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

Gene name - wingless

Synonyms - Dint-1

Cytological map position - 28A1-28A3

Function - secreted signaling protein

Keywords - segment polarity, oncogene

Symbol - wg

FlyBase ID:FBgn0284084

Genetic map position - 2-[22]

Classification - WNT family

Cellular location - extracellular and cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Li, X., Wu, Y., Shen, C., Belenkaya, T. Y., Ray, L. and Lin, X. (2015). Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26002470
Summary:
The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. A genetic screen was performed in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, were identified that are involved in the early secretory pathway of Wingless/Wnt. Genetic evidence is provided demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, it was shown that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, these data provide important insights into the early secretory pathway of Wg/Wnt.

Kumar, S. R., Patel, H. and Tomlinson, A. (2015). Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye. Dev Biol [Epub ahead of print]. PubMed ID: 26428511
Summary:
Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response.

Tian, A., Benchabane, H., Wang, Z. and Ahmed, Y. (2016). Regulation of stem cell proliferation and cell fate specification by Wingless/Wnt signaling gradients enriched at adult intestinal compartment boundaries. PLoS Genet 12: e1005822. PubMed ID: 26845150
Summary:
Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. This study utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. It was shown that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, it was demonstrated that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. The study concludes that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries.

Yamazaki, Y., Palmer, L., Alexandre, C., Kakugawa, S., Beckett, K., Gaugue, I., Palmer, R.H. and Vincent, J.P. (2016). Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26974662
Summary:
The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells. This study examines the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. It was found that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. Wingless transcytosis was shown to involve dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.

Trujillo, G. V., Nodal, D. H., Lovato, C. V., Hendren, J. D., Helander, L. A., Lovato, T. L., Bodmer, R. and Cripps, R. M. (2016). The canonical Wingless signaling pathway is required but not sufficient for inflow tract formation in the Drosophila melanogaster heart. Dev Biol [Epub ahead of print]. PubMed ID: 26983369
Summary:
The inflow tracts of the embryonic Drosophila cardiac tube, termed ostia, arise in its posterior three segments from cardiac cells that co-express the homeotic transcription factor Abdominal-A (abdA), the orphan nuclear receptor Seven-up (Svp), and the signaling molecule Wingless (Wg). To define the roles of these factors in inflow tract development, this study assessed their function in inflow tract formation. Using several criteria, it was demonstrated that abdA, svp, and wg are each critical for normal inflow tract formation. Wg acts in an autocrine manner to impact ostia fate, and it mediates this effect at least partially through the canonical Wg signaling pathway. By contrast, neither wg expression nor Wg signaling are sufficient for inflow tract formation when expressed in anterior Svp cells that do not normally form inflow tracts in the embryo. Instead, ectopic abd-A expression throughout the cardiac tube is required for the formation of ectopic inflow tracts, indicating that autocrine Wg signaling must be supplemented by additional Hox-dependent factors to effect inflow tract formation. Taken together, these studies define important cellular and molecular events that contribute to cardiac inflow tract development in Drosophila. Given the broad conservation of the cardiac regulatory network through evolution, these studies provide insight into mechanisms of cardiac development in higher animals.

Kim, G. W., Won, J. H., Lee, O. K., Lee, S. S., Han, J. H., Tsogtbaatar, O., Nam, S., Kim, Y. and Cho, K. O. (2016). Sol narae (Sona) is a Drosophila ADAMTS involved in Wg signaling. Sci Rep 6: 31863. PubMed ID: 27535473
Summary:
ADAMTS (a disintegrin and metalloproteases with thrombospondin motif) family consists of secreted proteases, and is shown to cleave extracellular matrix proteins. Their malfunctions result in cancers and disorders in connective tissues. This paper reports that a Drosophila ADAMTS named Sol narae (Sona; CG9850) promotes Wnt/Wingless (Wg) signaling. sona loss-of-function mutants are lethal and rare escapers had malformed appendages, indicating that sona is essential for fly development and survival. sona exhibited positive genetic interaction with wntless (wls) that encodes a cargo protein for Wg. Loss of sona decreased the level of extracellular Wg, and also reduced the expression level of Wg effector proteins such as Senseless (Sens), Distalless (Dll) and Vestigial (Vg). Sona and Wg colocalized in Golgi and endosomal vesicles, and were in the same protein complex. Furthermore, co-expression of Wg and Sona generated ectopic wing margin bristles. This study suggests that Sona is involved in Wg signaling by regulating the level of extracellular Wg.
Verghese, S. and Su, T. T. (2016). Drosophila Wnt and STAT define apoptosis-resistant epithelial cells for tissue regeneration after irradiation. PLoS Biol 14: e1002536. PubMed ID: 27584613
Summary:
Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. 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. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. 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.
Hall, E. T., Pradhan-Sundd, T., Samnani, F. and Verheyen, E. M. (2017). The Protein Phosphatase 4 complex promotes the Notch pathway and wingless transcription. Biol Open [Epub ahead of print]. PubMed ID: 28652317
Summary:
The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. 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.
Suresh, J., Harmston, N., Lim, K. K., Kaur, P., Jin, H. J., Lusk, J. B., Petretto, E. and Tolwinski, N. S. (2017). An embryonic system to assess direct and indirect Wnt transcriptional targets. Sci Rep 7(1): 11092. PubMed ID: 28894169
Summary:
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. The Wnt pathway is involved in a variety of these cellular functions, and its signals are transmitted in part through a beta-catenin/TCF transcriptional complex. This study reports an in vivo Drosophila assay that can be used to distinguish between activation, de-repression and repression of transcriptional responses, separating upstream and downstream pathway activation and canonical/non-canonical Wnt signals in embryos. 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.
Billmann, M., Chaudhary, V., ElMaghraby, M. F., Fischer, B. and Boutros, M. (2017). Widespread rewiring of genetic networks upon Wnt cancer signaling pathway activation. Cell Syst [Epub ahead of print]. PubMed ID: 29199019
Summary:
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. Using Wnt signaling in Drosophila as a model, >290,000 double perturbations were measured of the pathway in a baseline state, after activation by Wnt ligand, or after loss of the tumor suppressor APC. 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.
Apitz, H. and Salecker, I. (2018). Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9(1): 2295. PubMed ID: 29895891
Summary:
Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity.
Magri, M. S., Dominguez-Cejudo, M. A. and Casares, F. (2018). Wnt controls the medial-lateral subdivision of the Drosophila head. Biol Lett 14(7). PubMed ID: 30045903
Summary:
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. Loss of wg signalling transforms the dorsal/medial head into lateral head structures, including the development of ectopic CEs. This genetic analysis further suggests that wg signalling organizes the dorsal head medial-lateral axis by controlling, at least in part, the expression domains of the transcription factors Otd and Ey/Pax6.
Beaven, R. and Denholm, B. (2018). Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules. Elife 7. PubMed ID: 30095068
Summary:
Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. 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.
Huang, Y., Huang, S., Di Scala, C., Wang, Q., Wandall, H. H., Fantini, J. and Zhang, Y. Q. (2018). The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt. Elife 7. PubMed ID: 30355446
Summary:
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. MacCer positively regulates the synaptic level of Wnt1/Wingless (Wg) and facilitates presynaptic Wg signaling, whose activity is raft-dependent. Furthermore, a functional GSL-binding motif in Wg exhibiting a high affinity for MacCer is required for normal NMJ growth. These findings reveal a novel mechanism whereby the GSL MacCer promotes synaptic bouton formation via Wg signaling.
BIOLOGICAL OVERVIEW

A recurring, significant theme in insect development is the subdivision of the embryo into ever greater numbers of compartments within segments. 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. wingless, as a segment polarity gene, has a role in the establishment of different cell fates, working within and between the anterior and posterior compartments of segments.

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, 1991).

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. There is an altered distribution of Armadillo, and altered expression of shaggy/zeste-white3. 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. wg is expressed in one row of cells in each wild-type segment, roughly in the middle of the naked cuticle region. 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, 1999)?

Proper pattern formation requires temporal as well as spatial control of Wg activity (Bejsovec, 1991). Analysis of a temperature-sensitive wg allele that is wild type at 18oC and null for function at 25oC has shown that Wg activity between 4 and 5.5 hours of development generates diverse denticle types and stabilizes the expression of engrailed. en is a segment polarity gene expressed in the two rows of cells just posterior to the wg domain, at the posterior boundary of each segment. 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, 1999 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 shibirets (shits) mutation to block endocytosis (Bejsovec, 1995). shi encodes the fly dynamin homologue, a GTPase required for clathrin-coated vesicle formation. 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, 1999).

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, shiD 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 shiD 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, 1999).

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 shiD 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 shiD reduces endocytosis in a non-wg-expressing group of cells, and causes patterning defects in the cell population posterior to the en domain. Thus, reducing Wg transit through the en cells ‘casts a shadow’, producing patterning anomalies in an otherwise wild-type cell population. 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, 1999).

The results suggest that, during normal development, the temporal changes observed in directionality of Wg protein movement (Gonzalez, 1991) may correlate with the temporal changes in its apparent function (Bejsovec, 1991). 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, 1991). 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, 1991). 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, 1991; Gonzalez, 1991). 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, 1991) or for the generation of denticle diversity, and instead promotes specification of naked cuticle cell fate (Bejsovec, 1991, Moline, 1999).

It is unclear by what mechanism Wg is excluded from the posterior cells at stage 10. 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, 1999).

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, 1999 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, 1999).

Mutant Wg molecules that are secreted properly, but fail to signal, are transported as if by default (Bejsovec, 1995). 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, 1999). 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. Thus in the wing margin one can more easily observe the effect of the presence or the absence of wingless on bristle cell production and innervation, without having to contend with the effects of dpp production.

Both achaete and cut are involved in the specification of sensory bristles, the peripheral sense organs of the wing margin. wingless is expressed in a narrow band of cells. Adjacent cells which do not produce wingless serve as precursors of both sensory and non-sensory elements. Cut protein is expressed in a wingless dependent fashion in cells expressing wingless; achaete is expressed in the adjacent cells, those not expressing wingless. Both cut and achaete expression are dependent on wingless. The wings of flies carrying conditional lethal mutations of wingless show an absense of bristles; mechanoreceptors are transformed into chemoreceptors and the arrangement of chemoreceptors is altered. Thus the wingless signal modifies the production of achaete and cut resulting in altered sensory cell and bristle production (Couso, 1994). In summary, wingless critically regulates the production of bristles and sensory cells on the wing margin. It does this as a secreted molecule acting locally on adjacent cells, modifying the production of Cut and Achaete, two proteins involved in neurogenesis.

It has been suggested that wingless expression at the dorsal-ventral boundary of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch. Wingless expression is lost from the wing margin and the size of the wing is significantly reduced when Notch activity is removed from the third instar larva using a temperature sensitive allele of Notch. Therefore, it is likely that wingless is regulated by the Notch pathway acting through Suppressor of Hairless (Diaz-Benjumea, 1995).

Wingless has an earlier role in specification of the wing. Wing discs arise during embryonic development from a region of the epidermis devoid of wg expression. Ten to thirteen cells in each wing primordium express engrailed but not wingless. Thus, the obligitory role of wingless in leg disc formation does not appear to hold for wing disc formation.

During the second larval instar wg expression is first detected in the anterior compartment of wing discs. wingless appears to have a primary role in specifying the wing primordium. This conclusion is based on the observation that ectopic expression of wg can induce supernumary wings in the portion of the disc normally fated to give rise to body wall. Thus WG protein can reprogram cells in the notum to wing pouch identity very early in wing development. An important target of WG in this function is the gene pdm-1 which is involved in specifying the proximal-distal axis of the wing (Ng, 1996).

Thus, two distinct roles for wingless in wing morphogenesis have been identified: a primary role in specifying the wing primordium, and subsequent role mediating the patterning activities of the dorso-ventral compartment boundary (Ng, 1996).

Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).

Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations – in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).

The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP – e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation – rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).

Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).

It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).

What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).

One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk1 mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).

Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case – Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).

Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch (see Eisenmann's Wnt Signaling) mediating PCP and the canonical pathway playing no role in this. However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).

While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect – given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).

Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or 'HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. This study demonstrates that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals (Harris, 2016).

Many organisms lose the ability to regenerate damaged tissues as they mature. This change often occurs concurrently with a slowing of the growth of the organism, or a major transformation in its developmental state, e.g. metamorphosis in Drosophila and Xenopus. The loss of regenerative capacity is likely an important mechanism to balance the successful progression to reproductive adulthood at the cost of forming functionally complete tissue. Very few 'true' regeneration-specific genes have been identified (i.e. genes that are not required at any other time throughout the organism's life), but rather developmentally required pathways are often re-used during regeneration. Thus, how regenerative growth can be selectively inhibited without compromising cell proliferation or differentiation remains unknown. Here this study has shown that in the Drosophila wing disc this loss of regenerative capacity is achieved in part by the localized epigenetic inactivation of a damage-responsive enhancer that regulates the expression of wg and potentially Wnt6. This mechanism allows an organism to continue with its normal developmental program while shutting down its regenerative response to tissue damage (Harris, 2016).

Previous studies have demonstrated that the JNK pathway is robustly activated following tissue damage and has an important role in regenerative growth. The current data confirm that JNK is strongly activated following damage, but furthermore, it appears similarly activated in both day 7 and day 9 discs, as assessed by the expression of an AP-1 reporter. Thus, the loss of regeneration that occurs between day 7 and day 9 cannot be attributed to a failure to activate JNK. Despite the similar levels of AP-1 activity, the cellular responses and changes in gene expression elicited by tissue damage differ considerably as the disc matures. Importantly, genes that are known to be downstream targets of the JNK/AP-1 pathway such as Mmp1 have reduced expression on day 9 when compared to day 7. These changes in gene expression are likely to account for many of the differences in the cellular responses to tissue damage that we observe (Harris, 2016).

In addition to the aforementioned genes, the WNT genes wg and Wnt6 also exhibit a significant decline in damage-induced expression with disc maturity. The data shows this is due to the highly localized epigenetic silencing of a damage-responsive WNT enhancer, BRV118, that prevents their expression specifically in response to injury in mature discs, but still allows expression from nearby developmentally regulated enhancers. This mechanism ensures that the contribution of both genes to a regeneration program can be shut off in mature tissues independently of their essential roles in growth and development of the disc. An inability to detect expression of the BRV118-GFP reporter in unablated discs suggests that the BRV118 enhancer does not have a role in normal development. However, the wg1 allele, which results in an incompletely-penetrant phenotype characterized by a failure to specify the wing pouch, is a deletion whose breakpoints are very close to the boundaries of the BRV118 fragment that we have studied. This suggests that a separate element, possibly very close to, but not fully contained within the boundaries of BRV118, may also be disrupted by the wg1 deletion (Harris, 2016).

The expression profile of regenerating discs suggests the regulation of multiple genes is required during regeneration, and that a significant number of these genes are also involved in developmental processes. Thus, equivalent regeneration specific enhancers, like BRV118, might also exist for these genes, such as DILP8 and Mmp1. Both genes are known to be activated by JNK, although damage-responsive enhancers have not yet been characterized. Notably though, the Mmp1-lacZ reporter we used to investigate Mmp1 activation, which accurately reflects Mmp1 protein expression following injury, consists of a ~5 kb intronic region upstream of a lacZ reporter, which, based on its pattern of expression on days 7 and 9, must possess regulatory regions that allow both damage-induced activation and maturity-dependent silencing. Sequence comparison with BRV118 reveals several AP-1 binding sites that are identical to those found in BRV118, and multiple consensus sites for PcG repression. This combination of regulatory motifs could therefore reflect a molecular signature of genes that function in regeneration, and thus could potentially be used to identify genes that comprise a regeneration program through genome-wide analyses in the future (Harris, 2016).

These studies of the regulation of wg expression have shown that, despite similar levels of JNK activation, increased levels of PcG-mediated epigenetic silencing can override the effect of JNK activation and suppress gene expression in late L3. PcG-mediated silencing is best characterized for its role in the epigenetic silencing of Hox genes during embryonic development in Drosophila, but also has important functions in imaginal disc development and during regeneration. Indeed inappropriate cell fate switching following damage in imaginal discs (transdetermination) is associated with changes in PcG gene expression, and in one instance JNK signaling reduced the extent of PcG mediated repression. A key property of epigenetic regulation by PcG is the ability to simultaneously silence multiple regions across the genome via the activity of a single master regulator complex, and, moreover, this silencing is heritable and thus its activation can maintain the locus in a repressed state through subsequent cell generations. Such a mechanism is ideally suited to the sustained and progressive silencing of a regeneration program during the ongoing growth and development of imaginal discs. However, unlike Hox genes, silencing of wg and Wnt6 does not involve the entire transcription unit, but rather, is restricted to a damage-responsive enhancer. A similar local mode of epigenetic regulation has been described for the Drosophila rpr locus, in which epigenetic blocking of an irradiation-responsive enhancer region through enrichment of H3K27me3 prevents rpr expression following irradiation in late embryogenesis. Importantly, the remainder of the rpr locus itself remains accessible, and is thus responsive to developmental signals required for programmed cell death to occur in the nervous system in late embryogenesis. Localized epigenetic silencing of individual regulatory elements is therefore likely an important and potentially pervasive mechanism by which gene expression can be selectively activated or repressed by distinct inputs (Harris, 2016).

But how is this epigenetic silencing limited to just the enhancer? Elements that are responsible for expression of the 'inner circle' of wg expression at the edge of the pouch and for expression in the leg disc are immediately adjacent to the BRV118 enhancer. Thus, while the BRV-C fragment nucleates PcG-mediated repression that then spreads over the remainder of the BRV118 enhancer, mechanisms must exist that limit spread beyond the borders of the enhancer and thus preserve the activity of the adjacent developmentally-regulated enhancers. Chromatin boundary elements that are able to block the spread of heterochromatin formation have previously been described and are found in a variety of organisms including Drosophila. Unlike other boundary elements such as insulators that inhibit enhancer-promoter interactions, these ‘chromatin barrier' elements can prevent the propagation of repressive histone marks separately from a role in enhancer blocking. Thus, a similar barrier element might be present within or near BRV118 to limit chromatin modifications to the damage responsive region, yet allow nearby developmental enhancers to remain active (Harris, 2016).

If multiple genes that function in regeneration have a similar bipartite mode of regulation, it is unlikely that expressing just one of these genes at a later stage of development can restore the ability to regenerate. Indeed, this study found that restoring wg expression in day 9 discs did not promote regeneration. In contrast, expression of Myc, which is able to increase the levels of expression of both wg and Mmp1, and possibly the expression of other genes that are similarly regulated, was able to enhance regeneration. However, it is likely that Myc does not promote the expression of all genes that have been silenced in late L3. Indeed, unlike wg and Mmp1, this study found that the JAK/STAT reporter is not reactivated in mature discs by the presence of Myc. In addition, the delay in pupariation is not restored, which possibly results from a failure to restore the damage-responsive DILP8 expression level to that of a younger disc. While this study has shown that Myc functions cell autonomously to reactivate BRV118-mediated expression of WNT genes, it is unclear whether Myc reverses the PcG-mediated repression of BRV118 or bypasses it completely. However, since the BRV-B-Myc transgene is only expressed in a small region of the disc, it is not easy to detect a change in the overall level of H3K27 methylation at the WNT locus in these cells with confidence. Additionally, even increasing Myc levels has little effect by day 10, suggesting that the silencing mechanism has become even more effective. It might be necessary to combine Myc overexpression with other manipulations to restore regeneration at even later stages. Previous studies have implicated Myc as a regulator of chromatin organization and also as a regulator of cellular reprogramming, and therefore studying the role of Myc in reactivating BRV118-mediated expression might provide a tractable way of understanding the role of Myc in these processes (Harris, 2016).

Overall, this investigation has revealed a mechanism by which genes required for both regeneration and development can be regulated to allow the age-dependent restriction of a regenerative response without affecting normal organismal growth and patterning of tissues. As PcG proteins are highly conserved from flies to vertebrates, as indeed are the targets they regulate, it would be of considerable interest to determine whether the loss of regenerative capacity in vertebrates also results from the selective epigenetic silencing of damage-responsive enhancers that regulate orthologs of Drosophila genes, such as matrix metalloproteases and WNT genes (Harris, 2016).

Drosophila VAMP7 regulates Wingless intracellular trafficking

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. This study showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, this study found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. It is hypothesized that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling (Gao, 2017).

There are two models describing how the apically secreted Wg encounters basolateral receptors at receiving cells. One suggests that Wg and receptors can be internalized separately, and then, endosome fusion results in Wg and receptor interaction in the receiving cells. Another model proposes that apically secreted Wg undergoes endocytosis and will be transported to the basolateral surface in the producing cells, then spread to the receiving cells for the interaction with receptors. Therefore, Wg is actively endocytosed in both receiving cells and producing cells (Gao, 2017).

This study found that Wg distribution was affected in both receiving and producing cells in vamp7-/- mutant background. Further investigation indicated that Wg double labeled puncta significantly increased, so did the percentage of Rab4 and Wg double staining puncta. Thus, it is suggested that VAMP7 is required for Wg endocytosis in the both receiving cells and producing cells in Drosophila wing disc, and its mutation leads to Wg accumulating in endocytic organelles but not degradation. Rab4 dependent recycling endosomes can recruit proteins from the early endocytic organelles, which may finally lead to increased level of Wg in Rab4 dependent recycling endosomes (Gao, 2017).

Although endocytosis has been demonstrated for Wg transport, there is still debate about whether endocytosis plays a direct role in the Wg signaling. Classically, the early step of endocytosis is thought to contribute positively to signaling, as early endosomes can recruit signaling components, while subsequent vesicle transport may downregulate signaling by sequestrating signaling components in endosomes or degradating them in lysosomes. This study found that the expression of the Wg target gene sens was reduced in vamp7 mutant cells. One possibility is that Rab4 recycling endosomes may recruit Wg from early endosomes. As a previous report found that the expression of activated forms of Rab4 suppressed the ability of Rab5 to enhance activation of Wg pathway, Wg accumulation in Rab4 recycling endosomes may affect Wg signaling. Another possible reason is that vamp7 mutation enhances the level of Wg signaling inhibitors (Gao, 2017).

Dlp is a membrane-associated glypican that can interact with Wg by its core protein on the cell surface, and suppresses Wg target gene sens. However, the functional significance of interaction between Wg and Dlp inside the cell has not been well elucidated. This study showed that Wg might encounter endogenous Dlp in Rab4 dependent recycling endosomes, and vamp7 mutation could improve the levels of Dlp and Wg in Rab4 dependent recycling endosomes. Previous studies proposed that Dlp competes with Wg receptors to interact with Wg, and the signaling activity may be determined by the relative levels of receptor and Dlp. It is suggested that competition between Dlp and receptors might not only occur on the cell surface but may have started from intracellular vesicles. The increased levels of Dlp and Wg in Rab4 dependent recycling endosomes may lead to Sens reduction (Gao, 2017).

In conclusion, this study showed that an endocytic pathway involving VAMP7 regulates Wg and Dlp trafficking. This route adds another layer of spatial regulation in the Wg signaling pathway. Additional work will be needed to determine the functional significance of this route in other Drosophila tissues and whether vamp7 is required for vertebrate Wnt trafficking (Gao, 2017).


GENE STRUCTURE

cDNA clone length - 2907

Bases in 5' UTR - 417

Exons - five

Bases in 3' UTR - 1085


PROTEIN STRUCTURE

Amino Acids - 468

Structural Domains

The WG protein has an N-terminal hydrophobic region characteristic of a signal sequence whose function is to expedite secretion. There is one potential N-linked glycosylation site. The protein is rich in conserved cysteine residues (Rijsewijk, 1987).


wingless continued: Evolutionary Homologs | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References
date revised:  2 December 2018 

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