Control of Drosophila wing growth by the vestigial quadrant enhancer

Following segregation of the Drosophila wing imaginal disc into dorsal (D) and ventral (V) compartments, the wing primordium is specified by activity of the selector gene vestigial (vg). In the accompanying paper, we present evidence that vg expression is itself driven by three distinct inputs: (1) short-range DSL (Delta/Serrate/LAG-2)-Notch signaling across the D-V compartment boundary; (2) long-range Wg signaling from cells abutting the D-V compartment boundary; and (3) a short-range signal sent by vg-expressing cells that entrains neighboring cells to upregulate vg in response to Wg. Furthermore, we showed that these inputs define a feed-forward mechanism of vg autoregulation that initiates in D-V border cells and propagates from cell to cell by reiterative cycles of vg upregulation. Here, we provide evidence that this feed-forward mechanism is required for normal wing growth and is mediated by two distinct enhancers in the vg gene. The first is a newly defined ;priming' enhancer (PE), that provides cryptic, low levels of Vg in most or all cells of the wing disc. The second is the previously defined quadrant enhancer (QE), which we show is activated by the combined action of Wg and the short-range vg-dependent entraining signal, but only if the responding cells are already primed by low-level Vg activity. Thus, entrainment and priming constitute distinct signaling and responding events in the Wg-dependent feed-forward circuit of vg autoregulation mediated by the QE. We posit that Wg controls the expansion of the wing primordium following D-V segregation by fueling this autoregulatory mechanism.     

A matrix metalloproteinase mediates long-distance attenuation of stem cell proliferation

Ligand-based signaling can potentiate communication between neighboring cells and between cells separated by large distances. In the Drosophila melanogaster ovary, Wingless (Wg) promotes proliferation of follicle stem cells located ∼50 µm or five cell diameters away from the Wg source. How Wg traverses this distance is unclear. We find that this long-range signaling requires Division abnormally delayed (Dally)-like (Dlp), a glypican known to extend the range of Wg ligand in the wing disc by binding Wg. Dlp-mediated spreading of Wg to follicle stem cells is opposed by the extracellular protease Mmp2, which cleaved Dlp in cell culture, triggering its relocalization such that Dlp no longer contacted Wg protein. Mmp2-deficient ovaries displayed increased Wg distribution, activity, and stem cell proliferation. Mmp2 protein is expressed in the same cells that produce Wg; thus, niche cells produce both a long-range stem cell proliferation factor and a negative regulator of its spreading. This system could allow for spatial control of Wg signaling to targets at different distances from the source.     

Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation

The Drosophila wing primordium is defined by expression of the selector gene vestigial (vg) in a discrete subpopulation of cells within the wing imaginal disc. Following the early segregation of the disc into dorsal (D) and ventral (V) compartments, vg expression is governed by signals generated along the boundary between the two compartments. Short-range DSL (Delta/Serrate/LAG-2)-Notch signaling between D and V cells drives vg expression in ;border' cells that flank the boundary. It also induces these same cells to secrete the long-range morphogen Wingless (Wg), which drives vg expression in surrounding cells up to 25-30 cell diameters away. Here, we show that Wg signaling is not sufficient to activate vg expression away from the D-V boundary. Instead, Wg must act in combination with a short-range signal produced by cells that already express vg. We present evidence that this vg-dependent, vg-inducing signal feeds forward from one cell to the next to entrain surrounding cells to join the growing wing primordium in response to Wg. We propose that Wg promotes the expansion of the wing primordium following the D-V segregation by fueling this non-autonomous autoregulatory mechanism.     

Drosophila Heparan Sulfate 6-O-Endosulfatase Sulf1 Facilitates Wingless (Wg) Protein Degradation

Heparan sulfate proteoglycans regulate various physiological and developmental processes through interactions with a number of protein ligands. Heparan sulfate (HS)-ligand binding depends on the amount and patterns of sulfate groups on HS, which are controlled by various HS sulfotransferases in the Golgi apparatus as well as extracellular 6-O-endosulfatases called “Sulfs.” Sulfs are a family of secreted molecules that specifically remove 6-O-sulfate groups within the highly sulfated regions on HS. Vertebrate Sulfs promote Wnt signaling, whereas the only Drosophila homologue of Sulfs, Sulf1, negatively regulates Wingless (Wg) signaling. To understand the molecular mechanism for the negative regulation of Wg signaling by Sulf1, we studied the effects of Sulf1 on HS-Wg interaction and Wg stability. Sulf1 overexpression strongly inhibited the binding of Wg to Dally, a potential target heparan sulfate proteoglycan of Sulf1. This effect of Drosophila Sulf1 on the HS-Wg interaction is similar to that of vertebrate Sulfs. Using in vitro, in vivo, and ex vivo systems, we show that Sulf1 reduces extracellular Wg protein levels, at least partly by facilitating Wg degradation. In addition, expression of human Sulf1 in the Drosophila wing disc lowers the levels of extracellular Wg protein, as observed for Drosophila Sulf1. Our study demonstrates that vertebrate and Drosophila Sulfs have an intrinsically similar activity and that the function of Sulfs in the fate of Wnt/Wg ligands is context-dependent.     

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.     

The Sno Oncogene Antagonizes Wingless Signaling during Wing Development in Drosophila

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates.     

Extracellular spreading of Wingless is required for Drosophila oogenesis

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.     

JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc

JAK/STAT signaling is localized to the wing hinge, but its function there is not known. Here we show that the Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. We show that reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. We report negative interactions between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, we find that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. We conclude that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development.     

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation

Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.     

Cell degeneration and elimination in the imaginal wing disc,caused by the mutationsvestigial andUltravestigial ofDrosophila melanogaster

Summary The mutationsvestigial (vg; recessive) andUltravestigial (vg ^U; dominant) ofDrosophila melanogaster give rise to identical mutant adult phenotypes in which much of the cases this results from cell death in the presumptive wing margin of the wing disc in the third larval instar, but the process of cell degeneration is quite different in the two mutants. Invg cell death occurs continuously throughout the third larval instar, while invg ^U it occurs only in the early third instar. Cells fragment and some of the fragments condense, becoming electron dense (apoptosis). Both condensed and ultrastructurally normal cell fragments are extruded to the basal side of thevg disc epithelium. They accumulate under the basal lamina in the wing pouch area until they are phagocytosed by blood cells entering the wing pouch during the six hours following pupariation. Fragments are not extruded from thevg ^U epithelium but are apparently phagocytosed by neighboring epithelial cells. The basal lamina undergoes mophological changes following pupariation and is phagocytosed by blood cells in both wild-type andvestigial, but investigial the degenerated cell fragments are also engulfed by the same blood cells.     

Sphingolipid depletion impairs endocytic traffic and inhibits Wingless signaling

Sphingolipids are an important part of the plasma membrane and implicated in a multitude of cellular processes. However, little is known about the role of sphingolipids in an epithelial context and their potential influence on the activity of signaling pathways. To shed light on these aspects we analyzed the consequences of changing ceramide levels in vivo in the Drosophila wing disc: an epithelial tissue in which the most fundamental signaling pathways, including the Wnt/Wg signaling pathway, are well characterized.We found that downregulation of Drosophila’s only ceramide synthase gene schlank led to defects in the endosomal trafficking of proteins. One of the affected proteins is the Wnt ligand Wingless (Wg) that accumulated. Unexpectedly, although Wg protein levels were raised, signaling activity of the Wg pathway was impaired. Recent work has spotlighted the central role of the endocytic trafficking in the transduction of the Wnt signal. Our results underscore this and support the view that sphingolipid levels are crucial in orchestrating epithelial endocytic trafficking in vivo. They further demonstrate that ceramide/sphingolipid levels can affect Wnt signaling.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. Here, we characterize the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigate whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. We demonstrate a role for Arrow and DFz2 in Wg internalization. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, we find that Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, we suggest that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event.