Wingless

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This mutual antagonism between the Wg and EGFR pathways segregates the early wing disc into notum and wing regions and provides an explanation for the wing-to-notum transformation that occurs in the absence of wg function. Multiple pathways contribute to the refinement of the wg expression pattern as larval development proceeds.

During the late second instar, the expression of the selector gene apterous ap overlaps ventrally with that of wg , to divide the developing wing field into two regions Williams et al. Subdivision of the wing field is initiated by the Notch-induced expression of vestigial vg from its boundary enhancer vg BE. After the establishment of the expression of wg and vg in the wing primordium, the wing blade begins to grow during the third instar. The expression of vg is induced in cells of the wing blade that lie outside of the domain of Notch signaling through its quadrant enhancer vg QE Kim et al.

Flies deficient for wg do not develop wings, and mutant patches of cells that cannot respond to the Wg ligand are eliminated from the wing field in the wing disc Chen and Struhl This suggests that there is an absolute requirement of Wg for growth of the wing. However, until recently it had been unclear whether cells of the wing disc required Wg signaling as an instructive signal or a permissive signal in order to proliferate.

Early studies suggested that although the misexpression of wg has mitogenic effects in the hinge region, it does not induce cell proliferation within the wing pouch but rather respecifies these cells to assume a wing margin fate Neumann and Cohen The ability of uniform expression of moderate levels of Wg to indeed promote growth throughout the presumptive wing region was shown only recently Baena-Lopez et al.

In this study, the investigators propose a model in which Vg-expressing cells expand their numbers by inducing their neighboring, non-wing cells to express Vg, provided these cells also receive the Wg signal, and thereby be recruited into the wing field through a feed-forward mechanism. In this scenario, Wg is proposed to act as a permissive signal to enable the recruitment of cells into the wing field to promote growth of the wing disc Zecca and Struhl a , b.

For a secreted signaling ligand such as Wg to qualify as a morphogen, it must form a graded distribution away from its source, directly act on cells at a distance rather than indirectly through a relay mechanism, and induce the commitment of cells in the field, as a function of their distance from the morphogen source, to distinct developmental fates through the expression of different sets of genes. Experiments performed in the wing disc suggest that Wg does indeed behave as a morphogen in this tissue. The modulation of Wg signaling through clonal analyses or ectopic expression of arm and dsh , positive regulators of the pathway, shows a cell-autonomous change in Wg-responsive target gene expression, regardless of the position of the cell from the source of the Wg signal Zecca et al.

These data suggest that the Wg ligand acts directly on cells distant from its source, and the expression of target genes is not activated via a secondary or relay signal. In addition, in contrast to the wild type secreted Wg, a membrane-tethered form of the ligand that is unable to diffuse away from its source, activates expression of target genes only in its immediate neighbors Zecca et al.

Furthermore, through the use of a temperature-sensitive allele, wg ts , it has been shown that the level of Wg activity minimally required to activate expression of ac , a high-threshold target gene; dll , a medium-threshold target gene; and vg , a low-threshold target gene; is progressively lower. Because the activity of Wg ts is decreased by raising the temperature , the expression of ac is lost at a temperature at which dll expression is retained, and as the temperature is further increased, the expression of dll is lost with no effect on vg expression.

In both cases, there is a concomitant reduction of the expression domains of target genes toward the source of the ligand as the activity level of Wg is reduced. Consistent with this result, low levels of ectopically expressed Wg are able to activate dll but not ac Neumann and Cohen These data suggest that Wg activates different target genes in a concentration-dependent manner and defines their expression domains through different activation thresholds. This does not exclude the possibility that activation of target genes requires other permissive signals, but it does argue that the level of the Wg ligand is the instructive signal.

Morphogen gradient of Wingless. The expression of distalless is graded throughout its domain, whereas vestigial expression is graded only at its edges. Wingless has been proposed to behave as a morphogen additionally in the embryonic epidermis and midgut, but whether it actually does so in these developmental contexts is unclear.

In the embryonic ectoderm, Wg secreted from the parasegment boundary adopts a graded distribution in the anterior direction up to four cells away to specify naked cuticle Bejsovec and Martinez Arias Although in this context Wg fulfills the criteria of having a localized source, forming a gradient, and acting at long range, there is no evidence for a concentration-dependent induction of target genes. In fact, the epidermal phenotype of wg mutant embryos can be rescued through the ubiquitous expression of a wg transgene Sampedro et al.

Additionally, in a wg mutant background, the overexpression of a membrane-tethered form of Wg from cells that normally express the ligand can recapitulate the normal range of signaling, confirming that the restricted diffusion of Wg is dispensable for patterning of the embryonic cuticle Pfeiffer et al. In the embryonic midgut, wg is expressed in the visceral mesoderm and directs the neighboring endoderm to differentiate into two different cell types: As the level of Wg is modified, there is a concomitant change in the domains of the large, flat cells and copper cells.

Additionally, the labial gene is expressed in a graded manner in the region of copper cells, consistent with the proposed concentration-dependent effect of Wg. However, the effects of Wg have not been shown to be direct in this tissue Hoppler and Bienz Thus, in the embryonic ectoderm and midgut, it is possible that Wg signaling does not define the pattern of the response but, rather, stabilizes patterns of gene expression that have been specified through other mechanisms. Secreted Wg is theoretically capable of passive diffusion at a relatively fast rate in all directions over a long range.

However, the graded distribution of Wg forms at a slower rate over a shorter range than that predicted through free diffusion and directionally along the epithelial surface. Moreover, the contrasting range of its activity during embryogenesis 4 cell diameters Bejsovec and Martinez Arias ; Pfeiffer et al. Recent experimental and theoretical studies favor a model in which the Wg gradient is formed and maintained through the combined effects of restricted diffusion and endocytosis.

Wnt signaling pathway - Wikipedia

Once secreted, the Wg molecule undergoes restricted diffusion, as opposed to free diffusion, because of its interactions with lipid-based transport proteins, receptors on the surface of membranes, and heparan sulfate proteoglycans of the extracellular matrix Lin and Perrimon ; Baeg et al. We here review the evidence for the endocytosis-mediated regulation of the Wg gradient in the Drosophila embryo and wing disc. At the subcellular level, Wg protein can be detected not only in the extracellular environment, but also within intracellular vesicles and multivesicular bodies in non-secreting cells.

This clearly indicates that the Wg ligand is internalized and suggests that its gradient is regulated through endocytosis. The endocytic regulation of the Wg gradient has been investigated using mutations in components of the endocytic machinery. The formation of the Wg gradient in the embryo is a result of asymmetric endocytosis and trafficking to lysosomes, and perhaps transcytosis.

As previously described, during embryogenesis, the initially symmetrical Wg gradient becomes asymmetrical due to increased Wg degradation in cells posterior to its source Fig. Evidence for this model comes from experiments performed using an HRP horse radish peroxidase —Wg fusion protein expressed under the control of the endogenous wg promoter. Unlike the Wg portion of the fusion protein, the HRP moiety is stable throughout the endocytic pathway and thus serves as a tool to monitor Wg degradation in vivo.

In cells anterior to the HRP-Wg source, both HRP and Wg can be detected in intracellular vesicles presumed to be early endosomes , whereas only HRP can be detected in vesicles presumably late endosomes in cells posterior to the source. Moreover, posterior to the Wg source, many more HRP-positive intracellular compartments can be detected that extend beyond the Wg protein gradient, confirming that the degradation of Wg through the endocytic pathway limits the range of its gradient Dubois et al. In deep orange dor mutants that have impaired trafficking to the lysosome, Wg accumulates in multivesicular bodies Piddini et al.

Lastly, in clathrin heavy chain chc mutants that cannot initiate clathrin-mediated endocytosis, the Wg gradient extends in the posterior direction, suggesting that clathrin is normally required for establishing and maintaining the asymmetric distribution of Wg Desbordes et al. However, there is also contradictory evidence that suggests a role for endocytosis in Wg dispersal in the embryo. In temperature-sensitive shibire ts which encodes Dynamin, an essential component of both clathrin and caveolin-mediated endocytosis mutant embryos that are shifted to the restrictive temperature, the Wg gradient is reduced to a narrow range around the Wg-expressing cells Moline et al.

This narrowed Wg gradient in shibire ts embryos is not due to any effect on the secretion of the Wg ligand or its stability in non-secreting cells but, rather, can be attributed to an effect on the transport of the Wg ligand extracellularly Moline et al. The visualization of the trafficking of a Wg-GFP fusion protein in live embryos reveals that Wg can be internalized and recycled back to the cell surface Pfeiffer et al. In the wing disc, the Wg protein can indeed traverse a shibire mutant clone and can be detected extracellularly both proximal and distal to the clone Strigini and Cohen This suggests that in the wing disc, the Wg ligand once secreted is able to spread to non-expressing cells in the absence of endocytic trafficking.

In fact, the inhibition of endocytosis leads to an extension of the Wg gradient, indicating that endocytosis serves to down-regulate the levels of Wg in the wing disc Strigini and Cohen ; Piddini et al. In accordance with this hypothesis, more extracellular Wg is present within the shibire mutant clone than outside the clone Strigini and Cohen Notably, the majority of evidence from the embryonic epidermis and wing disc support different models of Wg transport and stability that contribute to the Wg gradient.

Further studies need to be performed to resolve which mechanism of Wg distribution is predominant under physiological conditions. Although the mechanistic details through which cell signaling pathways ultimately regulate gene expression to control the development of metazoans may differ, they all share at least three common, conserved features: Developmental signaling pathways regulate gene expression by a switch mechanism, whereby from an actively repressed state in the absence of the signal, genes are transcribed in the presence of the signal.

The combination of these three features allows a signaling pathway to robustly activate specific target gene expression in response to the signal, in a context-dependent manner, while preventing target gene expression in the absence of the signal for review, see Barolo et al. In the case of Wg signaling, default repression is exerted on the same pathway response element using the same signal-regulated transcription factor, Tcf, as is used in the presence of active signaling.

In the absence of signaling, Tcf binds Wg-responsive elements within target genes along with other corepressors to suppress gene expression. When the Wg ligand is present, the same transcription factor Tcf binds Arm and recruits other coactivators to direct the expression of target genes Brunner et al. However, it has recently become apparent that target genes of the pathway are not only directly activated by the Wg signal, but can be directly repressed as well. Signal-induced gene repression conflicts with the principle of default repression.

If gene transcription is silenced through default repression before pathway activity, there is no opportunity to repress gene transcription following signaling. Indeed, for Wg signaling to repress transcription of a gene, default repression of the gene would have to be circumvented before signaling with a switch to a default activation state. Conceptually, the reversal of this feature would allow signaling pathways to robustly repress target gene expression in the presence of the signal, in a context-dependent manner, while activating the target gene in its absence.

Several target genes including fz2 , svb , rho , Ultrabithorax Ubx , stripe sr , and dpp have been proposed to be directly repressed by Wg signaling. However, in the cases of fz2 , svb , and rho Cadigan et al. Only in the cases of sr in the embryonic epidermis Piepenburg et al.

Both of these genes contain functional Tcf-binding sites in their response elements that are required for repression, and mutation of these sites results in a failure of Wg-mediated gene silencing. In a recent study, several genes that are repressed by Wg signaling were identified from cultured Drosophila hemocytic cells. Surprisingly, the characterization of the cis regulatory element of one of these genes, Ugt36Bc, revealed that the Tcf recognition site is markedly different from a typical consensus Tcf binding site.

Furthermore, the novel Tcf binding sites were not only required for Wg-induced repression, but were also essential for the default transcription of Ugt36Bc in the absence of signaling Blauwkamp et al. This suggests that the nature of the Tcf binding site within the Wg-responsive element can determine the nature of the transcriptional output, both in the absence and presence of signaling, and is one possible mechanism through which Wg signaling can switch from activation to repression of certain genes. Over the last 30 years, the use of genetic analyses in Drosophila has elucidated both the function, in various contexts of development, and the molecular mechanism of the Wg pathway.

Owing to the vast array of genetic techniques available in Drosophila and its high degree of conservation, no doubt, novel components and Wg-regulated processes identified in future studies using this model system are likely to be directly applicable to vertebrate development. We apologize to those colleagues whose work was not cited because of length restrictions.

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Additional Perspectives on Wnt Signaling available at www. National Center for Biotechnology Information , U. Cold Spring Harb Perspect Biol. Sharan Swarup and Esther M.

FUNCTION OF WINGLESS SIGNALING IN THE EMBRYO

Author information Copyright and License information Disclaimer. This article has been cited by other articles in PMC. Abstract The Wingless Wg pathway represents one of the best-characterized intercellular signaling networks. Wnt further ensures the development of these tissues through proper regulation of cell proliferation and migration.

Wnt signaling functions can be divided into axis patterning, cell fate specification, cell proliferation and cell migration. In early embryo development, the formation of the primary body axes is a crucial step in establishing the organism's overall body plan. The axes include the anteroposterior axis, dorsoventral axis, and right-left axis. Wnt signaling is implicated in the formation of the anteroposterior and dorsoventral DV axes.

Wnt signaling activity in anterior-posterior development can be seen in mammals, fish and frogs. In mammals, the primitive streak and other surrounding tissues produce the morphogenic compounds Wnts, BMPs , FGFs , Nodal and retinoic acid to establish the posterior region during late gastrula. These proteins form concentration gradients. Areas of highest concentration establish the posterior region while areas of lowest concentration indicate the anterior region. Wnt involvement in DV axis formation can be seen in the activity of the formation of the Spemann organizer , which establishes the dorsal region.

Wnt signaling activated by FGFs is responsible for this movement. Wnt signaling is also involved in the axis formation of specific body parts and organ systems later in development. In vertebrates, sonic hedgehog Shh and Wnt morphogenetic signaling gradients establish the dorsoventral axis of the central nervous system during neural tube axial patterning. High Wnt signaling establishes the dorsal region while high Shh signaling indicates the ventral region.

Wnt proteins guide the axons of the spinal cord in an anterior-posterior direction. Specifically, Wnt7a helps produce the dorsal patterning of the developing limb. In the embryonic differentiation waves model of development Wnt plays a critical role as part a signalling complex in competent cells ready to differentiate. Wnt reacts to the activity of the cytoskeleton, stabilizing the initial change created by a passing wave of contraction or expansion and simultaneously signals the nucleus through the use of its different signalling pathways as to which wave the individual cell has participated in.

Wnt activity thereby amplifies mechanical signalling that occurs during development. Cell fate specification or cell differentiation is a process where undifferentiated cells can become a more specialized cell type. Wnt signaling induces differentiation of pluripotent stem cells into mesoderm and endoderm progenitor cells. Specifically, Wnt3 leads to mesoderm committed cells with hematopoietic potential. This allows for regeneration of nervous system cells, which is further evidence of a role in promoting neural stem cell proliferation. In order to have the mass differentiation of cells needed to form the specified cell tissues of different organisms, proliferation and growth of embryonic stem cells must take place.

Entry into the S phase causes DNA replication and ultimately mitosis , which are responsible for cell proliferation. This allows for overall growth and development of specific tissue systems during embryonic development. This is apparent in systems such as the circulatory system where Wnt3a leads to proliferation and expansion of hematopoietic stem cells needed for red blood cell formation.

The biochemistry of cancer stem cells is subtly different than that of other tumor cells. These so-called Wnt-addicted cells hijack and depend on constant stimulation of the Wnt pathway to promote their uncontrolled growth, survival and migration. In cancer, Wnt signaling can become independent of regular stimuli, through mutations in downstream oncogenes and tumor suppressor genes that become permanently activated even though the normal receptor has not received a signal.

LF3 strongly inhibits this binding in vitro, in cell lines and reduced tumor growth in mouse models. It prevented replication and reduced their ability to migrate, all without affecting healthy cells. No cancer stem cells remained after treatment. Cell migration during embryonic development allows for the establishment of body axes, tissue formation, limb induction and several other processes. Wnt signaling helps mediate this process, particularly during convergent extension. Signaling from both the Wnt PCP pathway and canonical Wnt pathway is required for proper convergent extension during gastrulation.

Wnt signaling also induces cell migration in later stages of development through the control of the migration behavior of neuroblasts , neural crest cells, myocytes , and tracheal cells. Wnt signaling is involved in another key migration process known as the epithelial-mesenchymal transition EMT. This process allows epithelial cells to transform into mesenchymal cells so that they are no longer held in place at the laminin. It involves cadherin down-regulation so that cells can detach from laminin and migrate.

Wnt signaling is an inducer of EMT, particularly in mammary development. Insulin is a peptide hormone involved in glucose homeostasis within certain organisms. Specifically, it leads to upregulation of glucose transporters in the cell membrane in order to increase glucose uptake from the bloodstream. In particular, Wnt10b is a Wnt protein that increases this sensitivity in skeletal muscle cells. Since its initial discovery, Wnt signaling has had an association with cancer.

When Wnt1 was discovered, it was first identified as a proto- oncogene in a mouse model for breast cancer.

The fact that Wnt1 is a homolog of Wg shows that it is involved in embryonic development, which often calls for rapid cell division and migration. Misregulation of these processes can lead to tumor development via excess cell proliferation. Canonical Wnt pathway activity is involved in the development of benign and malignant breast tumors. Wnt signaling has been implicated in the development of other cancers. Increased expression of Wnt ligand-proteins such as Wnt 1, Wnt2 and Wnt7A were observed in the development of glioblastoma , oesophageal cancer and ovarian cancer respectively.

This study has identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. These findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells.

Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue. The Protein Phosphatase 4 complex promotes the Notch pathway and wingless transcription. Biol Open [Epub ahead of print]. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, subunits of the serine threonine phosphatase Protein phosphatase 4 PP4 were identifed. Knockdown of the catalytic and the regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less.

PP4 regulates the Wg pathway by controlling Notch -driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized.

This study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, it was shown that PP4 regulates proliferation independent of its interaction with Notch. An embryonic system to assess direct and indirect Wnt transcriptional targets. Sci Rep 7 1: During animal development, complex signals determine and organize a vast number of tissues using a very small number of signal transduction pathways. These developmental signaling pathways determine cell fates through a coordinated transcriptional response that remains poorly understood.

Specific sets of genes were found downstream of both beta-catenin and TCF with an additional group of genes regulated by Wnt, while the non-canonical Wnt4 regulates a separate cohort of genes. Transcriptional changes were correlated with phenotypic outcomes of cell differentiation and embryo size, showing the model can be used to characterize developmental signaling compartmentalization in vivo. Widespread rewiring of genetic networks upon Wnt cancer signaling pathway activation.

Cell Syst [Epub ahead of print]. Cellular signaling networks coordinate physiological processes in all multicellular organisms. Within networks, modules switch their function to control signaling activity in response to the cellular context. However, systematic approaches to map the interplay of such modules have been lacking.

This study generated a context-dependent genetic interaction network of a metazoan's signaling pathway.

Genetic interactions within the Wnt network were found to globally rewire after pathway activation. Between-state networks were derived that showed how genes changed their function between state-specific networks. This related pathway inhibitors across states and identified genes required for pathway activation. For instance, it was predicted and confirmed that the ER-resident protein Catsup is required for ligand-mediated Wnt signaling activation. Together, state-dependent and between-state genetic interaction networks identify responsive functional modules that control cellular pathways.

Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9 1: Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. Their axons innervate one of the four lobula plate layers. This study shows that diffusible Wingless Wg between adjacent neuroepithelia induces its own expression to form secondary signaling centers. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind Omb , serving as a relay to postmitotic neurons.

Wnt controls the medial-lateral subdivision of the Drosophila head.

Biol Lett 14 7. In insects, the subdivision of the head into a lateral region, harbouring the compound eyes CEs , and a dorsal medial region, where the ocelli localize, is conserved. This organization might have been already present in the insects' euarthropodan ancestors. In Drosophila, the Wnt-1 homologue wingless wg plays a major role in the genetic subdivision of the head. To analyse specifically the role of wg signalling in the development of the dorsal head, this pathway was attenuated specifically in this region by genetic means.

Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. This study demonstrates that release and spread of Wingless is required to pattern the proximo-distal P-D axis of Drosophila Malpighian tubules.

Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending the understanding about the functional modes by which Wnts shape animal development, it is anticipated that this mechanism is relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.

The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt. Lipids are structural components of cellular membranes and signaling molecules that are widely involved in development and diseases, but the underlying molecular mechanisms are poorly understood, partly because of the vast variety of lipid species and complexity of synthetic and turnover pathways.

From a genetic screen, this study identified that mannosyl glucosylceramide MacCer , a species of glycosphingolipid GSL , promotes synaptic bouton formation at the Drosophila neuromuscular junction NMJ. Pharmacological and genetic analysis shows that the NMJ growth-promoting effect of MacCer depends on normal lipid rafts, which are known to be composed of sphingolipids, sterols and select proteins.

At the earliest stages of development segments are defined by pair rule genes , and subsequently, each segment is subdivided into anterior and posterior compartments by the action of segment polarity genes.

Protein wingless

Normally, each thoracic and abdominal segment contains an anterior denticle band, and a more posterior region of naked cuticle. In wingless mutants, the naked cuticle is absent, replaced by a disordered array of denticles Bejsovec, The effects of wingless mutation on morphology are mirrored by events inside the embryonic cells. Wingless is secreted by cells in each of 14 posterior compartments of parasegments embryonic segments. Wingless secretion is dependent on Hedgehog , produced in adjacent compartments.

Lack of functional posterior parasegmental compartments due to a failure to secrete Wingless results in altered activity just underneath the outer cell membrane. Armadillo is associated with adherens junctions, structures that bind one cell to another, and Shaggy is involved in the transmission of the wingless signal inside the cell. Mutation of wingless also alters the secretion of cuticle and the regulation of denticle production both in the posterior cells of each compartment, and in adjacent cells that would otherwise have responded to wingless signaling.

Wg influences two distinct cellular decisions in patterning the larval ventral epidermis. This segmentally repeating pattern consists of six rows of uniquely shaped denticles arranged in a belt at the anterior of the segment, anterior to the cells that secrete Wingless protein, and an expanse of smooth, naked cuticle form in the posterior portion of the segment.

In the absence of wg both the generation of diverse denticle types and the specification of naked cuticle are disrupted, resulting in a lawn of uniform denticles. Thus Wg activity influences cell fate decisions many rows of cells away from its source. What then accounts for the two cell fate regulated by Wg signaling in the ectoderm Moline, ? Proper pattern formation requires temporal as well as spatial control of Wg activity Bejsovec, Analysis of a temperature-sensitive wg allele that is wild type at 18 o C and null for function at 25 o C has shown that Wg activity between 4 and 5.

After 6 hours, Wg activity no longer produces these cellular responses, but instead promotes the naked cuticle-secreting cell fate. Thus the population of cells responding to Wg activity changes during development Moline, and references therein. Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces.

Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibire ts shi ts mutation to block endocytosis Bejsovec, Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg -expressing cells, shi mutant embryos show high level accumulation of Wg around the wg -expressing cells Moline, Reducing endocytosis in defined domains within the segment, through moderate-level expression of a dominant negative form of Shibire, alters the normal distribution of Wg and changes the domain of cells that respond to Wg.

When expressed using the prd-Gal4 , shi D reduces both anterior and posterior movement of Wg protein, causing it to accumulate in and around the wg -expressing row of cells. Driving expression of shi D with the en-Gal4 reduces movement only in the posterior direction, since the en -expressing cells are a non-overlapping cell population just posterior to the wg -expressing row of cells Moline, The effects on cuticular pattern elements indicate that Wg moving in an anterior direction from the row of wg -expressing cells defines the domain of cells destined to secrete naked cuticle, whereas posterior movement of Wg is required for correct specification of denticle types in the anterior of the adjacent segment.

The patterning defects caused by shi D expression are reversed by co-expression with wg plus, suggesting that the primary effect of reducing endocytosis in the embryonic epidermis is a disruption of Wg protein transport. Moreover, en-Gal4 -driven shi D reduces endocytosis in a non- wg -expressing group of cells, and causes patterning defects in the cell population posterior to the en domain. This supports the idea that Wg ligand is moved by active cellular processes through cells to arrive at distant target cell populations in the embryo Moline, The results suggest that, during normal development, the temporal changes observed in directionality of Wg protein movement Gonzalez, may correlate with the temporal changes in its apparent function Bejsovec, In wild-type embryos prior to stage 10, Wg protein is detected over many cell diameters both anterior and posterior to the wg -expressing row of cells Gonzalez, Disrupting posterior movement of Wg alters patterning of at least the first three rows of denticles in the segment posterior to the affected source of Wg.

Thus, posterior movement of Wg is detectable during the early time period when Wg activity is required in these cells for the generation of diverse denticle types and for the stabilization of en expression Bejsovec, At and after stage 10, Wg protein is no longer detected in cells posterior to the wg -expressing row, including the en -expressing cells of that segment, and shows an asymmetric distribution toward the anterior of the segment Bejsovec, ; Gonzalez, The results reported here correlate this anterior movement with specification of the correct expanse of naked cuticle-secreting cells, presumably through Wg-mediated antagonism of the EGF pathway.

This is consistent with previous reports that, after stage 10, Wg is no longer required for maintenance of en expression Bejsovec, or for the generation of denticle diversity, and instead promotes specification of naked cuticle cell fate Bejsovec, , Moline, It is unclear by what mechanism Wg is excluded from the posterior cells at stage It is proposed that wild-type naked gene function may contribute to the change in direction of Wg protein movement. Reducing Wg movement through the en -expressing cells eliminates Wg-mediated specification of excess naked cuticle and substantially rescues the nkd mutant phenotype.

Thus, posterior movement of Wg from the adjacent segment, and not anterior movement of Wg within the segment, appears to be responsible for the naked mutant phenotype. This observation suggests a role for nkd gene function in restricting posterior Wg transport Moline, Although some aspects of Wg transport appear to be independent of Wg signal transduction, the two processes cannot be completely separated. Overexpression of Dfz2 , a Wg signaling receptor, appears to restrict the distribution of the Wg protein, suggesting that it has the capacity to sequester ligand.

In contrast, Dfz2 overexpression in the imaginal disc has been shown to enhance the transport of Wg protein and consequently increase its range of activity. This dramatic change in the role of Dfz2 from embryo to imaginal disc suggests that mechanisms controlling Wg distribution may differ between these two developmental stages of Drosophila. For example, recent work has revealed that imaginal disc cells project cytoplasmic extensions, called cytonemes, toward the source of signaling molecules at the center of the discs.

These extensions may assist in the broad distribution and long-range activity documented for Wg in the imaginal discs Moline, and references therein. Such cytoplasmic extensions have not been detected in vivo in embryonic epidermal cells. If embryonic cells do produce cytonemes, they may not be functionally relevant to the distribution of Wg signaling activity. Reducing endocytosis in the two rows of en -expressing cells produces Wg-related pattern disruptions in the cells posterior to the affected domain.

This suggests that Wg must physically move through the en cells in order to influence cell fate decisions in the posterior cell population. Such an effect would not be predicted if the posterior population were able to extend cytoplasmic projections through the affected 2 cell diameters and directly contact the cells expressing wg Moline, Mutant Wg molecules that are secreted properly, but fail to signal, are transported as if by default Bejsovec, Initially, these mutant embryos show a wild-type distribution of Wg protein, but over time they begin to accumulate Wg-containing vesicles in tissues that do not express the gene and in which the protein is not normally detected.

This indicates that most, if not all, embryonic cells have the ability to internalize Wg, and that this process does not require signal transduction. Moreover, it suggests that the mutant Wg ligand is able to bind to a cell surface receptor that does not transduce signal. This is consistent with a multiple-receptor model for Wg, where some Wg-binding receptors are dedicated exclusively to the transport process. Thus the dynamic distribution of Wg during development may reflect an interplay between signaling receptors and other cell surface molecules essential for ligand transport Moline, These results suggest that a single signaling molecule, in this case Wingless, can determine multiple cell fates.

These alternate cell fates depend on cell autonomous temporal changes in responsiveness to the Wg ligand and on regulated transport across adjacent cell populations that facilitate or interfere with this transport differently. The effects of wingless signaling in the margin of the wing are fairly well understood.

Here decapentaplegic is not expressed adjacent to Wingless producing cells, as is the case in embryonic segmentation. Any possible compounding effects attributable to DPP are removed, due to its absence, thus demonstrating a pure wingless effect. In the case of the wing, wingless expression is independent of hedgehog while dpp expression remains dependent on hh. The anterior edge of the wing is marked by stout, slender, and chemosensory bristles, all three types of which are innervated.

Bristles and epidermal hairs are not innervated.

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