armadillo
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).
The phenotype and molecular lesions generated by different arm
mutations have been compared. Severely truncated proteins retain some function; the degree of function is
strictly correlated with the length of the truncated protein, suggesting that the internally repetitive ARM protein is modular in function (Peifer 1990).
Analysis of double mutants demonstrates that Armadillo's role in wingless
signaling is direct, and that Armadillo functions downstream of both wingless and zeste-white 3 (Peifer, 1994a).
In embryos mutant for
armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in
wingless 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).
In
embryos mutant for hedgehog, fused, cubitus interruptus and gooseberry, expression of
engrailed is affected to varying degrees. However wingless expression in the latter group decays in
a similar way earlier than engrailed expression, indicating that these gene products might function in
the maintenance of wingless expression (van den Heuvel, 1993).
Programmed cell death plays an essential role in the normal embryonic development of Drosophila. One region of the embryo where cell death occurs, but has not been studied in detail, is the abdominal epidermis. Because cell death is a fleeting process, time-lapse, fluorescence microscopy was used to map epidermal apoptosis throughout embryonic development. Cell death occurs in a stereotypically striped pattern near both sides of the segment border and to a lesser extent in the middle of the segment. Approximately three-quarters of the dying cells appear in or immediately adjacent to the en stripe. The rest of the apoptotic nuclei are located in the middle of the segment. It appears that two rows of cells on either side of the segmental border die in each segment of the ventral ectoderm during stages 12-14. There is also an apparent clustering of apoptotic nuclei at specific locations along the dorsal-ventral axis. The number of cell deaths occurring within the 2-3 cells either side of the segment boundary was counted and this was compared with 5-6 cells in the middle of the segment. Considering that a segment is 10-12 cells across, this accounting partitions the segment into two equal-sized groups: the segment border cells and mid-segment cells. The segment border cells includes the en cells plus one or two rows of cells anterior and two or three cell rows posterior to the en stripe. Analysis of four time-lapse recordings of wild-type embryos, where eight segments were scored per embryo, shows that 73% of cell death occurs in the segment border cells and the remaining 27% occurs in the mid-segment cell (Pazdera, 1998).
This map of wild-type cell death was used to determine how cell death patterns change in response to genetic perturbations that affect epidermal patterning. Previous studies have suggested that segment polarity mutant phenotypes are partially the result of increased cell death. Mutations in wingless, armadillo, and gooseberry lead to dramatic increases in apoptosis in the anterior of the segment while a naked mutation results in a dramatic increase in the death of engrailed cells in the posterior of the segment. When wg function is disrupted during stage 11 (the fate specification phase of epidermal development) approximately two rows of cells die in the anterior-most portion of each segment during stages 12-14. These dying cells are approximately 6 rows of cells away from the Wg-secreting cells. An arm mutation that disruptes Wg signaling also eliminates the same rows of cells. These results show that Wg signaling is required at a distance to promote the survival of cells in the anterior of the segment. It is important to note that the cells expressing Wg prior to the temperature shift and those cells immediately anterior to Wg-secreting cells do not die and, therefore, may be more resistant to apoptosis than those cells at a distance. Mutations in the segment polarity genes gsb also lead to the death of cells in the anterior of the segment. However this death is more restricted to the ventral surface (Pazdera, 1998).
Strong mutations in nkd results in a cuticle phenotype opposite that of wg mutations. Previous genetic studies have suggested that the nkd gene product is necessary to suppress the domain of Wg function. nkd mutants also evince increased cell death, of which the majority is located in the expanded Engrailed-expressing domain. There is however no increase in cell death in the anterior of the segment similar to that seen in Wg mutants. Taken together these results suggest that two separate systems may be involved in promoting cell survival in the embryonic ectoderm: a nkd-dependent system that keeps posterior cells alive and a wg-dependent system that plays a similar role in the anterior of the segment. These two systems may not be mutually exclusive. It has been suggested that cell death in a wg mutant is suppressed by a nkd mutation (Bejsovec, 1993). These results provide evidence that segment polarity gene interactions play an intimate role in epidermal cell survival.
However, much more work is needed to further understanding of these processes (Pazdera, 1998).
An intermediate mutant allele of armadillo was used to explore the requirement for ARM in adherens junction assembly, cell polarity and morphogenesis in Drosophila. Adherens junctions cannot assemble in the absence of ARM; this leads to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to one another, round up, and apparently become mesenchymal. In arm mutants, alpha-catenin no longer accumulates at the plasma membrane, but instead is found diffusely in the cytoplasm. Shotgun, the Drosophila E-cadherin, is normally tightly localized to the plasma membrane and enriched in adherens junctions, but in arm mutants, Shotgun accumulation at the plasma membrane is reduced. Much of the remaining Shotgun accumulates within cells, presumably in the ER, Golgi, or endosomes. This may be a result of endocytosis. Mutant cells also lose their normal cell polarity. These disruptions to the integrity of the epithelia constitute a block to the appropriate morphogenetic movements of gastrulation. There is little or no germ band extention, and the ventral furrow and posterior midgut fail to invaginate normally. Crumbs protein does not appear to be required for initial assembly of adherens junctions, or for early cell polarity. Genetic interactions between armadillo and crumbs are additive, with no dosage sensitive interactions, suggesting that they may not be required together early in development. In contrast, reducing Shotgun levels suppresses the armadillo segment polarity phenotype. It has been suggested that this suppression reflects the fact that Armadillo's roles in adherens junctions and Wingless signaling are separable, and that under conditions where ARM is limiting, the reduction in the number of junctional complexes frees up some of the wild-type ARM, allowing it to function in the Wingless signaling. Armadillo is also required in oogenesis. The earliest defect seen in arm null mutant egg chambers is failure in adhesion between follicle and germ cells. Centripetal follicle cells frequently fail to migrate and separate the oocyte from the nurse cells, while nurse cells often fail to transfer their contents into the oocyte (Cox, 1996).
The zonula adherens (ZA) belongs to a family of actin-associated cell junctions called adherens junctions. Antibodies specific
to cellular junctions and nascent plasma membranes have been used to study the formation of the
zonula adherens in relation to the establishment of basolateral membrane
polarity. The same approach was then used as a test system to identify X-linked
zygotically active genes required for ZA formation. ZA formation
begins during cellularization; the basolateral membrane domain is established
at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome,
genes have been identified that are required for the formation of the ZA and the
generation of basolateral membrane polarity. Embryos mutant for both
stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to
establish the ZA, the formation of the monolayered epithelium is disrupted after
cellularization, resulting by mid-gastrulation in formation of a multilayered cell sheet.
Electron microscope analysis of mutant embryos reveals a conversion of cells exhibiting epithelial
characteristics into cells exhibiting mesenchymal characteristics. To investigate how
mutations that affect an integral component of the ZA itself influences ZA formation, embryos with reduced maternal and zygotic supply of wild-type Arm
protein were studied. These embryos, like embryos mutant for both sdt and baz, exhibit an early
disruption of ZA formation. These results suggest that early stages in the assembly of
the ZA are critical for the stability of the polarized blastoderm epithelium (Müller, 1996).
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 Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array, with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work has shown that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave. Evidence is presented for a second and orthogonal axis of polarity: this signal can be functionally separated from the A/P axis. The polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps -- the activity of one signaling molecule functions to establish the graded activity of a second signal. Ectopic Wg expression results in two significant effects. (1) Clones are generated with associated polarity inversions. (2) Although significant changes in retinal polarity are associated with the clones, the distance over which the effect is exerted is restricted to from between 7 to 2 ommatidial rows. Ectopic Wg clones have two distinct features with respect to their polarity effects: (1) the aberrant polarity is asymmetrically distributed in relation to the clone (greater changes in polarity occur in polar positions relative to the center of the clone), and (2) the potency of the Wg-expressing clones to induce polarity reversals show maximial polarity-reversal effects at the equator and minimal effects at the pole (Wehrli, 1998).
Other genes downstream of wingless also appear associated with eye Eq/Pl polarity. The product of the arrow (arr) gene has been placed in the Wingless pathway based on a number of criteria: Third instar larval eye imaginal discs (the precursors of the adult eye) from homozygous APC-like mutants were analyzed prior to development of the photoreceptor phenotype, to determine whether photoreceptor cell loss is a consequence of defects in the initial formation of neurons or from defects in their subsequent differentiation. The mutant eye discs were examined for expression of Neurotactin, a neuronal-specific transmembrane protein that is used as a marker for photoreceptor cells. Both at the level of patterning of the ommatidial arrays and at the level of individual photoreceptor cells, the Neurotactin antigen staining is normal in the Apc mutant. As well, retinal axonal projections to the optic lobe are intact. This indicates that the initial photoreceptor cell formation proceeds normally in Apc mutants and that the defect seen in the mutant adult is the result of neuronal degeneration. Further studies have determined that the neuronal degeneration in the Apc mutant is a result of programmed cell death (apoptosis). Expression of p35, a baculoviral protein that interferes with apoptosis by inhibiting the function of caspase proteases, rescues the Apc mutant phenotype. Despite the dramatic rescue from death of Apc mutant retinal neurons by p35, there is a striking feature that distinguishes the rescued photoreceptors. In wild-type adult eyes, the retinal neurons extend the entire length of the ommatidia, tapering gradually in diameter from their apical to basal regions. In contrast, while the rescued Apc mutant photoreceptors display an intact morphology at the
apical surface of the eye, at more basal levels, their diameters are dramatically shrunken, and they lose contact with their neighbors. This abnormal morphology is thought to reflect an arrest in differentiation and that this arrest accompanies the apoptotic death observed in the Apc mutant eye (Ahmed, 1998).
To test for Apc regulation of Arm activity genetically, Arm levels in the Apc mutant were lowered by replacing one wild-type copy of the arm gene with an null allele. When the
wild-type arm gene dosage is reduced by one-half in the Apc mutant, many neurons survive. The overall efficiency of rescue
achieved by halving the Arm dosage is similar to that obtained by ectopic p35 expression; however, one striking difference between the
two is that the neurons rescued in the arm heterozygotes appear completely normal from apex to base. Thus,
an inactivating arm mutation is a dominant suppressor of both the differentiation defect and the cell death of retinal neurons in the Apc
mutant. These findings provide genetic evidence that Apc functions to regulate Arm negatively and suggest that in the absence of Apc, an
increase in Arm activity results in both a differentiation defect and apoptotic cell death (Ahmed, 1998).
To test this idea, the effects of elevated levels of Arm on photoreceptor differentiation were analyzed using the UAS/GAL4 system to overexpress Arm in retinal neurons. Neuronal-specific overexpression of Arm under control of the elav-GAL4 transactivator results in photoreceptor loss that is phenotypically similar to, but weaker than, that seen in Apc mutants, with some ommatidia losing all photoreceptor cells, and most others having a reduction in their number. This reveals that death of photoreceptors is sensitive to Arm dosage and suggests the requirement for titration of Apc activity. To examine further the effects of Arm levels on photoreceptor death, mutations in the amino terminus of Arm were used that reduce the rate at which it is degraded. Neuronal-specific overexpression of an amino-terminal deletion of Arm that results in its stabilization, directed by the elav-GAL4 transactivator, results in the loss of all neurons in all ommatidia, and only pigment cells remain. Overexpression of stabilized Arm under control of the sevenless-GAL4 transactivator, which directs strong expression in 3 of the 8 photoreceptor cells, and weak expression in 2 others, results in photoreceptor loss of only a fraction of cells per ommatidium. Together, these findings demonstrate that overexpression of Arm within retinal cells committed to a neuronal fate results in their death and suggest that the Apc mutant phenotype is mediated by elevation of Arm activity (Ahmed, 1998).
Since Arm is a multifunctional protein, specific mutants of arm that allow a dissection of its distinct functions were examined in a Apc mutant background to delineate regions that are required in the induction of cell death. The armH8.6 mutant allele creates a truncation of Arm's carboxyl terminus that reduces Arm's ability to mediate Wingless signaling in vivo. Flies heterozygous for the armH8.6 allele were examined to determine whether deletion of the carboxyl terminus reduces Arm's ability to induce apoptosis in a Apc mutant background. Reduction of the wild-type gene dosage of arm by one-half due to the introduction of the null allele armYD35 rescues many photoreceptors from death and thereby dominantly suppresses the Apc mutant phenotype. In contrast, the mutant allele armH8.6 acts like a wild-type copy of the gene; all photoreceptors in all ommatidia degenerate completely. This unexpected finding suggests that the carboxyl terminus of Arm is not required for Arm's ability to induce photoreceptor death in the Apc mutant. A similar assay was utilized to analyze the requirement of other regions of Arm for the induction of cell death. A series of transgenes containing deletions within Arm have differential effects on Arm's roles in cell adhesion and Wingless signal transduction. These transgenes were expressed under control of the Arm promoter, and protein levels of these mutated forms of Arm were shown to be equivalent to wild-type Arm levels (Orsulic, 1996). Flies heterozygous for these mutated arm genes were examined to determine whether they retain the ability to induce apoptosis in a Apc mutant background. A series of deletions in Arm, termed S5, S15, and S12, eliminate Armadillo repeats 5, 8, and portions of 10 and 11, respectively. Each deletion severely disrupts Arm's ability to induce cell death in the Apc mutant. In contrast, a deletion in which Arm's alpha-catenin binding site is eliminated retains the ability to induce cell death. This finding is consistent with the results obtained by overexpression of stabilized Arm. Although this stabilized mutant Arm protein lacks amino acids required for binding to alpha-catenin, it retains the ability to induce photoreceptor death. Together, these findings suggest that while the carboxyl terminus and alpha-catenin binding site are dispensable for Arm-induced cell death, Arm's central repeats are required for this activity (Ahmed, 1998).
When a constitutively active from of armadillo is expressed in pangolin mutants, its action is largely inhibited. Such double mutants resemble pangolin mutants in that they have alternating denticles and naked cuticle, with portions of the lateral naked cuticle converted to denticles. However, they often have regions of naked cuticle intruding into the normal denticle belt at the ventral midline (van de Wetering, 1997).
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).
Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. This study reports a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. The cellular defects are described that underlie these problems, focusing on dorsal closure as an example. Further, it is shown that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. Ena localizes to adherens junctions of most epithelial cells, and it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. Possible models for Abl function during epithelial morphogenesis are discussed in light of these data (Grevengoed, 2001).
Several lines of evidence support the possibility that the morphogenetic defects of ablMZ mutants result, at least in part, from Abl action at adherens junctions. (1) The effects on dorsal closure, germband retraction, and head involution are strongly enhanced by reducing the dose of DE-cadherin. (2) The defects in cell shape during dorsal closure resemble, in part, those of arm mutants. (3) The defects in morphogenesis are suppressed by mutations in ena, which is primarily found at adherens junctions. (4) A reduction in junctional Arm and alpha-catenin is seen in ablMZ mutants. It is important to note, however, that any role for Abl at adherens junctions would be a modulatory one. It is not absolutely essential for adherens junction assembly or function. Of course, it remains possible that other tyrosine kinases may act redundantly with Abl. The relationship between the cadherin-catenin system, Abl, and Ena that may occur in epithelial cells could also exist in the CNS. Arm and DN-cadherin play roles in axon outgrowth in Drosophila, and in this role arm interacts genetically with abl (Grevengoed, 2001).
One target of Abl might be Ena, which could regulate actin dynamics in the actin belt underlying the adherens junction. Just as local modulation of actin dynamics likely regulates growth cone extension or stalling, the cell shape changes and cell migration characteristic of morphogenesis will require modulation of actin dynamics and junctional linkage. The idea that Ena may regulate cell-cell adhesion recently received strong support from work in cultured mammalian keratinocytes, where inhibiting Ena/VASP function prevented actin rearrangement upon cell-cell adhesion. This model is further supported by the demonstration that both Ab1 and Ena regulate actin polymerization at the adherens junctions of ovarian follicle cells in Drosophila (Grevengoed, 2001).
The JNK and Wg signaling pathways have been thought to function
in distinct domains, with JNK regulating dorsal closure and Wg
regulating segment polarity. In this study it has been demonstrated that Wg
signaling is critical for normal dorsal closure and that a
negative regulator of the JNK pathway, Puc, plays an
unexpected role in ventral patterning. This connection emerged
from the observation that reduction in Puc function suppresses
both the dorsal closure and ventral segment polarity
phenotypes of non-null mutations in armadillo
mutants, which exhibit dorsal closure defects. Mutations in puckered, known from previous work to antagonize Jun
N-terminal kinase in dorsal closure, also suppress, in a dose-sensitive manner,
both the dorsal and ventral armadillo cuticle defects during segmentation.
Activation of the Jun N-terminal
kinase signaling pathway suppresses armadillo-associated defects in segmentation. Jun N-terminal kinase signaling
promotes dorsal closure, in part, by regulating
decapentaplegic expression in the dorsal epidermis. Wingless signaling is also required to
activate decapentaplegic expression and to coordinate cell
shape changes during dorsal closure. Together, these results
demonstrate that MAP-Kinase and Wingless signaling
cooperate in both the dorsal and ventral epidermis, and
suggest that Wingless may activate both the Wingless and
the Jun N-terminal kinase signaling cascades (McEwen, 2000).
The simplest
hypothesis to explain these results is that puc suppresses arm by
hyperactivating the JNK pathway. Consistent with this, the puc
enhancer trap, a JNK target gene, is ectopically activated in
certain ventral epidermal cells in puc mutants. In addition,
activation of JNK signaling suppresses arm in a fashion very
similar to that resulting from reduction in Puc function, and
activation of a JNKKK in a wild-type embryo mimics weak
activation of the Wg pathway (McEwen, 2000).
Zygotic loss-of-function mutations in the JNK pathway fail
to appreciably affect ventral segment polarity,
however. This is reminiscent of the role of JNK signaling in
planar polarity. Loss-of-function
mutations in the JNK pathway suppresses dominant activation of
Fz or Dsh, but these mutations fail to exhibit planar polarity
defects themselves. This may result from functional
redundancy and/or cross-talk between different MAPKKs
and/or MAPKs. Such crosstalk occurs:
both DMKK4 and Hemipterous can activate Basket, while Drosophila p38
orthologs can phosphorylate Djun and ATF2, both known
targets of Bsk. Thus, the JNK signaling
pathway may function redundantly with other MAPK
pathways, both in planar polarity and in segment polarity. As
JNK-independent expression of puc has also reported, additional studies will be required to
assess the ability of Puc to antagonize other MAPK signaling
pathways. While these circumstantial arguments are consistent
with a role for the JNK pathway in ventral patterning, the
caveats raised by the lack of effects of loss-of-function JNK
mutations leave open the possibility that Puc has a role in
ventral patterning that is independent of its role in regulating
JNK activity - for example, it could directly regulate the
canonical Wg pathway (McEwen, 2000).
These data, combined with previous studies of JNK signaling, further suggest that Wg and JNK signaling act in parallel during dorsal closure. Both
pathways regulate dpp expression in dorsal epidermal cells and
are required for the proper coordinated cell shape changes to
occur. These data are compatible with several different models.
It may be that the two pathways both impinge on the same
process and the same target gene, but that they do so in
response to independent upstream inputs. However a potential direct
connection between the Wg and JNK pathways is suggested.
Using both genetics and in vitro studies, it has been demonstrated
that JNK pathway kinases act downstream of Frizzled and Dsh
in planar polarity and that Dsh can activate the JNK signaling
cascade directly. This
suggests that Dsh may function as a binary switch, deciding
between the canonical Wg pathway and the JNK pathway
during the establishment of segment polarity and planar
polarity, respectively. Both the
canonical Wg and the JNK pathways are required for proper
dorsal closure, and both pathways affect expression of the
same target gene, dpp. One plausible model accommodating
these data is that Wg, acting via Frizzled receptors and Dsh,
activates both the JNK pathway and the canonical Wg pathway
simultaneously and in parallel during both dorsal closure
and ventral patterning. The possibility that Wg activates
both pathways, while exciting in principle, remains quite
speculative, and must now be tested by more direct
biochemical and cell biological means (McEwen, 2000).
It also is possible that Wg functions as a permissive signal
required to allow other effectors to promote dpp expression.
For example, dTCF (Pangolin) could repress dpp expression in the
absence of Wg signaling by recruiting Groucho, a transcriptional repressor, to the dpp promotor. Wg
signaling might relieve this repression by displacing Groucho
with stabilized Arm. Consistent with this hypothesis,
constitutive activation of Arm fails to rescue the dorsal closure
defects of kayak/Fos-related antigen mutants. Thus activation of the canonical Wg
signaling pathway is necessary but not sufficient to promote
dpp expression. Wg signaling may thus only amplify JNK-dependent
expression of dpp in the dorsal epidermis.
One possible intersection between MAPK signaling
cascades and TCF-mediated repression has been reported. Transcriptional
repression of Wnt target genes in C. elegans depends upon
POP-1, a TCF family member. POP-1 repressor activity is
regulated by Mom-4, a Tak1-like kinase, and Lit-1, a Nemo-like
MAP kinase relative (Nlk: see Drosophila nemo). In mammalian cells, the
transcriptional activity and DNA-binding properties of TCF
can be repressed by Tak1/Nlk activation. Therefore, the
canonical Wg and MAPK/JNK pathways might converge at
dTCF, with MAPK kinase signaling affecting dTCF activity.
Additional studies will be required to assess the mechanism by
which these pathways interact (McEwen, 2000).
The current model suggests that a sequential series of cellular
events drive dorsal closure. Leading edge cells are thought to
initiate closure by elongating in the DV axis and upregulating
Dpp, thus signaling lateral cells to initiate similar cell shape
changes. The events of dorsal closure apparently do not proceed in
lockstep, with each event requiring the successful completion
of the previous event. The stereotypical cell shape changes are
lost in wg mutants; however, the lateral epidermal sheets
usually meet at the dorsal midline. In contrast, while cell shape
changes are initiated in arm mutants, though not in a
coordinated fashion, the epidermis does not close. Further, as
dpp expression in leading edge cells is lost in wg mutants, Dpp
may not be essential for dorsal closure. Finally, because dorsal
closure is more normal in wg than in JNK pathway mutants,
the JNK pathway likely depends upon activation by signals
other than Wg and must affect other processes in addition to
Dpp signaling. Further work is required to clarify the semi-redundant
mechanisms regulating dorsal closure (McEwen, 2000).
The Drosophila tracheal tree consists of a tubular network
of epithelial branches that constitutes the respiratory
system. Groups of tracheal cells migrate towards
stereotyped directions while they acquire specific tracheal
fates. This work shows that the wingless/WNT signaling
pathway is needed within the tracheal cells for the
formation of the dorsal trunk (DT) and for fusion of the
branches. These functions are achieved through the
regulation of target genes, such as spalt in the dorsal trunk
and escargot in the fusion cells. The pathway also aids
tracheal invagination and helps guide the ganglionic
branch. Moreover the wingless/WNT pathway displays
antagonistic interactions with the Dpp
pathway, which regulates branching along the dorsoventral
axis. Remarkably, the wingless gene itself, acting through
its canonical pathway, seems not to be absolutely required
for all these tracheal functions. However, the artificial
overexpression of wingless in tracheal cells mimics the
overexpression of a constitutively activated Armadillo
protein. The results suggest that another gene product,
possibly a WNT, could help to trigger the wingless cascade
in the developing tracheae (Llimargas, 2000).
arm has been shown to play dual but separable roles: one in
Wg signal affecting gene expression and the other in cell
adhesion. Null arm mutants have a tracheal phenotype that
is, in part, due to loss of its adhesive function. This phenotype can not be simply explained by
a decrease in cell adhesion.
The results of this work provide several indications that at least part of arm
function in the trachea is indeed due to its action in Wg
signaling: (1) an arm allele that specifically impairs
Wg signal produces a similar phenotype to that of an
arm null allele; (2) some tracheal defects are related to a
direct or indirect regulation of tracheal target genes; (3)
other members of the Wg pathway participate in the
same tracheal events (Llimargas, 2000).
Which part of the arm tracheal phenotype is due to its
adhesive role? arm has a role in cell adhesion by interacting
with shotgun, which encodes DE-cadherin. shg and arm
mutants are both defective in branch fusion. However, the cause of these two phenotypes does not
appear to be the same, since esg (a marker of fusion fate) is
normally expressed in shg mutants but
not in arm mutants. This indicates that the fusion cells are
normally specified in shg mutants, whereas arm is required
within the fusion cells themselves to regulate fusion markers.
Therefore, arm might have a dual function during branch
fusion: it is first required to activate the fusion fate through the
Wg signaling and later, through its interaction with shg,
it is required for the cell reorganizations that lead to branch
fusion (Llimargas, 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).
The abdomen of adult Drosophila consists of a chain of alternating anterior (A) and posterior (P) compartments which are themselves subdivided into stripes of different types of cuticle. Most of the cuticle is decorated with hairs and bristles that point posteriorly, indicating the planar polarity of the cells. This study has focused on a link between pattern and polarity. Previous studies have shown that the pattern of the A compartment depends on the local concentration (the scalar) of a Hedgehog morphogen produced by cells in the P compartment. Evidence is presented in this study that the P compartment is patterned by another morphogen, Wingless, which is induced by Hedgehog in A compartment cells and then spreads back into the P compartment. Both Hedgehog and Wingless appear to specify pattern by activating the optomotor blind gene, which encodes a transcription factor. A working model that planar polarity is determined by the cells reading the gradient in concentration (the vector) of a morphogen 'X' which is produced on receipt of Hedgehog, is re-examined. Evidence is presented that Hedgehog induces X production by driving optomotor blind expression. X has not yet been identified and data is presented that X is not likely to operate through the conventional Notch, Decapentaplegic, EGF or FGF transduction pathways, or to encode a Wnt. However, it is argued that Wingless may act to enhance the production or organize the distribution of X. A simple model that accommodates these results is that X forms a monotonic gradient extending from the back of the A compartment to the front of the P compartment in the next segment, a unit constituting a parasegment (Lawrence, 2002).
In clones mutant for arm or arrow, the expectation was that the Wg pathway in these two types of clones would be blocked. Two effects were noted. (1)The clones in the dorsal epidermis differentiated cuticle characteristic of the ventral epidermis: they made pleural hairs, and patches of sternite. Clones in all portions of the tergite, in both the A and P compartments, were transformed in this manner, indicating a general requirement for Wnt signaling to specify dorsal as opposed to ventral structures. Thus, in the wild type, all dorsal cells are probably exposed to at least low levels of Wg or some other Wnt protein.
(2) Such clones affect polarity: in the tergites, the mutant clones were normal at the rear of the clone but reversed in the front, with reversal extending outside the clone. One explanation for these polarity changes could be that, in the tergites, Wg normally acts to enhance the production of X. Thus cells deficient in the Wnt pathway would produce less X than normal, giving a dip in the concentration landscape for X, causing reversed polarity at the front of the clone. In the eye, both arm- and arrow- clones cause equivalent polarity reversals and a similar resolution has been offered: it is suggested that Wg might regulate the production of a secondary polarizing factor also dubbed X (Lawrence, 2002).
Thus, it is proposed that Wg helps to produce X, but that Wg itself is not X. If Wg were X, both arm- and arrow- clones should not be able to transduce it, and hence, should have random polarity within the clone. Moreover, the effects on polarity should be cell autonomous. Yet, as has been seen, these clones behave as if they have caused an altered distribution of X, rather than any failure to transduce X. Similar arguments apply to sgg- clones. In this case, the Wg pathway should be constitutively activated in all cells within the clone, preventing them from detecting a gradient of Wg protein. However such clones are not randomly polarized, indicating that they can still respond to graded X activity (Lawrence, 2002).
It is useful to compare the roles of Omb and Wg on X production. Omb is apparently essential for X production: omb- clones at the back of A show reversed polarity that extends all the way to the posterior edge of the compartment. By contrast, in arm- and arrow- clones, reversal occurs only in the anterior portions of such clones. Thus, it is inferred that arm- and arrow- cells located at the back of A can produce some X, even though they cannot activate the canonical Wnt pathway. Thus, it could be that Hh drives X production mainly through Omb, but also adds to the level of X produced through the induction and action of Wg. The combination of both Omb and Wg activity might extend the reach of the X gradient to encompass the whole A compartment, and possibly also further forward into the neighboring P compartment (Lawrence, 2002).
None of the previous studies has helped gain an understanding of how the P compartment is patterned or how its cells are polarized. smo- clones have no phenotype in the P compartment, confirming that Hh has no function there. In the embryo and imaginal discs, Hh crossing over from the P compartment induces the expression of Wg and Dpp in line sources along the back of A. Both proteins then spread back into the P compartment where they act as gradient morphogens to control P growth and pattern. Wg and Dpp are also produced at the back of the A compartment in each abdominal segment (albeit in distinct dorsal and ventral domains). Hence, by analogy with the embryo and imaginal discs, these morphogens seem to be the most likely candidates to pattern the P compartment here as well. If so, it would be supposed that in the tergites, Hh induces Wg and this Wg moves posteriorly across the AP compartment boundary into the P compartment where it activates expression of omb, thus specifying the zone of hairy cuticle (p3) and distinguishing it from p2 cuticle, which is bald. This hypothesis was tested in the following experiments (Lawrence, 2002).
If Wg activates omb in anterior regions of the P compartment, blocking the Wnt pathway in cells in the P compartment should block expression of omb. Expression of omb was therefore monitored in arrow- clones. omb is sometimes, but not always, turned off autonomously in the clone. Conversely, ectopic activation of the Wnt pathway should transform bald cuticle (p2) at the back of P into hairy cuticle (p3) normally found at the front of P. Indeed, some clones lacking the sgg gene become hairy if situated in the bald areas of P, apparently causing a transformation from p2 to p3 cuticle. But, clones expressing either tethered Wg or activated Arm, which should behave similarly, have no clear effects. Even so the positive results with arrow and sgg give support to the hypothesis that Wg stratifies the P compartment by working through Omb (Lawrence, 2002).
Human ß-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in
transduction of Wingless/Wnt signals. It has been hypothesized that ß-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of ß-catenin was expressed in MDCK cells, where it altered their
migratory properties and their ability to send out long cellular processes. The generality of this hypothesis was tested in vivo
in Drosophila. Three model systems in which cell migration and/or process extension are known to play key roles during development were examined: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able
to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/ß-catenin, and that Armadillo/ß-catenin does not play a general role in inhibiting cell migration or process extension (Loureiro, 2001).
The murine Nemo homolog Nlk has been implicated in
regulating Wnt signaling by repressing the Arm/ß-Catenin-TCF complex, a component of Wingless signaling. Whether such an interaction occurs in flies during wing vein formation was examined. The extra vein phenotypes observed in nmo mutant wings are very similar to those that have been previously described as resulting from overexpression of
Armadillo and both vertebrate ß-catenin and plakoglobin
in the wing. In addition, ectopic veins are produced as a result of ectopic wg and dsh expression. Constitutively active Armadillo (UAS-Arms10) expressed using the 1348-Gal4 driver leads to the
formation of moderate ectopic veins emanating from the
PCV, in addition to more severe ectopic veins along LII. These are both regions of the wing that are sensitive to nmo mutations and where similar
ectopic veins are observed in nmoadk. The phenotypes seen with overexpression of wg and arm are consistent
with the theory that Nemo is a negative regulator of Wingless signaling since loss of nemo mimics extra veins seen with overexpression of arm and wg (Verheyen, 2001).
Overexpression of both wildtype mouse Lef-1 (a TCF homolog) and a constitutive repressor form of dTCF (Pangolin) results in dominant negative phenotypes. The constitutive repressor form of dTCF (UAS-dTCFDeltaN) is unable to
bind Arm and represses wg-dependent gene expression. These findings suggest that expression of wildtype dTCF (UAS-dTCFwt) somehow interferes with a wingless-targeted transcription factor in a dominant negative way. Consistent with this, it is found that ectopic expression of UAS-dTCFwt using vestigial-Gal4 results in defects in the posterior wing margin, a phenotype seen with loss of wg signaling. Ectopic expression of the UAS-dTCFDeltaN using the 1348-Gal4 driver is lethal. However, homozygosity for nmoadk is able to rescue the lethality and the flies that emerged had reduced ectopic wing veins. This finding can be interpreted by taking into account the dual roles TCF plays in the nucleus. In nmoadk mutants, the negative regulation of endogenous dTCF may be reduced, leading to more wg-dependent signaling and the induction of extra veins similar to those seen with constitutive Arm expression. Expression of UAS-dTCFDeltaN in the nmoadk background most likely interferes with the de-repressed endogenous dTCF and block the induction of extra veins seen in nmoadk (Verheyen, 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 (1) Mutant embryos display a segment polarity phenotype.
To a variable extent, clones of armadillo and dishevelled induce polarity inversions on their equatorial side. The critical observation is that mutations in these recognized transducers of the Wg signal induce non-autonomus effects, consistent with their regulating the activity of a sendary signaling factor. This secondary signal is termed factor-X. Not only do arr, arm and dsh clones specifically affect the equatorial side, they are also more potent in achieving this at the pole than the equator. Thus it is inferred that factor-X activity is graded in the Eq/Pl axis but there is insufficient information to determine whether the activity is high at the equator and low at the poles, or vice-versa (Wehrli, 1998).
(2) Not only does the segment polarity phenotype appear like wingless, but in all other tissues examined (gut, leg, wing) arr phenotypes phenocopy wg mutants.
(3) An epistasis experiment performed in the eye places arr downstream of Wg. arrow clones cause non-autonomous polarity inversion in the Eq/Pl axis.
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).
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).
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).
Armadillo function during leg development
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).
Wg has been shown to be required for the induction of the thoracic limb primordium and other imaginal discs. To investigate a late role of Wg signaling, the functions of intracellular signal transducers of Wg were studied. The Drosophila homolog of ß-catenin encoded by armadillo (arm) plays dual roles, one as a mediator of Wg signaling by regulating transcription of various target genes, and the other as a component of Cadherin-dependent cell adhesion. Two alleles of arm were analyzed, one being null allele armYD35, and the other armH8.6, which is specifically defective in Wg signaling. Since both armYD35 and armH8.6 show the same phenotype, the function of Arm in Wg signaling, but not in cell adhesion, is required for leg disc development (Kubota, 2003).
Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).
Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galphao subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).
The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).
Further evidence for the role of Go in transducing Wg comes from the overexpression experiments. When Go is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If Go achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of Go should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, Go overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).
In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-Go wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of Go overexpression lose Dll expression to a level comparable with clones in which Go is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of Go requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).
β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, mouse strains were generated where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, the role of Wnt/β-catenin signaling in dorsal neural tube development was probed. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. The model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity (Valenta, 2011).
The development of the signaling-defective form of β-catenin built on understanding of Armadillo. The epithelium of the Drosophila wing imaginal disc served as a validation system for the separation of the signaling and the adhesive function. Clones of cells null mutant for Arm are eliminated 20-24 h earlier than clones expressing the double-mutant form of Arm. The loss of Arm's adhesive activity led to the destabilization of cadherin-based adherens junctions, as indicated by the delocalization of E-cadherin from the membrane. Clones with affected cadherin-based adhesion are rapidly extruded from the wing disc epithelium and eliminated. Cells expressing the double-mutant form of Arm, which preserves the adhesion function, survive longer than null clones, but are also smaller than clones rescued by the wild-type form of Arm, supporting previous observations that Wg/Arm signaling activity is essential for proper cell proliferation and survival. Hence, in the wing disc, loss of Arm affects the behavior of mutant clones on two levels: adhesion and signaling (Valenta, 2011).
After validating mutant mammalian β-catenin in biochemical and cell-based signaling assays, the mutations were introduced into the mouse germline. Using the resulting alleles in combination with tissue-specific knockout of the β-catenin gene, cell populations could be generated that correspond to the arm clones in the Drosophila wing disc. With the Wnt1-Cre driver, corrupted adherens junctions associated with morphological changes were observed in the dorsal neural tube that would prevent studying the fate of cells lacking canonical Wnt output. Double-mutant β-catenin, however, restores the cell and tissue structures and serves now as an adequate basis to assess the role of Wnt/β-catenin signaling as well as its cross-talk with other signaling pathways (Valenta, 2011).
Embryos with transcriptionally inactive β-catenin die at E7.5, just like embryos with total loss of β-catenin. Therefore, β-catenin's role of mediating cadherin-dependent cellular adhesion does not seem to play an important function during gastrulation, although it is a dynamic process dominated by epithelial-to-mesenchymal transitions (EMT) and cell migrations. Moreover, at these early stages, β-catenin expression is more pronounced in areas where no E-cadherin is detected (E-cadherin is prominent in extraembryonic tissues and β-catenin in the developing embryo). It appears, therefore, that mutant embryos are not significantly affected by the lack of β-catenin's adhesive role; rather, their phenotype results from the inability to form mesoderm. This, in turn, can be attributed to the loss of Wnt signaling-dependent expression of transcription factors that are important for mesoderm formation. Indeed, embryos lacking MED12 die at E7.5, also from a failure to develop mesoderm (Rocha, 2010), and MED12 is an important coactivator of β-catenin (Valenta, 2011).
Using Wnt1-Cre to remove wild-type β-catenin revealed situations in which the consequences of total lack of β-catenin do not correspond to those of substituting it with the adhesion-competent form. Striking differences were observed in craniofacial and mesencephalic structures. Lack of β-catenin results in the absence of the midbrain and anterior hindbrain structures, yet when the signaling function is specifically ablated, the dorsal part of the midbrain develops. More posteriorly in the CNS, the specific removal of Wnt/β-catenin signaling seems to affect the ground state and the differentiation potential of precursors in the dorsal neural tube. As expected when Wnt signaling is blocked, expression of its downstream target, CyclinD1, is lost. However, the cell cycle of dorsal neural tube progenitors does not seem to be halted. Previous studies using the same Wnt1-Cre driver in conjunction with a constitutively active form of β-catenin indicated that Wnt signaling promotes proliferation, while BMP signaling induces differentiation. Indeed, decreased differentiation of spinal cord precursors to sensory neurons was observed, based on reduced pSmad1/5/8 activity in those cells. Thus, Wnt signaling not only plays an essential role for maintaining dorsal neural tube precursors in a proliferative state, it also affects their differentiation (Valenta, 2011).
This analysis of the developing neural tube demonstrates how the double-mutant β-catenin allele can be used to specifically block canonical Wnt signaling and that it represents a powerful new tool to discriminate between the structural and signaling function of β-catenin. Additionally, single-mutant alleles will help to analyze the needs for individual β-catenin transcriptional coactivators during normal development and in disease and could thus be invaluable to validate therapeutic strategies targeting the interaction of β-catenin with its coactivators (Valenta, 2011).
Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).
JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).
In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).
The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).
Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).
As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).
Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).
JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).
Cardiac valves serve an important function; they support unidirectional blood flow and prevent blood regurgitation. Wnt signaling plays an important role in the formation of mouse cardiac valves and cardiac valve proliferation in Zebrafish, but identification of the specific signaling components involved has not been addressed systematically. Of the components involved in Wnt signal transduction, pygopus (pygo), first identified as a core component of Wnt signaling in Drosophila, has not been investigated with respect to valve development and differentiation. This study took advantage of the Drosophila heart model to study the role of pygo in formation of valves between the cardiac chambers. Cardiac-specific pygo knockdown in the Drosophila heart was found to cause dilation in the region of these cardiac valves, and their characteristic dense mesh of myofibrils does not form and resembles that of neighboring cardiomyocytes. In contrast, heart-specific knockdown of the transcription factors, arm/beta-Cat, lgs/BCL9, or pan/TCF, which mediate canonical Wnt signal transduction, shows a much weaker valve differentiation defect. Double-heterozygous combinations of mutants for pygo and the Wnt-signaling components have no additional effect on heart function compared with pygo heterozygotes alone. These results are consistent with the idea that pygo functions independently of canonical Wnt signaling in the differentiation of the adult interchamber cardiac valves (Tang, 2014).
Obesity develops in response to an imbalance of energy homeostasis and whole-body metabolism. Muscle plays a central role in the control of energy homeostasis through consumption of energy and signaling to adipose tissue. MED13, a subunit of the Mediator complex, acts in the heart to control obesity in mice. To further explore the generality and mechanistic basis of this observation, this study investigated the potential influence of MED13 expression in heart and muscle on the susceptibility of Drosophila to obesity. This study shows that heart/muscle-specific knockdown of MED13 or MED12, another Mediator subunit, increases susceptibility to obesity in adult flies. To identify possible muscle-secreted obesity regulators, an RNAi-based genetic screen of 150 genes was performed that encode secreted proteins; Wingless inhibition was also found to cause obesity. Consistent with these findings, muscle-specific inhibition of Armadillo, the downstream transcriptional effector of the Wingless pathway, also evoked an obese phenotype in flies. Epistasis experiments further demonstrated that Wingless functions downstream of MED13 within a muscle-regulatory pathway. Together, these findings reveal an intertissue signaling system in which Wingless acts as an effector of MED13 in heart and muscle and suggest that Wingless-mediated cross-talk between striated muscle and adipose tissue controls obesity in Drosophila. This signaling system appears to represent an ancestral mechanism for the control of systemic energy homeostasis (Lee, 2014).
The results reveal a role of muscle in systemic regulation of obesity via the function of MED13 in Drosophila. A genetic screen identified muscle-secreted obesity-regulating factors, including Wg, and demonstrated that Wg signaling in muscle is necessary and sufficient to suppress obesity. Furthermore, it was shown that a skd-null mutation dominantly enhances the arm phenotype in muscle and that wg is epistatic to skd, suggesting that Wg is a downstream effector of MED13 in muscle (Lee, 2014).
The results reveal that MED13 in Drosophila muscle functions to suppress obesity based on several criteria, such as histology, measurement of whole-body triglycerides, tolerance to starvation stress, and susceptibility to high-fat diet. Similarly, muscle-specific knockdown of MED12 also increases fat accumulation, suggesting that MED12 and MED13 function similarly in the control of fat deposition in Drosophila. The finding that MED12 and MED13 modulate energy homeostasis adds to a growing number of examples in which components of the kinase module of the Mediator complex influence metabolic signaling on an organismal level. For example, the other two components of the kinase module, Cyclin-dependent kinase 8 and Cyclin C, have also been reported as negative regulators of fat accumulation in flies and mice. The finding that the activity of MED13 in cardiac muscle regulates fat accumulation in Drosophila is consistent with earlier observation with mice and suggests that the function of cardiac MED13 in systemic regulation of fat storage represents an ancestral mechanism conserved in metazoans. Although it seems most likely that the effect of MED13 on obesity is mediated by overall changes in metabolism, it is also conceivable that changes in feeding behavior contribute to the obesity phenotypes that were observed. Knockdown of MED12 and MED13 using drivers that are active specifically in the heart using Tin-Gal4 or generally in all muscles using Mef2-Gal4 or Mhc-Gal4 commonly evoked obesity and MED13 can control metabolic signaling from the heart, consistent with prior conclusions regarding the functions of MED13 in the mouse heart. However, these Gal4 drivers do not enable reaching of conclusions regarding the specific role of somatic or visceral muscle in this signaling process because Mhc-Gal4 and Mef2-Gal4 are active in diverse muscle-cell types. Given that MED12 and MED13 are ubiquitously expressed, it is possible that they also act in nonmuscle tissues to regulate metabolic homeostasis (Lee, 2014).
It is hypothesized that muscle-secreted factors mediate the function of MED13 in Drosophila muscle to suppress systemic fat accumulation. To identify such factors, a screen was carried out for muscle-secreted obesity-regulating proteins using two different muscle drivers, Mef2-Gal4 and Mhc-Gal4. Six genes were identified that increased fat accumulation of flies in both screens by >60%, including the genes encoding (1) an antimicrobial peptide, Diptericin B; (2) a Drosophila homolog of Angiotensin converting enzyme; (3) a G protein-coupled receptor ligand SIFamide; (4) one of seven Drosophila Insulin/IGF homologs, Insulin-like peptide 4; (5) a JAK/STAT signaling ligand, Unpaired 3; and (6) Wg. Interestingly, it has been shown recently that MED13 and MED12 are required for the expression of Diptericin B in response to Immune Deficiency (IMD) pathway activation, suggestive of additional regulatory functions of MED13 and the genes identified from the current screens beyond obesity control (Lee, 2014).
This study demonstrated that Wg and its autonomous signaling activity, controlled by Arm, in muscle are necessary and sufficient for systemic regulation of obesity in vivo. Previously, the correlation between obesity and the expression of genes involved in the Wnt signaling pathway in heart has been raised from transcriptome analyses using heart biopsies from obese patients. Similarly, correlations between obesity and differential expression of genes for Wnt signaling, as well as genes for insulin sensitivity and myogenic capacity, were also found in skeletal-muscle samples from obese rats. These findings suggest that Wg signaling activity in muscle serves as an intrinsic rheostat for obesity control (Lee, 2014).
Muscle-specific arm knockdown caused partial-patterning defects in the embryonic musculature, and a skd-null allele dominantly enhanced this phenotype to complete lethality. Given the central role of Arm in Wg target gene expression, the findings are consistent with the established function of wg in the development of mesoderm and the embryonic musculature. The findings reveal a close functional connection between MED13 and Arm, suggestive of the role of MED13 in Wg target gene expression. In fact, in the developing Drosophila eye and wing, MED13 and MED12 are essential for Wg target gene expression, and the MED13/MED12 complex physically interacts with Pygopus, a component of the Wg transcriptional complex. Furthermore, MED12 hypomorphic mutant mice are embryonic lethal with impaired expression of Wnt targets. Therefore, the genetic interaction data along with these previous reports suggest that MED13 is a general component of the canonical Wg/Wnt pathway (Lee, 2014).
epistasis experiments indicate that muscle-secreted Wg functions downstream of MED13 in muscle to suppress obesity. Because both wg and arm in muscle are crucial for obesity regulation, one function of muscle-secreted Wg might be to act on muscle. Accordingly, the nonautonomous function of Wg to suppress obesity may occur through autonomous Wg signal activity in muscle. However, if MED13 functions at the level of transcriptional control of Wg target genes and the sole function of muscle-secreted Wg ligand is to activate the Wg signal 'in' muscle, Wg should be upstream of MED13, which is contrary to the epistasis studies. Based on the data, it stands to reason that muscle-secreted Wg should also act directly on a tissue other than muscle for its nonautonomous effect. If so, which tissue may be the target? Ectopic expression of Wg using a fat body-specific Dcg-Gal4 decreased larval abdominal fat body mass, which demonstrates the role of Wg signaling in the fat body for fat-mass regulation. Similarly, in mammals, autonomous activation of the Wnt pathway in adipose tissue decreases fat mass. Wnt signaling blocks mammalian adipogenesis in vitro, and, in mice, activation of the canonical Wnt pathway in adipocytes by ectopic expression of Wnt10b, a Wnt ligand, inhibits obesity. Furthermore, autonomous activation of the Wnt pathway in adipose progenitors with constitutively active β-catenin expression decreases fat mass. Therefore, the reduced fat mass in Dcg > wg larvae indicates that autonomous Wg signaling activity in the fat body serves as a regulator of fat mass. Considered together with the data showing that muscle-secreted Wg contributes to nonautonomous regulation of adiposity in vivo, it is concluded that muscle serves as a source of Wg to regulate adiposity by modulating Wg signaling activity in fat body. However, the possibility cannot be ruled out that the systemic effect of Wg from muscle is mediated through an alternative tissue, such as nervous system (Lee, 2014).
Wg acts on short- and long-range targets. Wg is highly hydrophobic and has been shown to diffuse through the extracellular space and act on long-range targets by associating with solubilizing molecules such as lipoprotein particles and Secreted Wg-interacting molecule. Furthermore, Wnt-1 has been identified in serum, and decreased Wnt-1 levels in serum correlate with premature myocardial infarction and metabolic syndrome, suggesting that Wg may act on remote organs as an endocrine factor. Therefore, this study proposes a model in which muscle-secreted Wg is a downstream effector of MED13 and acts both to activate the signal in muscle and to act on the fat body ultimately to achieve systemic inhibition of obesity (Lee, 2014).
Armadillo Function in Oogenesis
Continued armadillo Effects of Mutation part 2/2
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