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: | 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.
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.
Tian, A., Duwadi, D., Benchabane, H. and Ahmed, Y. (2019). Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization. PLoS Genet 15(6): e1008111. PubMed ID: 31194729
Summary:
Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. This study tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. The critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all were disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease.
Chaudhary, V., Hingole, S., Frei, J., Port, F., Strutt, D. and Boutros, M. (2019). Robust Wnt signaling is maintained by a Wg protein gradient and Fz2 receptor activity in the developing Drosophila wing. Development 146(15). PubMed ID: 31399474
Summary:
Wnts are secreted proteins that regulate cell fate during development of all metazoans. Wnt proteins were proposed to spread over several cells to activate signaling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of the Wnt1 homolog Wingless (Wg) was observed extending over several cells away from producing cells. Surprisingly, however, it was also shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can largely replace endogenous Wg, leading to proper patterning of the wing. Therefore, the functional range of Wg and whether Wg spreading is required for correct tissue patterning remains controversial. In this study, by capturing secreted Wg on cells away from the source, it was shown that Wg acts over a distance of up to 11 cell diameters to induce signaling. Furthermore, cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain signaling. Frizzled2 expression is increased in the absence of Wg secretion and is required to maintain signaling and cell survival in NRT-wg wing discs. Together, these results provide insight into the mechanisms by which robust Wnt signaling is achieved in proliferating tissues.
Won, J. H., Kim, G. W., Kim, J. Y., Cho, D. G., Kwon, B., Bae, Y. K. and Cho, K. O. (2019). ADAMTS Sol narae cleaves extracellular Wingless to generate a novel active form that regulates cell proliferation in Drosophila. Cell Death Dis 10(8): 564. PubMed ID: 31332194
Summary:
Wnt/ Wingless (Wg) is essential for embryonic development and adult homeostasis in all metazoans, but the mechanisms by which secreted Wnt/Wg is processed remain largely unknown. A Drosophila Sol narae (Sona) is a member of A Disintegrin And Metalloprotease with ThromboSpondin motif (ADAMTS) family, and positively regulates Wg signaling by promoting Wg secretion. This study reports that Sona and Wg are secreted by both conventional Golgi and exosomal transports, and Sona cleaves extracellular Wg at the two specific sites, leading to the generation of N-terminal domain (NTD) and C-terminal domain (CTD) fragments. The cleaved forms of extracellular Wg were detected in the extracellular region of fly wing discs, and its level was substantially reduced in sona mutants. Transient overexpression of Wg-CTD increased wing size while prolonged overexpression caused lethality and developmental defects. In contrast, Wg-NTD did not induce any phenotype. Moreover, the wing defects and lethality induced by sona RNAi were considerably rescued by Wg-CTD, indicating that a main function of extracellular Sona is the generation of Wg-CTD. Wg-CTD stabilized cytoplasmic Armadillo (Arm) and had genetic interactions with components of canonical Wg signaling. Wg-CTD also induced Wg downstream targets such as Distal-less (Dll) and Vestigial (Vg). Most importantly, Cyclin D (Cyc D) was induced by Wg-CTD but not by full-length Wg. Because Sona also induces Cyc D in a cell non-autonomous manner, Wg-CTD generated by Sona in the extracellular region activates a subset of Wg signaling whose major function is the regulation of cell proliferation.
Chaudhary, V. and Boutros, M. (2019). Exocyst-mediated apical Wg secretion activates signaling in the Drosophila wing epithelium. PLoS Genet 15(9): e1008351. PubMed ID: 31527874
Summary:
Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the Drosophila wing epithelium, Wingless (Wg, the homolog of Wnt1) is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. This study performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo 'temporal-rescue' assay, these results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, these results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells.
Bell, K., Skier, K., Chen, K. H. and Gergen, J. P. (2019). Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo. Dev Dyn. PubMed ID: 31837063
Summary:
While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. This study investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. Two wg enhancers active at the blastoderm stage were identified: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.
Sui, L. and Dahmann, C. (2020). Wingless counteracts epithelial folding by increasing mechanical tension at basal cell edges in Drosophila. Development 147(5). PubMed ID: 32161062
Summary:
The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. This study shows that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. These results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.
Bakker, R., Mani, M. and Carthew, R. W. (2020). The Wg and Dpp morphogens regulate gene expression by modulating the frequency of transcriptional bursts. Elife 9. PubMed ID: 32568073
Summary:
Morphogen signaling contributes to the patterned spatiotemporal expression of genes during development. One mode of regulation of signaling-responsive genes is at the level of transcription. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. Although the effects of many gene regulatory mechanisms on transcriptional bursting have been studied, it remains unclear how morphogen gradients affect this dynamic property of downstream genes. This study adapted single molecule fluorescence in situ hybridization (smFISH) for use in the Drosophila wing imaginal disc in order to measure nascent and mature mRNA of genes downstream of the Wg and Dpp morphogen gradients. The experimental results were compared with predictions from stochastic models of transcription, which indicated that the transcription levels of these genes appear to share a common method of control via burst frequency modulation. These data help further elucidate the link between developmental gene regulatory mechanisms and transcriptional bursting.
BIOLOGICAL OVERVIEW

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).

Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila

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 (Apitz, 2018).

Visual signals received by the retina are generally not stationary because objects in the environment and/or the bodies of animals move. To detect motion, visual circuits perform complex spatio-temporal comparisons that convert luminance changes collected by photoreceptors into signals containing information about direction or speed. Despite the seemingly divergent anatomy of vertebrate and insect visual systems, they display remarkable parallels in the computations underlying motion vision and the neuronal elements performing them. In most sighted animals, this involves neurons that respond to motion signals in specific directions. Direction-selectivity emerges from differences in the connectivity of their dendrites. Motion-direction preferences by their axons are represented by layer-specific innervation. Thus, anatomical characteristics such as layer-specificity seem to be intricately linked with motion-directionality. However, how these are implemented during circuit development is poorly understood (Apitz, 2018).

The Drosophila visual system has emerged as a powerful model for elucidating the neural circuits and computations underlying motion detection. Photoreceptors (R-cells) in the retina extend axons into the optic lobe consisting of the lamina, medulla, lobula plate, and lobula. Neuronal projections in these ganglia are organized into retinotopically arranged columnar units. The medulla, lobula plate, and lobula are additionally subdivided into synaptic layers. They are innervated by more than a 100 neuronal subtypes that extract different visual features in parallel pathways. T4 and T5 lobula plate neurons are the first direction-selective circuit elements. Each optic lobe hemisphere contains ~5300 T4/T5 neurons. T4 dendrites arborize within medulla layer 10, and T5 dendrites in lobula layer Lo1. Their axons project to one of the four lobula plate layers, thereby defining four different neuron subtypes each. Axons segregate according to their motion-direction preferences. Thus, front-to-back, back-to-front, upward, and downward cardinal motion directions are represented in lobula plate layers. T4 neurons are part of the ON motion detection pathway reporting brightness increments, while T5 neurons are part of the OFF pathway reporting brightness decrements. Distinct neuron sets in the lamina and medulla relay ON and OFF information to T4 and T5 neurons. Direction-selectivity emerges within T4/T5 dendrites and involves the non-linear integration of input from these upstream neurons for enhancement in the preferred direction and suppression in the null-direction. Dendritic arbors of the four T4 neuron subtypes have characteristic orientations, that correlate with the direction preferences of lobula plate layers innervated by their axons. Thus, direction-selectivity involves the establishment of neuron subtypes, each with distinct spatial connectivities. This study addresses when and how T4 and T5 neuron subtypes with different layer identities are specified during development (Apitz, 2018).

Optic lobe neurons originate from two horseshoe-shaped neuroepithelia, called the outer and inner proliferation centers (OPC and IPC). These are derived from the embryonic optic lobe placode and expand by symmetric cell divisions during early larval development. At the late 2nd instar larval stage, neuroepithelial (NE) cells from the medial OPC edge begin to transform into medulla neural stem cells, called neuroblasts (Nbs). These undergo asymmetric divisions to self-renew and give rise to ganglion mother cells (GMCs), which divide to generate two neurons or glia. Apposing the OPC, two dorsal and ventral NE domains, called the glial precursor cell (GPC) areas, produce neuron subtypes associated with all ganglia. At the mid 3rd instar larval stage, the lateral OPC begins to generate lamina neurons (Apitz, 2018).

The IPC generates lobula and lobula plate neurons, including T4/T5 neurons from the early 3rd instar larval stage onward. Recent studies showed that NE cells in one domain, the proximal (p-)IPC, convert into progenitors in an epithelial-mesenchymal transition (EMT)-like process. Progenitors migrate to a second proliferative zone, the distal (d-)IPC, where they mature into Nbs. These transition through two competence windows to first produce C and T neurons, corresponding to C2 and C3 ascending neurons connecting the medulla and lamina, as well as T2/T2a and T3 neurons connecting the medulla and lobula, and then T4/T5 lobula plate neurons. Cross-regulatory interactions between Dichaete (D) and Tailless (Tll) control the switch in Nb competence defined by the sequential expression of the proneural bHLH transcription factors Asense (Ase) and Atonal (Ato). The latter is co-expressed with the retinal determination protein Dachshund (Dac). The molecular mechanisms that control layer-specific T4/T5 neuron subtype identities within this sequence of developmental events occurring at different locations have remained elusive (Apitz, 2018).

T4/T5 neuron diversity resulting in differential layer-specificity could be achieved by postmitotic combinatorial transcription factor codes upstream of distinct guidance molecules. Although not mutually exclusive, layer-specificity of T4/T5 neurons could also be determined by temporal differences in the expression of common postmitotic determinants, similar to the birth-order dependent R-cell growth cone segregation strategy described in the medulla. This study provides evidence for another mechanism, whereby layer-specific T4/T5 neuron subtype identity is determined early in the p-IPC neuroepithelium. Their specification depends on two relay mechanisms involving Wnt and Bone morphogenetic protein (Bmp) signaling and transcription factor interactions. These establish and translate the spatial patterning of NE cells into postmitotic neuronal subtype identities to bridge distances inherent to this particular neurogenesis mode (Apitz, 2018).

The spread of Wg is dispensable for patterning of many tissues. However, this study uncovered a distinct requirement for diffusible Wg in the nervous system, where it orchestrates the formation of T4/T5 neurons innervating lobula plate layers 3/4. Their generation depends on inductive mechanisms that are relayed in space and time. The spatial relay consists of a multistep-signaling cascade across several NE domains: Wg from the GPC areas induces wg expression in the s-IPC and Nb lineage adjacent to ventral and dorsal p-IPC subdomains; this secondary Wg source activates dpp expression. Dpp signaling mediates EMT of migratory progenitors from these subdomains. The p-IPC core produces Dac-positive layer 1/2 specific T4/T5 neurons. Dpp signaling in p-IPC NE subdomains triggers a temporal relay across intermediate cellular states by inducing omb. Omb in turn suppresses Dac, conferring layer 3/4 identity to postmitotic T4/T5 neurons (Apitz, 2018).

When Wg is membrane-tethered, the first step of this cascade is disrupted. This defect is not caused by decreased signaling activity of NRT-Wg protein in wg{KO;NRT-wg} flies. First, wild-type Wg signaling activity inside the GPC areas and the adjacent OPC was not affected. Second, in allele switching experiments, ectopic expression of a highly active UAS-NRT-wg transgene in the GPC areas was unable to rescue. By contrast, restoring wild-type wg function in the GPC areas was able to rescue, supporting the notion that Wg release and spread from the GPC areas are required to induce its own expression in the s-IPC and the Nb clone (Apitz, 2018).

Although Wg release is essential, the range of action is likely limited. Wg expression in the s-IPC commences in early 3rd instar larvae, when it is still in close proximity with the GPC. Half of the wg{KO;NRT-wg} flies showed residual dpp expression in one progenitor stream at the 3rd instar larval stage and a 25% reduction of T4/T5 neurons, correlating with three lobula plate layers in adults. The other half lacked dpp-lacZ expression and showed a 50% reduction of T4/T5 neurons correlating with two remaining layers. While this partial phenotypic penetrance is not fully understood, NRT-Wg likely partially substituted for Wg because of the initial close proximity of the GPC areas and the s-IPC and Nb clone. Occasional residual NRT-Wg expression in the s-IPC argues against an all-or-nothing inductive event and suggests a model, whereby cell-intrinsic signaling thresholds have to be reached. Theoretically, the dpp expression defect in the p-IPC of wg{KO;NRT-wg} flies could reflect the dependence on long-range Wg from the GPC areas. However, as this study has shown, IPC-specific wg knockdown leads to dpp loss in the p-IPC. Propagation of sequential Wnt signaling could explain long-range activities. Moreover, sequentially acting primary and secondary sources of Wg have been described in the developing Drosophila eye, suggesting that the regulatory mechanism observed in the optic lobe might be employed in several contexts. The different outcomes of early and late allele wg to NRT-wg allele switching indicate that Wg secretion is required for the induction but not long-term maintenance of wg expression in the s-IPC. The GPC areas become rapidly separated from the s-IPC and Nb clone by compact rows of newly generated neurons. As part of a relay system, diffusible Wg may therefore be required to bridge distances over a few cell diameters during the initial phase of neurogenesis. The s-IPC in wg{KO;NRT-wg} flies expressed Hth and generated two neuron clusters as in wild-type. Thus, the sole function of wg in the s-IPC is to relay the GPC-derived Wg signal to induce dpp expression in the p-IPC. Since Wg release is not required in the GPC areas to induce dpp in the adjacent OPC, this secondary wg function in the s-IPC is most likely juxtacrine (Apitz, 2018).

Compared to approximately 80 medulla neuron subtypes derived from the OPC, the specification of 13 distinct subtypes originating from the p-IPC appears simple. However, the distinct mechanisms employed are surprisingly complex. Previous work has shown that cross-regulatory interactions between D and tll regulate a Nb competence switch from generating early-born C2, C3, T2, T2a, and T3 neurons to eight distinct layer-specific T4/T5 subtypes. Ato and Dac are expressed in the second Nb competence window and depend on tll. Functional studies showed that dac mutant T4/T5 neurons adopted early-born T2/T3 neuron-like morphologies. Similarly, ato mutant T4/T5 neurons displayed neurite connectivity defects. Notably, simultaneous knockdown of dac and ato resulted in the absence of T4/T5 neurons, demonstrating that both are required together for the ability of d-IPC Nbs to produce new neuron subtypes in the second competence window (Apitz, 2018).

Dac is initially expressed in all T4/T5 neurons but only maintained in layer 1/2 innervating subtypes. This suggests that an essential step for the specification of layer 3/4 innervating neurons is the downregulation of Dac and the suppression of the T4/T5 default neuron fate, i.e., layer 1/2 identity. Although the mode of this inhibitory mechanism depends on the outcome of the Nb-specific switching mechanism in the d-IPC, it is already primed in p-IPC NE cells. Thus, layer-specificity and therefore motion-directionality are determined early in the NE precursors of T4/T5 neurons. Molecularly, it involves the Omb-mediated relay of Dpp-signaling-dependent NE cell patterning information across intermediate cell states to postmitotic T4/T5 neurons resulting in the repression of Dac. In contrast to the OPC, this study found no link between NE patterning in the p-IPC and Notch-dependent differential apoptosis of region-specific T4/T5 subtypes. Instead, Notch controls the choice between T4 and T5 identity, likely during the second competence window, indicating that the distinction between layer 1/2 and 3/4 fates precedes T4 and T5 neuron specification (Apitz, 2018).

The mechanisms controlling the maintenance of omb expression, and Omb-mediated downregulation of Dac are unclear. Hypotheses regarding the latter have to be reconciled with the fact that dac, together with ato, is required for the formation of all T4/T5 neurons and hence is expressed in all d-IPC Nbs during the second competence window. Omb and Dac are initially co-expressed in Nbs and young T4/T5 neurons, suggesting that Omb does not directly repress dac transcription. Yet, expression of the dacp7d23 enhancer trap Gal4 line showed that dac is only transcribed in layer 1/2 neurons in adults. A possible scenario is that Omb could break Dac autoregulation by triggering degradation of Dac. Since T-box genes can act as transcriptional activators and repressors and their effects are influenced by various co-factors, future studies will need to explore the molecular details underlying Omb-mediated repression of Dac. It will also be important to determine whether layer 3/4 specification is mediated solely by Dac downregulation, or whether omb has additional instructive roles (Apitz, 2018).

Consistent with the observation that C2 and C3 neurons have distinct developmental origins, this study found that Nbs derived from the Dpp-expression domain produce C2 and possibly T2a neurons during the first Nb competence window, while the core p-IPC generates C3, T2, and T3 neurons. dac mutant T4/T5 neurons adopt T2/T3-like morphologies suggesting that this is the default neuron fate in this neuron group. While Omb is maintained in C&T neurons derived from the Dpp-expression domain, Dac is not expressed, suggesting that Omb interacts with other molecular determinants in these neurons. While this study did not explore how layer 1 and 2 neurons or layer 3 and 4 neurons become distinct from each other because of the lack of specific markers, the data suggest a possible contribution of Ato/Dac and Notch signaling, as these are active within the d-IPC. Findings in a concurrent study of Pinto-Teixeira (2018) align with the current data concerning the role of Dpp and Notch signaling. Furthermore, a second study of Mora (2018) reported an additional role for Ato in controlling the transient amplification of d-IPC Nbs by symmetric cell division to ensure that the correct number of T4/T5 neurons is produced. It will be fascinating to identify the transcriptional targets of Notch, Ato/Dac, and Omb that mediate ganglion- and layer-specific targeting of T4/T5 dendrites and axons, respectively. Finally, future behavioral studies of layer 3/4-deficient flies will address to what extent direction selectivity is affected or compensatory mechanisms are in place (Apitz, 2018).

Signaling centers, also called organizers, pattern tissues in a non-autonomous fashion. The vertebrate roof plate and the cortical hem, for instance, both release Wnts and Bmps to pattern NE cells in the developing dorsal spinal cord and in the surrounding forebrain, respectively. In the Drosophila visual system, the GPC areas express wg and pattern the OPC by inducing dpp expression in adjacent dorsal and ventral OPC subdomains. Together with the current insights into the function of GPC-derived wg in IPC patterning and neurogenesis, this firmly establishes the GPC areas as local organizers of optic lobe development. At the onset of neurogenesis, wg is first expressed in the GPC areas followed by the s-IPC, explaining the well-established delay in neurogenesis between the IPC and OPC. Wg release from the GPC areas could coordinate the timely onset of neurogenesis in the OPC and IPC to safeguard the alignment of matching partner neurons across several retinotopically organized neuropils. The intercalation of new-born neurons between both neuroepithelia may have driven the need for a relay system using primary and secondary sources of Wg. Wg induces Dpp to subdivide the adjacent OPC and p-IPC NE into distinct regions as basis for generating neuronal diversity. The temporal relay mediated by Omb represents an efficient strategy to pass the memory of spatial NE patterning information by Dpp signaling on to postmitotic neurons generated at a distance. It is thus intricately tuned to the distinct neurogenesis mode of the p-IPC essential for spatially matching birth-order-dependent neurogenesis between the OPC and IPC. Interestingly, the progressive refinement of NE patterning by the induction of secondary signaling centers plays a central role in vertebrate brain development. Furthermore, similar signaling cascades have been recently identified in mammalian optic tissue cultures where sequential Wnt and Bmp signaling induces the expression of the Omb-related T-box transcription factor Tbx5 to specify dorsal retinal NE cells. Hence, such cascades could represent conserved regulatory modules that are employed repeatedly during invertebrate and vertebrate nervous system development (Apitz, 2018).

Carrier of Wingless (Cow) regulation of Drosophila neuromuscular junction development

The first Wnt signaling ligand discovered, Drosophila Wingless (Wg; Wnt1 in mammals), plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like Protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). This study investigated synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, Cow secreted from the presynaptic motor neuron was found to act to limit synaptic architecture and neurotransmission strength. In cow null mutants, this study found increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle (SV) fusion frequency based both on GCaMP imaging and electrophysiology recording. Membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It has been shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ. This study shows that Cow and Notum work through the same pathway to limit synaptic development. It is concluded Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix (Kopke, 2020).

The developing nervous system requires the coordinated action of many signaling molecules to ensure proper synapse formation and function. One key class of signals is the Wnt ligands. The first discovered Wnt, Drosophila Wingless (Wg), is secreted from presynaptic neurons and glia at the developing glutamatergic neuromuscular junction (NMJ) to bind to the Frizzled-2 (Fz2) receptor in both anterograde and autocrine signaling. In the postsynaptic muscle, Wg binding to Fz2 activates the noncanonical Frizzled Nuclear Import (FNI) pathway, which leads to Fz2 endocytosis and cleavage of the Fz2 C terminus (Fz2-C). The Fz2-C fragment is trafficked to the nucleus to control translation of synaptic mRNAs and glutamate receptors (GluRs). In presynaptic neurons, Wg binding to Fz2 activates a divergent canonical pathway inhibiting glycogen synthase kinase 3β (GSK3β) homolog Shaggy (Sgg) to control microtubule cytoskeletal dynamics via the microtubule-associated protein 1B (MAP1B) homolog Futsch, resulting in synaptic bouton growth. The Wg signaling ligand must be tightly regulated in the synaptic extracellular space (synaptomatrix) to ensure proper NMJ development (Kopke, 2020).

One critical category of proteins regulating Wg ligand in the synaptomatrix is heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein to which heparan sulfate (HS) glycosylphosphatidylinositol (GAG) chains are covalently attached. HS GAG chains are composed of repeating disaccharide subunits expressing variable sulfation patterns (the "sulfation code"). These GAG chains bind secreted extracellular ligands to regulate intercellular signaling. There are three HSPG families: transmembrane; glycerophosphatidylinositol (GPI) anchored; and secreted. The Drosophila genome encodes only five HSPGs, with the following three known to affect NMJ development: transmembrane syndecan; GPI-anchored Dally-like protein (Dlp); and secreted perlecan. A second secreted HSPG recently characterized in Drosophila was named Carrier of Wingless (Cow; Chang, 2014). In the developing wing disk, Cow directly binds secreted Wg and promotes its extracellular transport in an HS-dependent manner. Cow shows a biphasic effect on Wg target genes. Removing Cow results in a Wg overexpression (OE) phenotype for short-range targets, and a loss-of-function phenotype for long-range targets (Chang, 2014; Kopke, 2020 and references therein).

The mammalian homolog of Cow, Testican-2, is highly expressed within the developing mouse brain, and inhibits neurite extension in cultured neurons, although the mechanism of action is not known. This study therefore set out to characterize Cow functions at the developing Drosophila NMJ. The larval NMJ model is used because it is large, accessible and particularly well characterized for HSPG-dependent Wg trans-synaptic signaling (Sears, 2018). Each NMJ terminal consists of a relatively stereotypical innervation pattern, with consistent axonal branching and synaptic bouton formation. Boutons are the functional unit of the NMJ, containing presynaptic components required for neurotransmission including glutamate-containing synaptic vesicle (SV) pools and specialized active zone (AZ) sites for SV fusion. AZs contain Bruchpilot (Brp) scaffolds, which both cluster Ca2+ channels and tether SVs. AZs are directly apposed to GluR clusters in the postsynaptic muscle membrane. This spatially precise juxtaposition is critical for high-speed and efficient synaptic communication between neuron and muscle (Kopke, 2020).

This study sought to test Cow functions at the NMJ, with the hypothesis that Cow should facilitate extracellular Wg transport across the synapse. Structurally, cow null mutants display overelaborated NMJs with more boutons and more synapses, phenocopying Wg overexpression. This phenotype is replicated with targeted neuronal Cow knockdown, but not muscle Cow knockdown, which is consistent with Cow secretion from the presynaptic terminal. Functionally, cow null mutants display increased synaptic transmission strength. Both electrophysiology recording and postsynaptically targeted GCaMP imaging show increased SV fusion, indicating elevated presynaptic function. Replacing native Wg with a membrane-tethered Wg blocks secretion. Tethered Wg has little effect on NMJ development, but when combined with the cow null suppresses the synaptic bouton increase, indicating that Cow mediates only secreted Wg signaling. It was recently shown that Notum, a secreted Wg deacylase, also restricts Wg signaling at the NMJ (Kopke, 2017). This study shows that combining null cow and notum heterozygous mutants causes a synergistic increase in NMJ development, indicating nonallelic noncomplementation. Moreover, combining null cow and notum homozygous mutants did not cause an increase in NMJ development compared with the single nulls, indicating an interaction within the same pathway. It is concluded that Cow functions via negative regulation of Wg trans-synaptic signaling (Kopke, 2020).

The function of signaling ligands in the extracellular space is tightly regulated to ensure coordinated intercellular development, often via glycan-dependent mechanisms. The most recently discovered Drosophila HSPG, secreted Cow, was characterized with this role (Chang, 2014). In the developing wing disk, the Wnt Wg is produced in a stripe of cells at the dorsal/ventral margin boundary, and acts as an intercellular morphogen through Fz2 receptor signaling. The glypican HSPGs Dally and Dlp, bound to outer plasma membrane leaflets via GPI anchors, bind Wg to regulate both ligand distribution and intercellular signaling. It has been proposed that Dally/Dlp HSPGs are involved in the movement of extracellular Wg to form a morphogen gradient. However, in dally dlp double mutant clones, extracellular Wg is detected far away from Wg-secreting cells, suggesting that another extracellular factor can transport Wg. Cow was shown to fill this role by binding extracellular Wg to increase stability and rate of movement from producing to receiving cells (Chang, 2014). Supporting this model, cow mutants manifest Wg ligand gain-of-function/overexpression phenotypes for short-range targets, and loss-of-function phenotypes for long-range targets (Kopke, 2020).

At the NMJ, such a long-range Wg morphogen transport function is not seemingly required, except perhaps as a clearance mechanism, but Wg extracellular regulation and short-range Wg transport to cross the synaptic cleft is critical for NMJ development. At the forming of NMJ, Wg from neurons and glia signals both presynaptically (neuronal) and postsynaptically (muscle) via Fz2 receptors. In the motor neuron, Wg signaling inhibits the GSK3β homolog Sgg to regulate the MAP1B homolog Futsch to modulate microtubule dynamics controlling NMJ bouton formation. However, Futsch distribution and microtubule dynamics do not change with elevated Wg signaling, so this pathway alone does not explain the increased bouton formation with increased Wg signaling. In the postsynaptic muscle, Wg signaling drives Fz2 endocytosis and C-terminus cleavage, with transport to the nucleus regulating mRNAs involved in synaptogenesis, including postsynaptic GluR distribution. In wg mutants, GluRs are more diffuse; with clusters irregular in size/shape, increased receptor numbers and a larger postsynaptic volume. Thus, Wg trans-synaptic signaling controls both NMJ structure and function (Kopke, 2020).

Based on the findings from Chang (2014), it was hypothesized that Cow binds Wg to facilitate the transport across the synapse to Fz2 receptors on the muscle. If this is correct, a presynaptic Wg OE phenotype would be expected in the absence of Cow (Wg buildup at the source), and a postsynaptic Wg decrease/loss phenotype (failure of Wg transport). Presynaptically, increased synaptic bouton number was found in cow null mutants phenocopying the Wg OE condition (Kopke, 2017), consistent with this hypothesis. These results indicate that Cow normally inhibits NMJ bouton formation, consistent with the effects of inhibiting presynaptic Wg signaling. Postsynaptically, an increased number of GluR clusters were found due to elevated synapse formation in cow null mutants, but no evidence of diffuse GluR clusters of irregular size/shape and larger volume, as has been reported in wg mutants. Therefore, no strong support for the second prediction of the hypothesis. GluR changes within single postsynaptic domains are challenging to see even with enhanced resolution microscopy , but future studies could focus more on GluRIIA cluster size/shape/intensity in cow mutants. If GluR defects are detected in cow nulls, it would be interesting to test the Frizzled Nuclear Import (FNI) pathway (Kopke, 2020).

Wg signaling regulates multiple steps of NMJ development including branching, satellite bouton budding, and synaptic bouton maturation. None of the cow manipulations cause changes in branching, indicating that Cow does not regulate this Wg signaling, likely working in concert with other Wg regulators. Wg loss (wgts) decreases bouton formation, while neural Wg OE increases branching, satellite, and total bouton numbers. Satellite boutons represent an immature stage of development, with small boutons connected to the mature (parent) bouton or adjacent axon. Neuronal Cow OE does not change mature bouton number, but increases satellite bouton budding. Neuronal Cow RNAi also increases satellite boutons. Thus, changing neural Cow levels in either direction elevates satellite bouton numbers, suggesting different consequences on budding versus developmental arrest. It also appears that the cellular source of secreted Cow, or the balance between sources, may be important for proper Wg regulation. Importantly, glia-secreted Wg regulates distinct aspects of synaptic development, with loss of glial-derived Wg accounting for some, but not all, of wg mutant phenotypes. Similarly, cell-targeted cow manipulations cause different NMJ phenotypes. There is no evidence for normal Cow function in postsynaptic muscle, but it remains possible that Cow secreted from glia could regulate Wg trans-synaptic signaling (Kopke, 2020).

Increasing Wg signaling elevates evoked transmission strength and functional synapse number (Kopke, 2017), which is phenocopied in cow null mutants. Block of postsynaptic Wg signaling causes increased SV fusion frequency and amplitude of miniature excitatory junctional potentials (Speese, 2012). With neuronal cow RNAi, there is a similar increase in event frequency and amplitude. These results suggest a decrease in postsynaptic Wg signaling when cow is lost, supporting the Wg transport hypothesis. Blocking Wg secreted from neurons or glia increases muscle GluR cluster size, albeit with differential effects on neurotransmission efficacy. Reducing neuronal Wg has no effect on mEJC frequency, but reducing glial-derived Wg increases SV fusion frequency. Both nerve-evoked and spontaneous neurotransmission are increased in cow null mutants, together with increased Brp active zones and postsynaptic GluR clusters forming supernumerary synapses. SynapGCaMP is an exciting new tool to test function at individual synapses. With targeted neuronal cow RNAi, there is an increase in both the number of SV fusion events and the postsynaptic Ca2+ signal amplitude, which is consistent with both presynaptic and postsynaptic regulation of Wg signaling. These functional phenotypes, combined with coordinated changes in presynaptic and postsynaptic formation suggest Cow regulates trans-synaptic Wg transport (Kopke, 2020).

There were differences between spontaneous synaptic vesicle fusion findings between TEVC electrophysiological recordings and SynapGCaMP reporter (MHC-CD8-GCaMP6f-Sh) Ca2+ imaging. Motor neurons that presynaptically targeted cow RNAi showed stronger impacts on SV fusion frequency with imaging in contrast to recordings, comparable to effects in the cowGDP null mutants. Moreover, SynapGCaMP imaging revealed significantly larger SV fusion event magnitudes in contrast to the lack of change found with TEVC recording. While the basis of these differences is unknown, it is speculated that it is due to the differential nature or sensitivity of these two methods. The Ca2+ imaging is based on measuring the change in the fluorescence signal over the baseline NMJ fluorescence, and it may be that glutamate receptor Ca2+ permeability or intracellular Ca2+ signaling dynamics is changed in a way not directly related to detectable membrane current changes in the cow mutants. TEVC recordings capture whole NMJ activity, whereas with imaging type 1b bouton activity was only captured normalized to area. In future studies, SynapGCaMP imaging can be used to map spatial changes in synapse function by assaying quantal activity separately in convergent type 1s and 1b motor neuron inputs and within discrete synaptic boutons. Moreover, differences between cowGDP and cowGDP/Df conditions could be influenced by second site-enhancing mutations on the Df chromosome. Overall, it should be noted that the changes in spontaneous SV fusion frequency and amplitude in cow mutants are subtle and variable, and need to be further studied in the future (Kopke, 2020).

Wg is lipid modified via palmitoylation to become strongly membrane associated. The hydrophobic moiety is located at the interface of Wg and Fz2 binding, shielded from the aqueous environment by multiple extracellular transporters until signaling interaction with the receptor. There have been many modes of extracellular Wg transport demonstrated, primarily from work in the wing disk, including microvesicles, lipoproteins, exosomes, and cytoneme membrane extensions. These multiple mechanisms of transport are much less studied at the synapse; however, exosome-like vesicles containing the Wg-binding protein Evenness Interrupted (Evi) have been demonstrated at the Drosophila NMJ. Cow could be considered an alternative extracellular Wg transport method, acting to shield Wg while facilitating transport through the extracellular synaptomatrix. In addition, HSPGs have been shown to regulate ligands by stabilizing, degrading, or sequestering the ligand, or as bifunctional coreceptors, or as facilitators of transcytosis. Results presented in this study are consistent with the hypothesis that Cow is mediating Wg transport across the NMJ synapse, but also that Cow has an additional role in the negative regulation of Wg synaptic signaling (Kopke, 2020).

The need for secreted Wg has been recently challenged, with Wg tethering to the membrane (NRT-wg) showing Wg secretion to be largely dispensable for development. In contrast, other recent studies suggest that Wg release and spreading is necessary. This study finds that tethering Wg at the NMJ synapse increases extracellular Wg ligand levels, with no change in mature bouton numbers. This Wg accumulation shows that NRT-wg is more stable at the synaptic signaling interface, consistent with other studies. However, although Wg levels increase, Wg signaling is less effective. With NRT-wg, only the budding of new satellite bouton is increased, with no increase in mature bouton formation. Reducing Wg function causes Fz2 upregulation, so this study hypothesized that Wg signaling could be maintained by increased presynaptic Fz2 receptors. When Wg is tethered, Cow cannot mediate intercellular transport, so the hypothesis predicts a similar phenotype with Cow (NRT-wg) or without Cow (NRT-wg; cowGDP). Indeed, Cow removal in the NRT-wg condition does not impact synaptic bouton number, although it does block the increase in satellite boutons, consistent with a Cow role in greater Wg stability (Chang, 2014). These results show that Wg secretion is required for the elevated NMJ development characterizing cow mutant animals (Kopke, 2020).

To further test how Cow is working through the Wg pathway to negatively regulate NMJ development, genetic interaction tests were performed with the Wg-negative regulator Notum. At the NMJ, Wg trans-synaptic signaling is elevated in the absence of Notum, and null notum mutants display larger NMJs with more synaptic boutons, increased synapse number and elevated neurotransmission (Kopke, 2017). All these defects are phenocopied by neuronal Wg OE, showing that the positive synaptogenic phenotypes arise from lack of Wg signaling inhibition. Consistently, genetically correcting Wg levels at the synapse in notum nulls alleviates synaptogenic phenotypes (Kopke, 2017). This study shows that cow null mutants have the same phenotypes of expanded NMJs, supernumerary synaptic boutons, greater synapse number/function, and strengthened transmission, suggesting that Cow acts like Notum in regulating Wg signaling. A genetic test was performed to ask whether Cow and Notum work in this same pathway. While cow and notum null heterozygotes do not exhibit NMJ defects, cow/notum trans-heterozygotes display grossly expanded NMJs with excess boutons. This combined haplo-insufficiency (type 3 SSNC) of nonallelic noncomplementation suggests that Cow and Notum share related roles. When full double mutants were tested, there is no additive effect, showing that Cow and Notum restrict Wg signaling in the same pathway. However, this pathway convergence appears restricted only to the control of structural synaptogenesis but not of functional neurotransmission, although the control neurotransmission amplitude was elevated in these studies (Kopke, 2020).

Cow now joins the list of synaptic HSPGs with key roles in NMJ development. HSPGs have been implicated in vertebrate NMJ synapse formation for over 3 decades. The Agrin HSPG is secreted from presynaptic terminals to maintain postsynaptic acetylcholine receptor clustering. Another secreted HSPG, perlecan, regulates acetylcholinesterase localization. Drosophila NMJ analyses have begun to more systematically elucidate HSPG roles in NMJ formation and function. In particular, the glypican HSPG Dlp regulates Wg signaling to modulate both NMJ structure and function, including the regulation of active zone formation and SV release. Wg binds the core Dlp, with HS chains enhancing this binding, to retain Wg on the cell surface, where it can both compete with Fz2 receptors and facilitate Wg-Fz2 binding. This biphasic activity depends on the ratio of Wg, Fz2, and Dlp HSPG as expounded in the 'exchange factor model'. Cow may impact this exchange factor mechanism as a fourth player, acting with Dlp to modulate Wg transport and Wg-Fz2 binding at the synaptic interface. It will be important to test Dlp levels and distribution in cow nulls to see how Cow fits into this model (Kopke, 2020).

In addition to Cow, perlecan (Trol) is another secreted HSPG reported to regulate bidirectional Wg signaling at the Drosophila NMJ. Trol has been localized near the muscle membrane, where it promotes postsynaptic Wg accumulation. In the absence of Trol, Wg builds up presynaptically, causing excess satellite bouton formation. It is interesting to note that cow mutants enhance Wg signaling without increasing satellite boutons. In trol mutants, ghost boutons increase due to decreased postsynaptic Wg signaling. Note that cow mutants do not exhibit ghost boutons, which fails to support decreased postsynaptic Wg signaling. Other postsynaptic defects in trol mutants (e.g., reduced SSR, increased postsynaptic pockets) are NMJ ultrastructural features that could be a future focus using electron microscopy studies. Similar to cow mutants, extracellular Wg levels are decreased in the absence of Trol, speculated due to increased Wg proteolysis, since HS protects HS-binding proteins from degradation. In cow mutants, it is not yet known whether Wg is decreased due to elevated signaling (ligand/receptor endocytosis) or to increased degradation due to Cow no longer protecting/stabilizing the ligand. Given that synaptic Fz2 is internalized with Wg binding, future experiments could test internalized Fz2 levels in cow mutants as a proxy of Wg signaling (Kopke, 2020).

In summary, this study has confirmed new tools to study Cow HSPG function and has discovered that Cow from presynaptic motor neurons restricts NMJ bouton formation, glutamatergic synapse number, and NMJ functional differentiation. Cow acts within the same Wg trans-synaptic signaling pathway as Notum by regulating the Wg ligand in the extracellular synaptomatrix. Secreted Cow modulates extracellular Wg ligand levels, with additional functions controlling Wg signaling efficacy, which may be independent of or dependent on Wg transport. It will be interesting to determine whether Cow core protein and/or its HS chains are important for the synaptic structural and functional phenotypes. Wg must be secreted for Cow to act on it, as shown by the membrane-tethered interaction studies, showing that secreted Cow must work on the freely diffusible Wg ligand. Perhaps most informative for future studies will be dissection of the interactions, coordination or redundancy of the multiple synaptic HSPGs at the NMJ, to further the understanding of extracellular Wg trans-synaptic signaling regulation during synaptic development. Drosophila is a particularly well suited model to study HSPGs because of the relatively reduced complexity in this system (Kopke, 2020).

POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila

Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. This study identified pdm3m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that pdm3 acts upstream to wg. Furthermore, transient expression of pdm3 induced ectopic expression of wg-LacZ reporter and wg effector proteins in wing discs. It is proposed that pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles (Kim, 2020).

Transcription factors play essential roles by inducing genes during the formation of body plans, organ development, tissue specificity, and generation of diverse cell types. Numerous transcription factors are grouped based on similarity in their sequences and domain structures. Pituitary-specific positive transcription factor 1, Octamer transcription factor-1, Uncoordinated-86 domain (POU) transcription factors belong to a subfamily of homeodomain transcription factors, and are highly conserved in all metazoans. POU domain consists of two DNA binding domains, POU homeodomain and POU specific domain, and these two domains are linked by a flexible linker. Based on sequence homology of the POU domain and the linker, POU proteins are grouped into six classes. POU proteins are often expressed in spatiotemporally restricted patterns during development, implying that they may be specialized for differentiation of specific cells or tissues by activating required signal transduction pathways (Kim, 2020).

The class VI Drosophila POU domain motif 3 (Pdm3) protein is reported to function in olfactory receptor neurons (ORNs) by regulating olfactory receptor gene expression and axon targeting, and in ring (R) neurons by regulating the development of ellipsoid body (EB) and axon targeting to EB in the central brain. pdm3 is also important for the axon targeting of a type of tracheal dendrite (td) neurons. In particular, td neurons that normally form synapse in the nerve cord change their target to the central brain by ectopic expression of Pdm3. Besides the neuronal functions of Pdm3, pdm3 also acts as a repressor of abdominal pigmentation in D. melanogaster, and plays a role in female-limited color dimorphism in abdomen of D. montium. Despite these studies, it is still unknown how pdm3 performs these neuronal and non-neuronal functions (Kim, 2020).

pdm3f00828 and pdm31 homozygotes exhibit defects in axon targeting, odor perception, and locomotion. pdm3f00828 allele has insertion of a piggyback element in an intron near the 3' end of the pdm3 gene, and pdm31 has a premature stop codon in the middle of the coding region that results in the deletion of the POU domain. This study identified a new pdm3 allele, pdm3m7, as a suppressor of lethality induced by Sol narae (Sona) overexpression in a genetic screen. Sona is a fly ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) whose family members are secreted metalloproteases important for cell proliferation, cell survival and development. This study has shown that Sona positively regulates Wingless (Wg) signaling and is essential for fly development, cell survival, and wg processing. wg is a prototype of Wnt family that initiates signal transduction cascade as extracellular signaling proteins, and activation of Wnt signaling leads to transcriptional induction of multiple genes for regulation of cell proliferation, cell survival, cell fate decision, and cell migration. wg is important for the development of all appendages, and the wing imaginal disc has been a great tool to study wg signaling because wg secreted from its dorsal-ventral midline is crucial for growth and development of wings (Kim, 2020).

Wg also plays an essential role in the development of NMJ. During larval development, NMJs continue to form synaptic boutons that are specialized structures with axon terminals of motor neurons surrounded by reticular subsynaptical reticulum (SSR) formed by the plasma membrane of postsynaptic muscle19. Among multiple types of boutons such as type Ib, Is, II, and III, wg is secreted at a high level from the glutamatergic type Ib bouton known as the main localization site of wg protein and wg signaling components, and is absent or at very low levels in other types of boutons (Packard, 2002; Kim, 2020).

Type Ib boutons also have more extensive SSR compared to other bouton types, so are easily detected by the high level of Discs-Large (Dlg) as a postsynaptic marker. Type Ib boutons in NMJs of wg mutants show reduction in bouton number but increase in bouton size. Components in wg signaling such as Arrow (Arr) that positively regulates wg signaling as a coreceptor of wg also shows its mutant phenotype similar to wg, but Shaggy (Sgg)/GSK3β that negatively regulates wg signaling as a kinase shows opposite phenotype to wg. Thus, dynamic regulation of wg signaling is essential for the development of NMJ (Kim, 2020).

Secreted wg also signals to the presynaptic motor neuron to regulate Futsch, one of the microtubule-associated proteins (MAPs). Futsch is a homolog of mammalian MAP1B, and both Futsch and MAP1B are phosphorylated at a conserved site by Sgg/GSK3β. The phosphorylated MAP1B does not bind microtubules, which results in reduced stability of microtubules. Therefore, localization of Futsch at NMJ faithfully reflects the stability of microtubules that is dynamically regulated by wg signaling. Loss of futsch phenotype is similar to the loss of wg phenotype in NMJ (Kim, 2020).

This study reporta that pdm3 is identified as a suppressor of Sona-induced lethality. Based on the involvement of Sona in wg signaling and the neuronal role of Pdm3, the roles of pdm3 in NMJ were specifically studied. Similar to loss of wg, loss of pdm3 in NMJ caused decrease in number but increase in size of boutons. Lethality induced by overexpressed pdm3 was completely rescued by the knockdown of wg in motor neurons but not vice versa. This indicated that pdm3 functions upstream to wg, and prompted a test whether pdm3 can induce wg transcription. Indeed, transient expression of pdm3 in wing discs induced wg transcription and wg effector proteins. Based on these data, it is propose that one of the main functions of pdm3 in NMJ is to induce wg transcription (Kim, 2020).

This study reports that pdm3 regulates growth and development of NMJs. pdm3 mutants showed increase in bouton size and decrease in bouton number, which are similar to the phenotype of wg mutants. Lethality induced by the overexpression of pdm3 was rescued by knockdown of wg in NMJ, indicating that pdm3 functions upstream to wg. Furthermore, overexpression of pdm3 induced wg transcription in wing discs. It is proposed that a major function of pdm3 in motor neurons is to induce wg transcription, and secreted wg from motor neurons regulates growth, development, and maturation of both pre- and post-synaptic regions of NMJ (Kim, 2020).

The mammalian homolog of pdm3 is Brain-5 (Brn-5)/POU class 6 homeobox 1 (POU6F1) mainly expressed in brain and spinal cord. Brn-5 is heavily expressed in embryonic brain but also expressed in adult brain and multiple adult organs such as kidney, lung, testis, and anterior pituitary. In developing brain, Brn-5 is expressed in postmitotic neurons after neuronal progenitor cells exit cell cycle in the early process of terminal neuronal differentiation. Therefore, both pdm3 and Brn-5 function in differentiation of neurons. Interestingly, ectopic expression of Brn-5 inhibits DNA synthesis, which is similar to cell cycle arrest phenotype by wg overexpression. Given the homology between pdm3 and Brn-5 as well as functional similarities, Brn-5 may also induce wnt transcription (Kim, 2020).

Most of pdm3 functions identified so far are related to the maturation of neurons such as olfactory neurons, R neurons and td neurons as well as their postsynaptic partners. Ectopic expression of pdm3 induced lethality without exception, indicating that expression of pdm3 in fly tissues is generally repressed in vivo in order to express wg under the strict spatiotemporal control. An important question is whether pdm3 directly transcribe wg. This study found that wg transcription is induced only after 36 hours of transient overexpression of Pdm3. It is possible that the level of pdm3 needs to be over a threshold to induce wg transcription. Alternatively, pdm3 may need to turn on other components to indirectly induce wg transcription. DNA sequence of Brn-5 binding site has been reported, so analysis on wg and wnt regulatory regions will help understand the mechanism of wnt induction by pdm3 and Brn-5 (Kim, 2020).

This study consistently found more significant NMJ phenotypes in A2 than A3 in both pdm3 and wg mutants. Therefore, pdm3 and wg may play more prominent roles in the A2 than the A3 segment. In fact, the level of pdm3 was higher in the anterior region than the posterior region of ventral ganglion, which suggests that more wg may be present in the NMJs of anterior abdominal segments. Consistent with this idea, the number of type Ib boutons in the A2 segment was 1.8 times more than A3 segment. One difference between pdm3 and wg mutants is the lack of certain phenotypes in the A3 segment of pdm3 NMJs: the size of boutons and the number of axon terminals in A3 were not affected in pdm3 mutant. It is possible that pdm3 turns on both common and segment-specific genes besides wg, and A3 segment-specific components may alleviate the loss of wg phenotype in the A3 segment. Similarly, other proteins induced by pdm3 may also play important roles in NMJ growth, differentiation and maintenance. In fact, multiple signaling pathways including Glass-bottom-boat (Gbb) pathway also play roles in NMJ development. Gbb is secreted from muscles and induces development of both pre- and post-synaptic structures, similar to wg signaling (Kim, 2020).

This study identified a defective hobo element in the pdm3m7 allele. The hobo element belongs to Ac family found in maize and has short inverted terminal repeats. Laboratory and wild strains of D. melanogaster have average 28 and 22 copies of hobo elements in the genome that are either full-length or defective, respectively. Because other suppressors identified in the genetic screen using Sona overexpression did not have hobo element in the pdm3 gene, the transposition of the hobo element to the pdm3 gene may have occurred subsequent to the generation of a point mutation in the arr gene by EMS. Since both arr and pdm3 are positively involved in wg signaling, this hobo insertion may have helped the original arrm7 mutation to further decrease the activity of wg signaling under the condition of Sona overexpression (Kim, 2020).

Besides the neuronal roles of Pdm3, all pdm3 mutants show minor but consistent defects in planar cell polarity in a restricted region of the wing as well as adhesion between the dorsal and ventral wing blades. Other phenotypes such as wing drooping and premature death were also observed in all pdm3 mutants, but these may be due to malformation of synaptic structures. pdm3 also plays a role in female-limited color dimorphism in abdomen of D. montium. The authors found in sexually dimorphic females that the first intron of the pdm3 gene has four tandem sets with predicted binding sites for the HOX gene Abdominal-B (Abd-B) and the sex determination gene doublesex (dsx). Interestingly, it has been shown that wg expression is repressed by the combinatory work of Abd-B and Dsx proteins. Taken together, it is possible that transcription of wg and pdm3 is co-repressed by Abd-B and Dsx. Such co-repression of wg and pdm3 transcription may be also required for synaptic growth and differentiation in neurons. Further studies on pdm3 will help understand how this understudied transcription factor is involved in the final differentiation of various cell types (Kim, 2020).

Regulation of anisotropic tissue growth by two orthogonal signaling centers

The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, this study shows that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. The data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving the T cell factor (TCF) Pangolin (Barrio, 2020).

Two orthogonal signaling centers, corresponding to the AP and DV compartment boundaries and expressing the Dpp and Wg morphogens, regulate growth and patterning of the developing wing along the AP and PD axes, respectively. Whereas graded activity of these morphogens defines the spatial location of longitudinal veins and sensory organs that decorate the adult wing along these two axes, their graded activity is not an absolute requirement for its growth-promoting role. Despite the non-instrumental role of Wg and Dpp gradients in regulating tissue size, this study presents evidence that these two morphogens control the size of the adult wing along two orthogonal axes by mediating the growth-promoting activities of compartment boundaries in a non-interchangeable manner through the use of morphogen-specific molecular mechanisms. While Dpp regulates growth along the AP axis by maintaining the levels of the transcriptional repressor Brinker below a growth-repressing threshold, Wg regulates growth along the PD axis by counteracting the activity of TCF as a transcriptional repressor. At the time TCF was molecularly identified in flies, it was shown that clones of cells mutant for TCF are poorly recovered in the primordium of the wing pouch and proposed to be a consequence of TCF promoting proliferative growth. However, later studies identified cell competition as the mechanism to eliminate cells with steep differences in Wg signaling in the wing primordium. The Warts-Hippo signaling pathway governs organ size in animals, and the upstream regulators include the atypical cadherins Fat and Dachsous. Surprisingly, inactivation of the Warts-Hippo signaling pathway was unable to rescue the tissue size defects caused by morphogen depletion. These data indicate that for wing blade cells to grow along the PD and AP axes, cells need first to lose TCF and Brinker, and it is proposed that Hippo signaling can then modulate the amount of growth of those cells in which these two repressors are not active or expressed. The experimental data are consistent with a model whereby a minimal amount of signaling from the two morphogens, sufficient to maintain the activity levels of the two transcriptional repressors below a growth-repressing threshold, regulate the physical size of the adult wing primordium along the AP and PD axes. The mechanistic similarities of how Dpp and Wg morphogens, their gradients, and their range of activity regulate the patterning and growth of the fly wing are remarkable and might shed light on the role of morphogens in regulating proliferative growth and patterning in vertebrates (Barrio, 2020).

Experimental conditions in developing wings in which proliferation rates are either increased or reduced have shown that a perfectly normal-sized wing can be obtained with fewer or more cells. Similarly, experimental randomization of the orientation of cell divisions in the growing wing primordium can give rise to well-shaped adult wings. These results suggest that the ability of compartment boundaries, and their dedicated morphogens, to drive anisotropic growth and regulate the width and length of the adult wing blade does not rely only on the control of cell division or oriented cell divisions. Several experimental data indicate that it is the range of the morphogen and not the total amount of it that regulates the physical size, and not the number of cells, of each axis. How do Wg and Dpp regulate growth preferentially along a certain axis and not the other? Restricted expression of these two morphogens along the two existing orthogonal boundaries does not appear to be essential as their ability to drive anisotropic growth is still observed when they are ubiquitously overexpressed in all wing cells. The experimental data indicate that the capacity of Wg and Dpp to drive anisotropic growth relies on the existence of morphogen-specific and non-interchangeable molecular mechanisms mediating their growth-promoting activities and the requirement of the presence of the two of them to drive growth. In this regard, each morphogen promotes growth only along a particular axis, as the distance to the source of the other morphogen has to be maintained to get sufficient levels of the two of them to promote wing growth. The data also indicate that the Wg gradient contributes to orient growth along the PD axis. However, this contribution does not appear to play an essential role since well-shaped elongated wings can be obtained upon uniform expression of Wg (Barrio, 2020).

While the growth-promoting role of Dpp emanating from the AP compartment boundary has been experimentally validated and recently clarified, previous experimental characterization of the growth-promoting role of Wg emanating from the DV compartment boundary reached opposing conclusions. This study presents experimental evidence that Wg mediates the organizing activity of the DV boundary in terms of growth, as uniform expression of this morphogen rescues the extreme growth defects caused by the absence of a DV signaling center. Moreover, the data indicate that Wg is the main growth-promoting Wnt in the developing wing, the DV boundary is the main source of Wg driving proliferative growth of the primordium of the wing appendage, and boundary Wg regulates tissue growth and proliferation rates equally in distal and proximal regions of the developing wing appendage, throughout development and independently of its potential role as survival factor. This latter observation questions the proposal that Wg drives wing growth, at least in part, by promoting cell survival. This proposal was based on the ability of apoptotic inhibitors to rescue the poor recovery and growth of clones of cells unable to transduce the Wg signal, but cell competition was subsequently shown to be the mechanism used to eliminate cells with steep differences in Wg signaling. The experimental observation that even late depletion of Wg expression has an effect on wing size questions the proposal that continuous exposure to Wg is not an absolute requirement for wing cells to grow. Recently, a membrane-tethered form of the Wg protein was shown to be able to substitute for the endogenous Wg protein in producing normally patterned wings of nearly the right size. Either the activity of cellular extensions at a distance, higher stability of the membrane-tethered form of Wg, or emerging compensatory mechanisms should be able to facilitate or extend in time the exposure of all wing cells to the morphogen in the absence of secretion, thus fulfilling its continuous growth-promoting role (Barrio, 2020).

Earlier Description of Wingless Function

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).


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:  15 April 2020 

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