Axin
An examination was performed to see whether Drosophila Axin (Axn) produced by in vitro translation could
interact with fragments of Armadillo fused to glutathione S-transferase (GST). Axn specifically interacted with the Armadillo
repeat domain of Arm (amino acids 140 to 713), but not with its
NH2-terminal (amino acids 1 to 139) or its COOH-terminal (amino acids 714 to 843) domain. Pull-down assays with a
series of deletion fragments of Axn showed that a fragment of Axn
containing amino acids 459 to 538 binds to Arm. This region
corresponds to the beta-catenin-binding domain of Axin and
conductin/Axil and contains a small segment (amino acids 494 to 525)
that is highly conserved, suggesting that it may function in binding to
Arm (Hamada, 1999).
Mammalian Axin
family members interact not only with beta-catenin but also with GSK-3beta and
APC. In line with these findings, it has been found that the
RGS domain of Axn interacts with a fragment of D-APC (amino acids
757 to 1270). This region of D-APC corresponds to the region
of APC that interacts with beta-catenin, conductin, and Axin. However, Axn does not
bind to Zeste white-3/Shaggy (ZW3/Sgg; Drosophila GSK-3beta) (Hamada, 1999).
Immunoprecipitation of either Arm or Zw3 co-precipitates
the endogenous Axn protein from embryo lysates.
Immunoprecipitation of Axn also precipitates Zw3 protein; however, no Arm could be detected in Axn
immunoprecipitates from embryo lysates.
To confirm that Arm co-immunoprecipitates with Axn, Axn was overexpressed under the control of the heat-shock
promoter in Schneider 2 cells. Upon induction of the
Axn transgene, significantly more Zw3 and Arm co-immunoprecipitate
with Axn, thus demonstrating
that Axn forms a complex or complexes with Zw3 and Arm (Willert, 1999b).
The protein-serine kinase Shaggy(Zeste-white3) [Sgg(Zw3)] is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway; epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. The biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase has been investigated in two types of Drosophila cell lines and in embryos. Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. The level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of Drosophila Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins (Ruel, 1999).
Drosophila Armadillo plays two distinct roles during development. It is a component of adherens
junctions, and functions as a transcriptional activator in response to Wingless signaling. In the current model,
Wingless signal causes stabilization of cytoplasmic Armadillo allowing it to enter the nucleus where it can activate
transcription. However, the mechanism of nuclear import and export remains to be elucidated. Two gain-of-function alleles of Armadillo are shown to activate Wingless signaling by different mechanisms. The S10 allele localizes to the nucleus, where it activates transcription. In contrast, the DeltaArm allele localizes to the plasma
membrane, and forces endogenous Arm into the nucleus. Therefore, DeltaArm is dependent on the presence of a functional endogenous
allele of arm to activate transcription. DeltaArm may function by titrating Axin protein to the membrane, suggesting that Axin
acts as a cytoplasmic anchor keeping Arm out of the nucleus. In axin mutants, Arm is localized to the nuclei. Nuclear retention is
dependent on dTCF/Pangolin. This suggests that cellular distribution of Arm is controlled by an anchoring system, where various nuclear and
cytoplasmic binding partners determine its localization (Tolwinski, 2001).
Evidence is provided for the titration model, but focus is on potential cytoplasmic anchors that retain ß-catenin/Arm in the cytoplasm. Endogenous Arm accumulates in the nucleus in response to expression of DeltaArm, and the underlying mechanism appears to be independent of protein levels. DeltaArm functions downstream of zw3, and does not increase endogenous protein levels appreciably. These results point to a mechanism by which DeltaArm affects some component of the cytoplasmic retention machinery. axin may be this component, since its mutation leads to nuclear Arm accumulation, and its overexpression prevents it. Axin appears to be amenable to a titration model, because its function is highly dose dependent. Only maternal mutation of axin leads to a naked cuticle with a partial rescue by a paternal copy. Zygotic mutation doesn’t produce an embryonic phenotype. Overexpression leads to a wg phenotype only if expressed very early. Observations in tissue culture show that Axin is localized to the cytoplasmic membrane and the cytoplasm, but is excluded from the nucleus. Also, mutant forms of Arm lacking repeats that are required for Axin binding localize to the nucleus. Therefore, a model is favored in which DeltaArm directly titrates out Axin, leading to nuclear localization of endogenous Arm. DeltaArm retains arm repeats 3 through 8, shown to be required for Axin binding, and may sequester Axin away from endogenous Arm. This suggests a dual role for Axin, both as a scaffold for degradation and as a component of the cytoplasmic retention machinery (Tolwinski, 2001).
Studies have found that ß-catenin import is constitutive. They suggest a system of cytoplasmic and nuclear anchors that control the flow of ß-catenin into and out of the nucleus. However, prevention of import by cytoplasmic anchoring may be the regulated step, since export is probably controlled by APC. In resting cells, ß-catenin is observed mostly at the cell membrane, therefore it seems likely that localization of ß-catenin to this compartment prevents it from entering the nucleus. Axin has been observed to localize to the plasma membrane, as well as the cytoplasm, and is thus well positioned to function as an anchor. A strong nuclear localization of Arm is observed in experiments where no Axin protein is present. In contrast, overexpressed Axin prevents the nuclear accumulation of Arm normally associated with DeltaArm expression (Tolwinski, 2001).
A model is favored where the dynamic import and export of Arm is controlled by binding partners in the cytoplasm and the nucleus. Axin is involved in cytoplasmic anchoring, and dTCF/Pan is involved in nuclear retention. Arm retained in the cytoplasm is degraded unless it enters adherens junctions. In response to Wg, degradation stops, and Arm accumulates in the cytoplasm bound to Axin. Some Arm enters the nucleus where it binds dTCF/Pan. An equilibrium is reached as a result of active import and export, and inactive degradation. This is the situation in Arm stripes where diffuse staining throughout the cell is observed. However, the existence of anchoring offers a second level of signaling control that could induce a rapid and concentrated nuclear accumulation of Arm with no change in levels. Specific nuclear accumulation has been observed in Xenopus and sea urchin. Though levels were not measured, the striking lack of cytoplasmic ß-catenin is suggestive of a lack of cytoplasmic anchoring. Another response of this type may be what is observed in the epithelial to mesenchyme transition. Here, ILK is overexpressed in epithelial cells resulting in very high nuclear accumulation of ß-catenin without an increase in levels, suggesting the possibility of inhibition of cytoplasmic anchoring (Tolwinski, 2001).
Recently, two studies have suggested that APC is involved in the nuclear export of Arm/ß-catenin. APC contains a nuclear export signal (NES) which is required for efficient export of ß-catenin from the nucleus. Combining this result with the current data, it is proposed that there are at least two levels of control of Arm/ß-catenin localization involving cytoplasmic anchoring and active export. APC may play a role in preventing Arm/ß-catenin from accumulating in the nucleus due to dTCF binding. Both controls must be overcome to accumulate enough Arm/ß-catenin to activate transcription (Tolwinski, 2001).
Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).
The data argue for a different regulatory mechanism of Wg signal transduction, proceeding through the inhibition of the protein Axin, rather than through the inhibition of Zw3/GSK3β. Axin has been identified in both vertebrates and invertebrates as a negative component of the pathway. Later work established Axin as a critical scaffold protein required for the assembly and function of the degradation complex. This complex functions in the destruction of Arm/β-catenin by bringing the kinase Zw3 and Arm into close proximity, leading to the phosphorylation of Arm, and thereby targeting it to the proteasome for degradation. For efficient Arm degradation, both Axin and APC must be present in the complex. How Wg input controls activity of the degradation complex has never been properly established, although most models have focused on the inhibition of the kinase Zw3. It is also unclear whether Arm degradation always plays a central role in converting Wnt input into transcriptional responses. In sea urchins and mammals, the most obvious response to Wnt signaling is a relocalization of Arm protein from the cytoplasm to the nucleus; it has been shown that both Axin and APC have a profound effect on Arm localization that cannot be explained by their interaction with Zw3 or the degradation complex alone (Tolwinski, 2003).
Evidence is presented that the Wg signal can be transmitted through a posttranslational regulation of Axin accumulation. Despite uniform transcription of Axin using the UAS/GAL4 system, Axin accumulates to different levels in different cells across each parasegment. Cells with lower steady-state levels of Axin are those exposed to Wg input, and this was strictly dependent on Wg. Loss of Wg causes excess accumulation of Axin, whereas uniform Wg expression (and therefore signaling) lowers total Axin levels. The phenomena observed in embryos parallel earlier reports showing that Axin accumulation is affected by Wnt signaling in tissue culture cells. GSK3β phosphorylation of Axin leads to its stabilization. However, the actual role that phosphorylation plays appears to be more complex, since further work contradicted this finding. In the current experiments, the phosphorylation state of Axin was not examined in cells responding to Wg (those with low Axin levels), nor in those not exposed to Wg (high Axin levels). Therefore, whether modification may inactivate Axin or whether modification leads to removal of Axin by degradation cannot be distinguished. It was found, however, that Zw3 kinase activity is not necessary for the reduction in Axin accumulation that is observed; the Axin striping pattern is maintained in embryos that lack Zw3 function. These results argue for a link between Wg signaling and Axin accumulation that is independent of the Zw3-mediated degradation complex (Tolwinski, 2003).
Although Zw3 does not appear to be required for Axin degradation, the more upstream component, Arrow, appears to be important for this mode of Wg signal transduction. The cytoplasmic domain of Arrow interacts with Axin in the yeast two-hybrid system, an interaction also identified for one of the mammalian LRPs, mLRP5, whose rapid binding of mAxin is ligand stimulated. Binding data for Arrow are largely in agreement with the mammalian study, except that no contribution is found of the Zw3 binding region of Axin in binding of Arrow bait. Interestingly, full-length Axin fails to interact significantly with the Arrow C terminus in yeast and all the Axin clones isolated in the library screen lack sequences N-terminal to position 353. This finding suggests that an inhibitory domain is present in Axin, N-terminal to the Zw3 binding domain, and that this inhibitory domain prevents Axin from binding Arrow. It is possible that the Wnt signal necessary for the mouse Axin interaction with LRP5 induces a conformational change in Axin that removes, modifies, or otherwise displaces the inhibitory domain. In contrast, Armadillo bait significantly binds both full-length and truncated Axin. These data taken together with the demonstration in Drosophila that signaling leads to loss in Axin striping and a lowered steady-state level of Axin, suggest that the Arrow/LRP5 interaction with Axin induces a change in activity and/or stability of Axin (Tolwinski, 2003).
The prevailing view on Wnt reception states that Arrow/LRP5,6 function as coreceptors together with Frizzled proteins. It is well established that Frizzled proteins bind Wnt ligands and that this interaction is essential for Wnt signal transduction. Initial work on LRP6 extended this model in suggesting that Wnt provides a bridging function in assembling a complex of Frizzleds and Arrow, at least for the particular combination mFz8/mWnt-1/mLRP6. However, biochemically, such complex formation has also not always been confirmed. In addition, the functional significance for signaling of the observed ternary complex has not been demonstrated in vivo. Therefore an experiment was designed that tested whether, in vivo, physical proximity of Arrow and Frizzled-2 is sufficient for signaling. In fact, it was found that Frizzled-2 can initiate ligand-independent signal transduction. The constitutive activity of the Fz2-Arr[intra]chimeric protein is significant, since only expression of the fusion protein but not expression of the individual components (Fz2 and Arr[intra]) activate signal transduction. It is inferred that association of Frizzled2 with the Arrow C terminus is indeed a key step in signal initiation in vivo, and that the proximity afforded here by protein fusion also occurs during normal signaling. The Fz2-Arr[intra] chimera is uncoupled from the need for ligand to trigger the intracellular signal transduction cascade. Therefore, whether the Arrow extracellular domain participates in a true “reception” complex with Fz2 in Wg binding cannot be addressed. Nevertheless, Arrow, or at least its C terminus, likely interacts intimately with Fz2 during signal initiation at the membrane. In addition, activation of the pathway by the Fz2-Arr[intra] chimera proceeds through canonical pathway components, most notably requiring Disheveled, a result consistent with the finding that Dsh functions downstream of Arrow. In cultured vertebrate cells, one report has suggested that in some circumstances, LRP6 can induce Wnt signal transduction independently of Disheveled. In contrast, the experiment of overexpressing biologically active Arrow cytoplasmic sequences in the form of the Fz2-Arr[intra] chimera revealed a strict Dsh dependence, suggesting instead an obligate role for the Dsh protein at signal initiation by Arrow, and by extension, presumably by vertebrate LRP5,6 (Tolwinski, 2003).
Though the Fz2-Arr[intra] chimera clearly signals, it is not as active as a Wg-stimulated endogenous receptor complex. Presumably, and not surprisingly, the protein fusion will present a distorted topology to cofactors required in the signal initiation complex, and therefore is not optimally configured for initiating signal transduction. This may explain why the chimera retains some measure of reliance on endogenous Arrow, as is apparent from a reduced level of signaling in its absence (Tolwinski, 2003).
In order to study the Axin protein in embryos, antibodies were raised. Endogenous Axn protein is only detected with the Axin antibodies by immunoprecipitation followed by immunoblotting (IP-western). Two antibodies directed to distinct regions of Axin recognize the same set of bands with apparent molecular weight of 95 kDa, strongly suggesting that these antibodies detect the Axn protein. Furthermore, overexpressed Axn migrates at the identical position. At present, the difference in Axn giving rise to the two bands is not known. It is possible that various phosphorylation states generate the two species of Axn protein, as is the case for mouse Axin (Willert, 1999a). Alternatively, the two separately migrating Axn species could be the A1 and A2 forms of Axn. These forms of Axn consist of amino acid differences between two independent Axin isolates of Willert (1999b) and the previously published Axn sequence (Hamada, 1999). Form A2 includes the six amino acids SRSGSS while Form A1 does not (Willert, 1999b).
The time course of endogenous Axn expression in the fly embryo was examined. Immunofluorescence staining of whole embryos with the Axn antibodies produces weak staining. Thus, the Axn protein may be present at very low to undetectable levels or the Axn specific antibodies do not react efficiently in fluorescence assays. By IP-western, Axn protein was detectable at low levels during the earliest timepoints (0- to 1-hour old embryos), suggesting that some Axn protein is maternally contributed. At later timepoints, Axn protein accumulates to higher levels until it reaches a plateau at about 3-4 hours after egg laying (Willert, 1999b).
Wingless is critical for patterning and cell fate determination in embryonic
segmentation. Although embryos that are zygotically
mutant for Axn appear to have almost normal segment patterning, embryos devoid of
both maternal and zygotic Axn gene products are
completely naked, lacking all denticles on the ventral cuticle. Embryos that lack the maternal
Axn product but have received one paternal wild-type copy
of the gene have some denticles on the ventral cuticle, suggesting that
the zygotic Axn product can partially rescue the
Axn maternal deficiency. These phenotypes are
similar to those of embryos derived from homozygous zw3/sgg
female germ lines and to those of embryos ubiquitously expressing the
wild-type Wg or constitutively active Arm. Thus, Wg signaling is constitutively activated in embryos lacking maternal Axn (Hamada, 1999).
Wg is required for the organization of wing blade development,
especially for specification of the wing margin structure. Clones of Axn mutant cells
marked with a yellow mutation produce ectopic marginal
bristles cell autonomously. Wg also plays an essential role in organizing leg
structures; ectopic activation of Wg signaling induces supernumerary
outgrowth on the dorsal side of normal legs. The Axn clone also produces a supernumerary leg
from the dorsal side of the normal leg. Furthermore, Wg
signaling is required for the formation of sternites in the ventral
side of the adult abdomen, and its ectopic activation results in the
appearance of ectopic sternite structures. The same
phenotype is observed in an abdomen containing Axn clones. During wing disc development, Wg signaling is induced
along the dorsoventral compartment boundary in the wing imaginal disc.
Arm accumulates in the cytoplasm, associates with its partner,
Pangolin/DTcf, and activates expression of target genes such as
Distal-less (Dll). In Axn clones, the levels
of Arm are markedly enhanced in a strictly cell-autonomous manner. In addition, Arm is
localized predominantly in the cytoplasm and nuclei in the
Axn mutant clones, in contrast to the membrane
localization observed in wild-type cells. The levels of
Dll expression are also elevated in the Axn
clones in a cell-autonomous manner. These results suggest
that Axn negatively regulates Wg signaling by down-regulating
intracellular levels of Arm and that this regulatory mechanism is
essential for Wg signaling (Hamada, 1999).
To further examine the function of Axn, the
Axn gene was ectopically expressed using the GAL4/UAS system. In contrast to the phenotypes observed with
the Axn mutant clones, ectopic
expression of Axn induces notches in the wing, generation of a supernumerary leg
from the ventral side of the normal leg, and loss of the
sternite structure in the abdomen. In addition, when
Axn is expressed in the posterior compartment under the
control of engrailed-GAL4, Dll expression is
severely repressed in the posterior region of the dorsoventral
compartment border. Thus, ectopic expression of Axn exerts an inhibitory effect on Wg signaling (Hamada, 1999).
It is concluded
that Axn is required in vivo for the negative regulation of Wg
signaling. Of particular interest is the finding that the levels of
cytoplasmic Arm are highly and uniformly elevated wherever Axn clones are located in the wing discs. For
example, the accumulation of Arm in Axn clones is
observed not only around the region where Wg is secreted but also in the region where Wg is not supposed
to reach.
Together with the fact that Axn is ubiquitously expressed, these findings suggest that Wg activity is not required for
the effect of Axn. It is speculated that the Axin family of proteins
functions to establish a threshold to prevent premature signaling
events caused by Wg/Wnt and to restrict areas that are capable of
responding to Wg/Wnt (Hamada, 1999).
naked cuticle (nkd) is an embryonic lethal recessive zygotic mutation that produces
multiple segmentation defects, the most prominent of which is the replacement
of denticles by excess naked cuticle. This phenotype is also seen in embryos
exposed to excess Wg, as well as in embryos lacking both maternal
and zygotic contributions from any of three genes that antagonize Wg:
zeste-white3/glycogen synthase kinase 3beta (zw3/gsk3beta), D-axin
and D-Apc2. In nkd embryos,
hh and en transcripts initiate normally but accumulate in broad
stripes, including cells further from the source of Wg, which suggests that these cells are hypersensitive to Wg. Next,
a stripe of new wg transcription appears just posterior to the expanded
Hh/En stripe. This extra wg stripe
requires both wg and hh activity and is required
for the excess naked cuticle seen in nkd mutants. The
death of cells producing Hh/En contributes to the marked shortening of
nkd mutant cuticles (Zeng, 2000).
The posteriorly expressed signaling molecules Hedgehog
and Decapentaplegic drive photoreceptor differentiation
in the Drosophila eye disc, while at the anterior lateral
margins Wingless expression blocks ectopic differentiation.
Mutations in axin prevent photoreceptor
differentiation and leads to tissue overgrowth; both
these effects are due to ectopic activation of the Wingless
pathway. In addition, ectopic Wingless signaling causes
posterior cells to take on an anterior identity, reorienting
the direction of morphogenetic furrow progression in
neighboring wild-type cells. Signaling
by Dpp and Hh normally blocks the
posterior expression of anterior markers such as Eyeless.
Wingless signaling is not required to maintain anterior
Eyeless expression and in combination with
Dpp signaling can promote Ey downregulation,
suggesting that additional molecules contribute to anterior
identity. Along the dorsoventral axis of the eye disc,
Wingless signaling is sufficient to promote dorsal
expression of the Iroquois gene mirror, even in the absence
of the upstream factor pannier. However, Wingless
signaling does not lead to ventral mirror expression,
implying the existence of ventral repressors (Lee, 2001).
Two characteristics distinguish anterior from
posterior behavior in the eye disc: growth occurs in the
anterior, with the exception of the second mitotic
wave, and differentiation occurs in the posterior. Wg signaling regulates both of these
properties. Wg signaling promotes the growth of eye disc cells.
Loss of axin causes dramatic overgrowth
and outgrowth of cells in the eye disc, and this phenotype is due only to excessive Wg pathway activity, since it can be blocked by a dominant negative form of
dTCF/Pangolin. The strength of the phenotype may reflect higher levels
of Wg signaling than are induced by loss of sgg; perhaps Axin
contributes to retaining Arm in the cytoplasm, in addition to
promoting its phosphorylation. Vertebrate Axin has been
shown to associate with mitogen-activated protein kinase
kinase kinase 1 and activate the c-jun N-terminal kinase (JNK)
pathway. However, JNK signaling does not appear to be essential for the growth or differentiation of cells in the Drosophila eye disc,
and it does not contribute to the axin mutant phenotype in the eye. The ability of Wg signaling to promote overgrowth in the eye disc is
consistent with the reduction in the size of the eye
disc caused by loss of Wg signaling (Lee, 2001).
Loss of axin function at the posterior margin
results in outgrowths from the disc, over-riding the
normal control of organ size. axin mutant clones
also form smooth borders with surrounding cells,
suggesting that their ability to adhere to wild-type
cells is decreased. Growth control requires the
formation of normal junctions between cells, so it is possible that the outgrowth
results from this loss of adhesion. Because the
posterior margin is the site of dpp expression prior
to initiation, the outgrowth observed could also require Dpp signaling; in the
leg disc, overlap between dpp and wg promotes the
extension of a proximal-distal axis. However, punt;axin
double mutant clones show a similar degree of
overgrowth, suggesting that Dpp signaling does not contribute to this (Lee, 2001).
Early transplantation experiments and other studies have suggested that the region of the eye disc anterior to the morphogenetic furrow contains
intrinsic positional information; the subsequent
finding that Hh is essential for furrow movement has
been taken to mean that all this information
originates posterior to the furrow. The observations presented here challenge
this view by showing that Wg signaling can
generate a source of anterior positional information
that appears to attract the morphogenetic furrow
toward itself. Cells mutant for axin autonomously
express ey and other markers for the region anterior
to the furrow, including hth, tsh and mirr. Cells
adjacent to an axin mutant clone show a
reorientation of the pattern of Atonal expression and
cells at the internal border of the clone express Hairless,
which is likely to be activated by Dpp signaling
from adjacent wild-type cells. This suggests that axin mutant cells
produce a non-autonomous signal that maintains
nearby cells in an Ato-expressing state; since wg-lacZ
expression is activated in axin mutant clones, the
signal could itself be Wg (Lee, 2001).
In addition to providing anterior information to the eye disc,
Wg acts early in development to define its dorsal domain.
Dorsal wg expression is controlled by pnr, and ectopic eye
formation caused by loss of pnr. This can be blocked by restoring
wg, suggesting that wg is a downstream effector of pnr. In addition, Wg signaling is necessary to maintain the expression of mirr and
can induce ectopic mirr expression along the ventral margin. axin mutation has been used to clarify the relationships between pnr, wg
and mirr. Small clones of cells mutant for pnr maintain mirr
expression, showing that pnr does not have a direct effect on
mirr, but acts through one or more non-autonomous factors.
This is consistent with the expression of pnr in a smaller
domain than mirr. Restoring Wg signaling to pnr mutant eye discs, by making pnr;axin double mutant clones in a Minute background, allows
the expression of mirr; thus, no other factor downstream of pnr
can be essential for mirr expression (Lee, 2001).
hh was expressed dorsally in early eye discs and activation of the Hh pathway in ventral ptc clones leads to ectopic mirr expression. The results are consistent with two possible roles for Hh. Dorsal hh expression could be independent of pnr and contribute to mirr activation in the absence of both pnr and axin. The dorsal domain of hh expression in the eye disc is indeed not stably established until the second larval instar,
while wg and pnr are expressed dorsally from late embryonic
stages. Alternatively, Hh could act downstream of pnr but upstream of
wg to activate mirr. In support of this hypothesis, anterior
ventral ptc clones activate mirr non-autonomously, and have also been shown to activate wg expression. Because ventral
axin clones do not activate mirr expression, this mechanism
would imply that Hh activates factors in addition to wg that
allow ventral expression of mirr (Lee, 2001).
The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the
DNA-binding protein TCF (Pangolin
If pygo is a core component of Wg signaling in the fly, where does it act in the pathway? This question was approached using epistasis analysis. Initially, this was achieved via overexpression. In the absence of Wnt signaling, ß-catenin (and by extension Arm) is believed to be phosphorylated at serine and threonine residues at its N terminus via the GSK3ß/Axin/APC complex. If these residues are deleted or substituted, ß-catenin becomes resistant to degradation. In flies, these mutant forms of Arm (Arm*) activate Wg signaling independently of Wg. When placed under the control of the GMR promoter, Arm* causes a small eye phenotype similar to that of GMR-wg. Co-expression of pygo severely suppresses this phenotype. This strongly suggests that pygo overexpression blocks Wg signaling downstream of Wg-induced Arm stabilization (Parker, 2002).
To examine the position of pygo in the pathway using loss-of-function genetics, Axin;pygo double mutants were created. In Axin mutants, the signaling pathway is constitutively activated because of stabilization of Arm. As found in vertebrate systems, Axin functions in a complex with Sgg to phosphorylate Arm. The Wg target gene Senseless (Sens) was used as a readout in wing imaginal discs. In pygo clones, Sens expression adjacent to the dorsal/ventral Wg stripe is lost. In Axin clones, Sens is activated, no matter where in the presumptive wing blade the clones are located, since loss of Axin constitutively activates Wg signaling. In Axin;pygo double mutant clones, Sens expression is always lost. Thus, pygo acts downstream of Axin in this assay (Parker, 2002).
Epitasis analysis was also performed in the eye. At the beginning of the third larval instar, a wave of apical constriction of the columnar epithelial cells, called the morphogenetic furrow (MF) sweeps across the presumptive eye from the posterior to the anterior. Behind the MF, ordered clusters of photoreceptors develop. When Wg signaling is activated in the primordial eye, such as in Axin mutant clones, no photoreceptors are specified. Thus, the eye offers another test of whether pygo is epistatic to Axin (Parker, 2002).
Photoreceptor development, as judged by Elav staining, appears normal in pygo mutant cells. Even at higher magnification, no detectable difference was observed in the photoreceptor clusters between pygo-positive and pygo mutant cells. Clones that lacked Axin lack any evidence of photoreceptor development. This dramatic phenotype is completely rescued in Axin;pygo double mutant clones, clearly demonstrating that pygo is epistatic to (acts downstream of) Axin. This is consistent with the overexpression studies that suggest pygo acts downstream of Arm stabilization (Parker, 2002).
When Wg signaling is activated, Arm is stabilized and translocates to the nucleus. In Drosophila, it has proved very difficult to detect nuclear Arm, even in cells receiving high levels of endogenous Wg. However, Axin maternal and zygotic mutant embryos display high levels of nuclear Arm. Because attempts to make Axin;pygo germline clones were unsuccessful, clones in the wing disc were generated to investigate Arm levels and localization. In clones of cells lacking pygo, Arm is present at low levels at the cell periphery, consistent with its role in adherence junctions. In Axin clones, Arm protein levels are greatly increased in both the nucleus and cytoplasm. Axin;pygo double mutant clones also have high levels of cytosolic and nuclear Arm, though the nuclear levels of Arm appear slightly less than in Axin clones. These data are interpreted to mean that Arm is still stabilized in the absence of pygo (as would be expected if pygo acts downstream of Axin) and that, for the most part, pygo is not required for Arm nuclear import (Parker, 2002).
These experiments indicate that pygo acts downstream of Axin, an activated form of Arm and Arm nuclear import. Consistent with this, a tagged form of Pygo is nuclear. Taken together these data strongly suggests that Pygo acts in the nucleus, probably at the transcriptional level (Parker, 2002).
How pygo influences transcription of Wg target genes in the nucleus could occur in several ways. Simple explanations include the possibility that pygo could simply be required for the interaction of Arm with TCF, or for TCF to bind to DNA. However, the fact that Arm still accumulates to high levels in the nuclei of Axin;pygo mutant cells may indicate that the Arm/TCF/DNA complex still forms in the absence of pygo. It has been shown that expression of a dominant-negative version of TCF (which lacks the Arm-binding domain but retains its ability to bind DNA) prevents Arm nuclear accumulation. This supports the idea that TCF acts as a nuclear tether for stabilized Arm. Using this line of reasoning, Arm is still found in the nuclei of Axin;pygo mutant cells because it is still bound by TCF, which is still localized properly on the DNA. It should be noted a subtle reduction in nuclear Arm accumulation is seen in Axin;pygo versus Axin mutant cells. However, the small difference suggests that this effect may be indirect (Parker, 2002).
Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, genetic screens were performed to isolate additional dsh alleles and the potential role of protein phosphorylation was examined by site-directed mutagenesis. Two alleles were identified with point mutations in the Dsh DEP (Dishevelled, Egl-10, Pleckstrin) domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, it was found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, a screen was carried out for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. Many alleles of the transgene containing missense mutations were recovered, including mutations in the DIX (Dishevelled, Axin) domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. Second-site-suppressing mutations were recovered in axin, mapping at a region that may specifically interact with overexpressed Dsh (Penton, 2002). In addition to mutations in the UAS-Dsh transgene itself, the UAS-dsh misexpression screen yielded second-site modifiers on the third and fourth chromosome. Modifiers on the first and second chromosome could not be recovered due to the strategy of the screen. Five of the second-site modifiers map near axin and were indeed found to contain mutations within the axin gene. They behave as dominant suppressors of Dsh misexpression phenotypes in both the wing and eye but do not modify Wg or DFz2 misexpression phenotypes. In addition, these alleles are homozygous viable and have no phenotype when they are recombined away from UAS-dsh. Since Axin normally suppresses Wg signaling, and null axin alleles do not interact with UAS-dsh, it is inferred that these alleles specifically suppress overexpressed forms of Dsh but do not affect Dsh that is regulated by Wg signaling. This would imply that overexpressed Dsh works through a mechanism that is different from Dsh when activated by Wg. For example, overexpressed Dsh may interact with Axin through binding to a domain that is different from the Axin domain that interacts with Wg-activated Dsh (Penton, 2002).
The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).
In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).
Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).
The expression patterns of Eyes absent, Sine oculis and Dachshund in axin- and/or arm* mutant clones were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin- M+ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin- M+ clones. Consistent with these results, in arm*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).
Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).
Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).
In addition to wg signaling, hh signaling is also essential for SSC maintenance and proliferation. Hyperactive hh signaling causes follicle cell over-proliferation and abnormal differentiation of follicle cells. Disrupting hh signaling in SSCs by removing the function of hh downstream components such as Smoothened and Cubitus interruptus results in rapid SSC loss. Similarly, reduction or elimination of wg signaling also causes rapid SSC loss. Removal of patched, a negative regulator of the hh pathway, stabilizes SSCs. However, SSCs mutant for negative regulators for the wg pathway, sgg and Axn, are destabilized. All the evidence indicates that wg and hh may use different mechanisms to regulate SSCs in the Drosophila ovary (Song, 2003).
Constitutive wg signaling increases the division rates of early follicle cell progenitors in the germarium. When Fz2, dsh and activated arm are over-expressed, extra follicle cells accumulate in the ovarioles, suggesting that hyper-activation of wg signaling causes over-proliferation of follicle cells. Furthermore, sgg or Axn mutations cause over-proliferation of follicle cells, resulting in the formation of extra follicle cells that accumulate outside egg chambers. These cells are not mitotically active and usually assume some stalk cell characteristics. These results suggest that production of extra follicle cells by excessive wg signaling is because of higher mitotic activities of progenitors and/or SSCs in the germarium. It is important to note that sgg mutations are more potent than Axn in stimulating the proliferation of follicle cell progenitors. The different potencies may be because of differences in how these mutations affect wg signaling. Alternatively, because sgg negatively regulates hh signaling, sgg could be involved in negatively regulating both hh and wg signaling in the ovary. It has been demonstrated that excessive hh signaling causes extra follicle cells to accumulate outside egg chambers. Therefore, it might be probable that sgg is involved in regulating both hh and wg signaling pathways in follicle cells of the Drosophila ovary (Song, 2003).
This study also demonstrates that constitutive wg signaling disrupts the normal differentiation of somatic follicle cells. Mutant Axn or sgg follicle cells in and outside the germarium express higher levels of Hts in their membranes and tend to accumulate between egg chambers. In ovarioles that contain a majority of mutant follicle cells, germline cysts fail to undergo normal morphological changes necessary for proper encapsulation by follicle cells, although they are wild type, suggesting that the mutant follicle cells are defective in their interactions with germ cells. Although some of them are recruited to egg chambers, these mutant follicle cells have abnormal morphologies (e.g. smaller and irregular sizes). Huli tai shao is present not only on spectrosomes in GSCs, cystoblasts and fusomes in early germline cysts, but also on the membranes of somatic follicle cells. The abnormal follicle cell phenotype may be because of abnormal levels of Hts, which may prevent follicle cells from shape changes and growth. The extra mutant follicle cells accumulating outside egg chambers express Lamin C and do not divide similar to stalk cells. However, unlike stalk cells, they express high levels of Fas3. Similar to the mutant follicle cells in the germarium, the mutant follicle cells that are recruited to egg chambers also express high levels of Hts. Unlike the follicle cells in the germarium, the cells fail to express high levels of Fas3. These results indicate that constitutive wg signaling in follicle cells disrupts proper follicle cell differentiation (Song, 2003).
Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).
To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).
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date revised: 15 April 2007
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