The endocytic pathway and formation of the Wingless morphogen gradient

Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. We have utilized short-term expression of dominant negative Rab proteins to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. Our results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As previously shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. We find that both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. We find that Dally-like is not required for Wingless spread when endocytosis is blocked, and propose that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient.     

Signalling at a distance: transport of Wingless in the embryonic epidermis of Drosophila.

Secreted signalling molecules affect the behavior of cells at a distance. Here we discuss how the Wnt family member Wingless reaches distant cells within the embryonic epidermis of Drosophila.We consider three possible mechanisms: free diffusion, restricted diffusion and active transport. We argue that free diffusion is unlikely to occur. However, a variant of restricted diffusion may account for Wingless transport. It may be that Wingless is carried from one side of a cell to the other by a drifting transmembrane protein such as a specific receptor or a glycosaminoglycan. Transfer from cell-to-cell would involve release from the donor cell and recapture in an adjacent cell. Alternatively, Wingless might be transported by a mechanism akin to transcytosis. This would involve the packaging of Wingless in specialized vesicles at one end of a cell, active transport across the cell, and vesicle fusion and Wingless release on the other side. We describe the evidence in favor and against these two alternatives.     

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.     

A screen for genes regulating the wingless gradient in Drosophila embryos

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport.     

The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages.     

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.     

Notch responds differently to Delta and Wingless in cultured Drosophila cells

Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane.     

HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient

The secreted signaling protein Wingless acts as a morphogen to pattern the imaginal discs of Drosophila. Here we report identification of a secreted repressor of Wingless activity, which we call Notum. Loss of Notum function leads to increased Wingless activity by altering the shape of the Wingless protein gradient. When overexpressed, Notum blocks Wingless activity. Notum encodes a member of the alpha/beta-hydrolase superfamily, with similarity to pectin acetylesterases. We present evidence that Notum influences Wingless protein distribution by modifying the heparan sulfate proteoglycans Dally-like and Dally. High levels of Wingless signaling induce Notum expression. Thus, Wingless contributes to shaping its own gradient by regulating expression of a protein that modifies its interaction with cell surface proteoglycans.     

Control of Drosophila eye specification by Wingless signalling

Organ formation requires early specification of the groups of cells that will give rise to specific structures. The Wingless protein plays an important part in this regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. We show that Wingless signalling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signalling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signalling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, we find that under conditions of excess Wingless signalling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures.     

Spatiotemporally modulated Vestigial gradient by Wingless signaling adaptively regulates cell division for precise wing size control

In animal development, the growth of a tissue or organ is timely arrested when it reaches the stereotyped correct size. How this is robustly controlled remains poorly understood. The prevalent viewpoint, which is that morphogen gradients, due to their organizing roles in development, are directly responsible for growth arrest, cannot explain a number of observations. Recent findings from studies of the Drosophila wing have revealed that the interpretation of the Wingless gradient requires signaling-induced self-inhibition and that cell proliferation is controlled by graded vestigial expression. These findings highlight a growth control mechanism that involves Wingless regulated vestigial expression, but a question is whether they can quantitatively explain the observed preciseness and robustness of wing size control. Quantitative and systematic investigation into Wingless signaling using a mathematical model has elucidated two points. First, negative regulation of the Vestigial gradient by Wingless signaling makes vestigial expression precise and robust. Second, weak Wingless signaling in a primarily small wing pouch causes a short and steep Vestigial gradient, which stimulates more cell divisions and leads to a significant expansion of the wing pouch; however, strong Wingless signaling in a primarily large wing pouch causes a long and smooth Vestigial gradient, which stimulates fewer cell divisions and results in a slight expansion of the wing pouch. These results substantially decipher an inherent mechanism of tissue and organ size control. Our model explains, and is supported by, a number of experimental observations.     

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.     

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.     

The ins and outs of Wingless signaling

Signaling through the highly conserved Wingless/Wnt pathway plays a crucial role in a diverse array of developmental processes, many of which depend upon the precise regulation of Wingless/Wnt signaling levels. Recent evidence has indicated that the intracellular trafficking of Wingless/Wnt signaling components can result in significant changes in the level of signaling. Here, we examine three mechanisms through which intracellular trafficking might regulate Wingless signaling--the degradation of Wingless, its transport and the transduction of its signal. The intracellular trafficking of several Wingless/Wnt signaling components, including LRP5, LRP6, Dishevelled and Axin, as well as the functional implications of protein localization on Wingless/Wnt signaling, will be discussed.