of armadillo and two are recessive alleles of a locus on the fourth chromosome, designated as pangolin. A stronger allele of pan was isolated which shows strong genetic interactions when trans-heterozygous with either of the two isolated armadillo mutations, causing adult phenotypes characteristic of reduced wg activity. These results suggest that pan, like arm, encodes a component of the wingless transduction pathway, and also raise the possiblity that these components may physically interact (Brunner, 1997).
The HMG-box protein Pangolin (Drosophila Tcf) can function as either an activator or a repressor of Wingless-responsive genes depending on the state of the Wingless signaling pathway and the availability of Armadillo, Pangolin's coactivator. Mutations of Tcf-binding sites in the promoters of Drosophila Ultrabithorax or Xenopus siamois reduce the level of gene expression in the normal expression domain of the animal, showing that Pangolin and its vertebrate homolog act as gene activators.
In Drosophila, signal
transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with Pangolin
and activates expression of Wingless-responsive genes. In the absence of Armadillo, Pangolin acts as a transcriptional repressor of
Wingless-responsive genes, and Groucho acts as a corepressor in this process.
Reduction of Pangolin activity partially suppresses wingless and
armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. wingless null mutants completely lose epidermal engrailed expression before stage 10, but in homozygous wg embryos that are heterozygous for pangolin, some cells maintain en expression. This corroborates a repressive role for Pangolin in cells in which the Wg signalling pathway is not active. Reduction of Armadillo levels causes Pangolin to act as a repressor. Dominant negative Pangolin, lacking the Armadillo-binding regions, acts as a constitutive repressor.
Furthermore, overexpression of wild-type Pangolin enhances the phenotype
of a weak wingless allele.
Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes since Groucho physically interacts with Pangolin and is required for its full repressor activity. When the N-terminal region of Groucho is expressed in cultured cells, it localizes to the cytoplasm. Coexpression of either human Tcf-1 or Pangolin results in the localization of this truncated Groucho to the nucleus, consistent with a physical association between the proteins. Full-length Gro is constitutively nuclear, and as such, is not informative in this assay. The recruitment of truncated Groucho by Pangolin is very similar to the recruitment of beta-catenin, a known Tcf-binding partner. groucho mutations show dose-senstive interactions with both wg and arm. Reducing the dose of maternal Gro suppresses the wg null phenotype, whereas reduction of paternal Gro has no effect. Pangolin repression is shown to requires Groucho. Deletion analysis defines a minimal region in hTCF-1 (amino acids 176-359) that is capable of binding to Grg-5; this domain is separable from the Armadillo (Arm)-interaction domain (amino acids 4-63). XGrg-5, which lacks the C-terminal WD40 repeats of the longer Grg proteins, enhances the transcriptional activity of suboptimal amounts of Arm-XTcf-3 complexes. mGrg-5 has no intrinsic transactivation properties when fused to a Gal4 DNA-binding domain. The enhancement of transcription by XGrg-5 could probably be attributed to its interference with the repressive effects of endogenous Gro proteins. A deletion mutant of XTcf-3 that lacks the Grg-interaction domain is a tenfold more potent transcriptional activator than its wild-type counterpart, confirming the activity of endogenous corepressors of Tcf factors. Therefore, it is proposed that the balance between the activity of Gro and Arm controls cell-fate choice by the Wnt pathway in both vertebrates and invertebrates (Cavallo, 1998).
Loss-of-function mutant
phenotypes can be mimicked by the injection of double
stranded RNA. This method, referred to as RNA interference
(RNAi), was initially successfully used in C. elegans and more
recently in Drosophila.
RNAi was used to disrupt the function of Axin during
patterning of the embryonic cuticle, a process that requires
Wg signaling. The wild-type embryo secretes a cuticle
consisting of a repeated pattern of denticle belts with
intervening naked regions. Loss of wg leads to a
cuticle covered with denticles -- one lacking any naked areas. Overexpression
of wg in the embryo leads to loss of all denticle structures, i.e.
a naked cuticle. Disruption of Axin expression by RNAi leads to a
similar naked cuticle, suggesting that Axn
functions to down-regulate Wg signaling. Virtually all injected
embryos had extra naked cuticle, ranging from partially
to nearly completely naked. Control
injection of either sense or antisense single stranded AXN
mRNA does not cause any phenotypic changes.
As expected, wg RNAi produces a partial wg-like cuticle. Thus,
mimicking loss of Axn function by RNAi leads to
phenotypes similar to overexpressing wg, consistent with the
model that Axn is a negative regulator of Wg signaling (Willert, 1999).
A P-element insertion near the
beginning of the Axn gene has been shown to disrupt expression of the gene to
produce a loss-of-function allele of Axn. Embryos lacking zygotic Axn are still wild type and
only upon removal of the maternally contributed Axn gene
product is a naked cuticle revealed. Thus, the RNAi
experiments successfully disrupt the maternally contributed
Axn gene product and produce a phenotype identical to that
of a loss-of-function mutation in the gene (Willert, 1999).
To address further whether Axn regulates Wg signaling, the UAS-Gal4 system was used to overexpress Axn in various
tissues. The Daughterless (Da)-Gal4 driver expresses early
during embryogenesis; when
combined with UAS-wg, it produces a completely naked cuticle. Overexpression of
Axn using the Da-Gal4 driver produces a loss of wg-like
phenotype, a cuticle covered with denticles, consistent with overexpression of Axn
blocking Wg signaling in the embryonic epidermis.
To extend this study Axn was misexpressed in the wing
using the 69B-Gal4 driver. wg is expressed in a narrow stripe
along the presumptive wing margin where it is required for
proneural achaete-scute complex gene expression and for the
formation of margin bristles. Loss of Wg signaling
along the wing margin, as in the case of dsh loss-of-function
clones, leads to loss of these margin bristles and notches along
the wing. Overexpression of Axn in
the wing also produces this wing notching effect, a
result consistent with Axn interfering with the Wg signaling
pathway at the wing margin (Willert, 1999).
A screen for identifying genes interacting with Armadillo, the
Drosophila homolog of ß-catenin. Two viable fly stocks have been generated by altering the level of Armadillo available for
signaling. Flies from one stock overexpress Armadillo (Armover) and, as a result, have increased vein material and bristles in the wings. Flies
from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Armunder).
These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly
modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that
dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of
adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).
Mutations in 17 genes (26 deficiencies) were characterized that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 1 consisted of wingless pathway genes: Four known members of the pathway were identified: wg, dsh, zw3, and nkd. All interact in the direction expected (wg, dsh, and sgg/zw3 had already been tested prior to the screen). naked (nkd) has also been identified as a suppressor of Armunder and an enhancer of Armover; however, these interactions are much weaker than those seen for zw3M11 (Greaves, 1999).
Mutations in 17 genes (26 deficiencies) were characterized that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 1 consisted of wingless pathway genes: Four known members of the pathway were identified: wg, dsh, zw3, and nkd. All interact in the direction expected (wg, dsh, and sgg/zw3 had already been tested prior to the screen). naked (nkd) has also been identified as a suppressor of Armunder and an enhancer of Armover; however, these interactions are much weaker than those seen for zw3M11 (Greaves, 1999).
Among the interactors identified was naked (nkd), a mutant that has long been associated with excess Wg activity. The embryonic phenotype of nkd mutants is characterized by an excess of naked cuticle, just like that of sgg/zw3 mutants or embryos overexpressing Wg. In the case of sgg/zw3, this phenotype clearly follows from overactivation of the pathway, irrespective of whether endogenous wg is present or not. In contrast, wg/nkd double mutants resemble the wg single mutant, suggesting that nkd is upstream of wg. More precisely, since nkd mutants have enlarged stripes of Engrailed [and concomitant Hh] expression, nkd has been proposed to be a negative regulator of Engrailed expression. Broader hh expression in nkd embryos (as a result of widened engrailed expression) is thought to induce ectopic stripes of wg expression; this would cause the naked cuticle phenotype. However, in wing imaginal discs, wg expression is not controlled by engrailed or hh and therefore the finding that nkd modifies the Armover and Armunder phenotypes in the wing implies a more widespread role of nkd in Wg signaling. Maybe absence of nkd function renders cells more responsive to Wg. This would explain why endogenous Wg is required for the nkd phenotype to arise. It would also be consistent with the genetic interactions that are detected in the wing. Note that, so far, no function has been ascribed to nkd in disc development (Greaves, 1999).
Epistasis analysis was carried out to position Adenomatous polypopsis coli tumor suppressor homolog 2 (Apc2) with respect to other components of the signal transduction pathway. wg; Apc2DeltaS double mutant embryos (with Apc2DeltaS mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle, suggesting that Apc2 is downstream of wg. There are two possible explanations for the fact that the double mutant does not show the same phenotype as the Apc2 single mutant: either Apc2DeltaS is not null, or the negative regulatory machinery remains partially active in the absence of Apc2. If Apc2DeltaS is not null, it was reasoned that repeating the epistasis test with Apc2DeltaS in trans to a deficiency removing Apc2 (Df(3R)crb87-4) might further reduce Apc2 function, producing a double mutant phenotype more similar to that of Apc2DeltaS alone. However, when this was done, there was no change in the double mutant phenotype, suggesting that Apc2DeltaS may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of Apc2. Embryos maternally and zygotically mutant for both dishevelled (dsh) and Apc2 show a phenotype indistinguishable from the dsh single mutant, as do embryos maternally mutant for both dsh and Apc2 that are zygotically dsh/Y; Apc2DeltaS/Df(3R)crb87-4. Likewise, arm; Apc2 and Apc2; dTCF double mutants (derived from Apc2 homozygous mothers) are indistinguishable from arm or dTCF single mutants. Thus, dsh, arm, and dTCF all act genetically downstream of Apc2; this was expected for arm and dTCF, but was surprising for dsh (McCartney, 1999).
naked cuticle (nkd), a Drosophila segment-polarity
gene, encodes an inducible antagonist for Wingless. nkd was identified by the inabilility of known naked mutants to complement the lethality of enhancer trap line l(3)4869, which exhibits a weak nkd phenotype. The l(3)4869 insert was used to positionally clone ndk. In
fly embryos and imaginal discs nkd transcription is induced by Wg.
In embryos, decreased nkd function has an effect similar to excess
Wg; at later stages such a decrease appears to have no effect. nkd encodes a protein with a single EF hand
(a calcium-binding motif) that is most similar to the recoverin family of
myristoyl switch proteins. Nkd may therefore link calcium ion fluxes to the regulation
of the potency, duration or distribution of Wnt signals. Signal-inducible
feedback antagonists such as nkd may limit the effects of Wnt proteins
in development and disease (Zeng, 2000).
Overproduction of Nkd in Drosophila and misexpression of Nkd in the
vertebrate Xenopus laevis result in phenotypes resembling those of
loss of Wg/Wnt function. When P[Hs-nkd] is used to overexpress
nkd in otherwise wild-type embryos, rare cuticles with weak wg-like
denticle belt fusion phenotypes are observed, similar to those
seen when zw3 is overexpressed. Nkd is more potent
in a sensitized wg/+ background. In wgIl114/+
embryos, induction of P[Hs-nkd] before 4 h AEL results in decreased
en and wg expression. wgIl114/+
embryos are patterned normally, but practically all wgIl114
/+ embryos exposed to high levels of Nkd secrete cuticles with
denticle belt fusions and an excess of the predominant denticle type made
by wg/wg embryos (Zeng, 2000).
Misexpressing nkd during larval development using UAS/Gal4 transgenes results in adult phenotypes that are indistinguishable from many
wg loss-of-function phenotypes. The observed phenotypes include
(1) wing-to-notum transformations; (2) leg truncations and duplications; (3) loss, lateral displacement and disorientation
of sternite bristles; (4) haltere loss; (5)
ventral eye reduction; (6) loss of wing margin
(7) extra wing anterior crossveins (C. Conley and S. Blair, personal communication to Zeng, 2000);
and (8) loss of antennae. The gene dosage of the Wg pathway
influences the effect of ectopic Nkd: loss of one wild-type copy of porcupine
(porc), wg, dishevelled (dsh) or arm enhances, and
loss of zw3 and nkd suppresses, the UAS-nkd overexpression
phenotypes (Zeng, 2000).
The mechanism by which the Wingless signal is received and transduced across
the membrane is not completely understood. The arrow gene function is essential in cells receiving Wingless input. arrow
acts upstream of Dishevelled and encodes a single-pass
transmembrane protein; this indicates that it may be part of a receptor complex
with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related
protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, a new and conserved function is suggested for the LRP subfamily in Wingless/Wnt
signal reception. The position of arrow in intracellular Wingless siganl transduction
cascade was examined. Smooth cuticle is restored to arrnull embryos
in alternate segments when Dsh is expressed using Prd-GAL4, indicating
rescue of Wg signal transduction. This contrasts
with the overexpression of Wg, which has no effect in arrnull
embryos. These data suggest that Arrow acts downstream
of Wg but upstream of Dsh, because signaling, once activated by Dsh, no longer
requires Arrow. It remains possible that Arrow might normally act as a scaffold
and concentrate Dsh to an appropriate subcellular location; in this model,
flooding the cell with Dsh simply bypasses the requirement for Arrow. In either
case, Arrow is unlikely to act in a pathway parallel to Wg signal transduction
since (1) the arrow mutant phenotype is rescued by Dsh, a canonical Wg
signal transducer, and (2) loss of arrow function blocks signaling
even when excess Wg is presented. In addition, the fact that the arrow
phenotype is not suppressed by excess Wg distinguishes arrow from
the genes involved in proteoglycan-assisted presentation of the Wg ligand.
Each of the mutants affecting this step, sugarless, sulfateless
and dally, is substantially suppressed by providing excess Wg
ligand, showing that glycosaminoglycans are not essential for reception of
the signal but only increase the efficiency of ligand presentation to the
receptor. Together,
these data argue that Arrow is absolutely essential for Wg to signal, and
are consistent with a role for Arrow in reception rather than presentation
of signal (Wehrli, 2000).
In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation.
nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using
ectopic expression, it has been found that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components
Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg
signal, and the role of Wg is to relieve this inhibition. Double-mutant analysis shows that, in contrast to Zw3, Nkd acts to restrain signal transduction when the Wg
pathway is active . Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially
timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. These results suggest that Nkd acts
directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/ß-catenin and activate downstream
genes (Rousset, 2001).
To determine how Nkd impinges on the Wg pathway, the
ability of Nkd to block the action of the positive regulators Wg, Dsh,
and Arm was tested. To do so, advantage was taken of a Drosophila eye
misexpression system. Production of Wg in a
subset of photoreceptor cells throughout the eye using a
sevenless promoter transgene (P[sev-wg]) prevents
formation of interommatidial bristles in a paracrine fashion;
otherwise, the eye is normal. Previous Nkd misexpression experiments did not indicate whether Nkd blocks Wg synthesis, Wg distribution, or cellular responses to received Wg. To distinguish between these possibilities, the GAL4/UAS binary expression system was used to evaluate the effect of
Nkd (UAS-nkd) on Wg-mediated eye bristle suppression. Misexpression of Nkd alone using multiple repeats of the eye-specific glass (gl) enhancer (GMR) to drive the yeast transcription factor GAL4 (P[GMR-GAL4]) has no visible effect on eye development. However, the combination of
sev-wg with nkd misexpression results in nearly
complete suppression of the P[sev-wg]-induced bristle-loss phenotype. Nkd misexpression did not alter the levels or distribution of Wg antigen, indicating that Nkd is probably blocking signaling events downstream from Wg (Rousset, 2001).
The effect of Nkd on the downstream Wg pathway components Dsh and
Arm was also tested using the GMR-GAL4 system. Dsh
misexpression (UAS-dsh) produces small, bristle-less eyes
devoid of ommatidia. Nkd strongly suppresses the Dsh
misexpression eye phenotype, restoring numerous bristles and ommatidia. If the Dsh misexpression eye phenotype is Wg-dependent, its
suppression by Nkd could be due to Nkd acting on Wg rather than on Dsh
or other downstream components. Previous work suggests that the Dsh
misexpression eye phenotype is Wg-independent. To confirm the Wg-independence of the Dsh phenotype, a dominant-negative form of Dfz2 (UAS-GPI-Dfz2) was coexpressed with either sev-wg or UAS-dsh.
UAS-GPI-Dfz2 effectively suppresses sev-wg-induced
bristle loss in the eye. Coexpression of UAS-GPI-Dfz2 and UAS-Dsh results in some eye necrosis, but it has negligible effects on the UAS-dsh eye phenotype. These results confirm that the Dsh misexpression effect in the eye is Wg-independent. Therefore, rescue of the UAS-dsh phenotype by Nkd is not an indirect effect due to suppression of Wg activity (Rousset, 2001).
GMR-driven expression of UAS-armS10, a
constitutively activated form of arm, also produces bristle
loss and failure of proper ommatidial development. Nkd coexpression had no effect on the Arm misexpression phenotype. Dsh and Arm misexpression phenotypes are not
affected by simultaneous expression of UAS-lacZ, indicating
that suppression of the dominant eye phenotypes by Nkd was not due to
GAL4 titration. The ability of Nkd to block effects of
Wg and Dsh but not Arm suggests that Nkd is acting at the level of, or
downstream from, Dsh but not downstream of Arm (Rousset, 2001).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
wingless
continued:
Biological Overview
| Evolutionary Homologs
| Transcriptional regulation
|Targets of Activity
| Protein Interactions
| mRNA Transport
| Developmental Biology
| References
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