Wingless signaling and the control of cell shape in Drosophila wing imaginal discs

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer β-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.     

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin (DE-Cad) regulates epithelial cell-cell adhesion at adherens junctions (AJs). Here, we show that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless (Wg), a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of beta-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, we show that Ft intersects Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing.     

Morphogen control of wing growth through the Fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.     

scalloped functions in a regulatory loop with vestigial and wingless to pattern the Drosophila wing

 The development of the Drosophila wing involves progressive patterning events. In the second larval instar, cells of the wing disc are allotted wing or notum fates by a wingless-mediated process and dorsal or ventral fates by the action of apterous and wingless. Notch-mediated signalling is required for the expression of the genes vestigial and scalloped in the presumptive wing blade. Later, wingless, Notch and cut are involved in cell fate specification along the wing margin. The function of scalloped in this process is not well understood and is the focus of this study. We show that patterning downstream of Notch and wingless pathways is altered in scalloped mutants. Reduction in scalloped expression results in a loss of expression of wing blade- and margin-specific markers. Misexpression of scalloped in the presumptive wing causes misexpression of scalloped, vestigial and wingless reporter genes. However, high levels of scalloped expression have a negative influence on wingless, vestigial and its own expression. Our results demonstrate that scalloped functions in a level-dependent manner in the presumptive wing blade in a loop that involves vestigial and itself. We suggest that wing development requires the regulated expression of scalloped together with vestigial–the ”wing formation” effects of Vestigial in other imaginal discs are probably due to its interaction with the scalloped gene product normally expressed in these discs. Received: 6 May 1998 / Accepted: 22 July 1998     

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Here, we show that three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. We also identify dachs as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. Our observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway.     

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.     

Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Previously, we have shown that Ubx represses dorsoventral (DV) signaling to specify haltere fate. Here we examine the mechanism of Ubx-mediated downregulation of DV signaling. We show that Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on DV boundary function of Wg, it maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions, at least, at two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3 beta to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations.     

Drosophila S2 Cells Secrete Wingless on Exosome‐Like Vesicles but the Wingless Gradient Forms Independently of Exosomes

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless‐expressing Drosophila S2 cells, Wingless is present on exosome‐like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless‐containing exosome‐like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome‐like vesicles. Using these exosome markers and a cell‐based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless‐expressing cells.     

Scalloped and strawberry notch are target genes of Notch signaling in the context of wing margin formation in Drosophila.

The Notch pathway regulates the differentiation of many cell types throughout development of higher metazoa. Different cellular responses are elicited through specific activation of distinct Notch target genes. In the Drosophila wing, for example, the cut gene is activated by Notch signaling along the dorso-ventral boundary but, as we show here, not in other cell types. We identify additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. Our data support a model, whereby context specific involvement of distinct co-regulators modulates Notch target gene activation.     

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. Here, we show that a temporally dynamic response to Wingless signalling sequentially elaborates the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, zfh-2, is initially activated by Wingless throughout the "wing primordium," but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. Our data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

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.