Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein

Wingless (Wg) is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. We show that dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix.     

Roles of N-glycosylation and lipidation in Wg secretion and signaling

Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modification at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo receptor involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.     

Wingless secretion promotes and requires retromer-dependent cycling of Wntless

Wnt ligands are lipid-modified, secreted glycoproteins that control multiple steps during embryogenesis and adult-tissue homeostasis. Little is known about the mechanisms underlying Wnt secretion. Recently, Wntless (Wls/Evi/Srt) was identified as a conserved multi-pass transmembrane protein whose function seems to be dedicated to promoting the release of Wnts. Here, we describe Wls accumulation in the Golgi apparatus of Wnt/Wingless (Wg)-producing cells in Drosophila, and show that this localization is essential for Wg secretion. Moreover, Wls localization and levels critically depend on retromer, a conserved protein complex that mediates endosome-to-Golgi protein trafficking in yeast. In the absence of the retromer components Dvps35 or Dvps26, but in presence of Wg, Wls is degraded and Wg secretion impaired. Our results indicate that Wg, clathrin-mediated endocytosis and retromer sustain a Wls traffic loop from the Golgi to the plasma membrane and back to the Golgi, thereby enabling Wls to direct Wnt secretion.     

Signaling Activities of the Drosophila Wingless Gene Are Separately Mutable and Appear to Be Transduced at the Cell Surface

The Drosophila segment polarity gene wingless encodes an intercellular signaling molecule that transmits positional information during development of the embryonic epidermis. We have explored the mechanism of wg signal transduction by perturbing cellular processes genetically and by performing structure/function analysis of the Wg protein. We present evidence that Wingless protein may transduce signal at the cell surface and that Wg may bind to its cell surface receptor without necessarily activating it. We demonstrate that two specific signaling activities of the Wg molecule can be disrupted independently by mutation. Sequence analysis indicates that these different signaling activities are not promoted by discrete functional domains, but rather that the overall conformation of the molecule may control distinct signaling functions. We conclude that wg signaling may involve complex interactions between the Wg ligand and its cell surface receptor molecule(s) and that some of this complexity resides within the Wg ligand itself.     

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Here, we show that patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overproliferation of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner.     

The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos

Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread is still unclear. It is generally assumed that they reach their target site either by diffusion or active transport (reviewed in [1] [2]). Here, we report an alternative mode of transport for Wingless (Wg), a member of the Wnt family of signaling molecules. In embryos of the fruit fly Drosophila, the wingless (wg) gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. We found that these cells or their progeny are free to roam towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. The cells leaving the expression domain retain inherited Wg protein in secretory vesicles, however, and carry it forwards over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg showed that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independently of protein inheritance, possibly by restricted diffusion. We suggest that both transport mechanisms operate in wild-type embryos.     

Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries

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. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence 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, we demonstrate 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. We conclude 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.     

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.     

<Emphasis Type="Italic">kuzbanian</Emphasis> is required cell autonomously during Notch signalling in the <Emphasis Type="Italic">Drosophila </Emphasis>wing

The kuzbanian gene encodes a metalloprotease of the ADAM family that is involved in Notch signalling. However, its precise role is a matter of controversy. While original reports concluded that kuz is required on the receiving side of the Notch signalling pathway, a more recent report suggests that Kuz is required on the signal-emitting side for the generation of an active secreted form of the ligand Delta. In this scenario, kuz should act cell non-autonomously. A third possibility is that Kuz is required on the signal-emitting as well as the receiving side. Here I present the clonal analysis of kuz in Drosophila wing. The results show that Kuz acts on the receiving side of the pathway and is not required for Delta signalling. This further confirms the hypothesis that Kuz is required for the release of the intracellular domain of Notch that transduces the signal to the nucleus. The presented results complement recent data that indicate that Kuz can perform the S2 proteolytic cleavage of the Notch receptor that is required for its activation.     

Arabidopsis SUC1 loads the phloem in suc2 mutants when expressed from the SUC2 promoter

Active loading of sucrose into phloem companion cells (CCs) is an essential process in apoplastic loaders, such as Arabidopsis or tobacco (Nicotiana sp.), and is even used by symplastic loaders such as melon (Cucumis melo) under certain stress conditions. Reduction of the amount or complete removal of the transporters catalysing this transport step results in severe developmental defects. Here we present analyses of two Arabidopsis lines, suc2-4 and suc2-5, that carry a null allele of the SUC2 gene which encodes the Arabidopsis phloem loader. These lines were complemented with constructs expressing either the Arabidopsis SUC1 or the Ustilago maydis srt1 cDNA from the SUC2 promoter. Both SUC1 and Srt1 are energy-dependent sucrose/H(+) symporters and differ in specific kinetic properties from the SUC2 protein. Transgene expression was confirmed by RT-PCRs, the subcellular localization of Srt1 in planta with an Srt1-RFP fusion, and the correct CC-specific localization of the recombinant proteins by immunolocalization with anti-Srt1 and anti-SUC1 antisera. The transport capacity of Srt1 was studied in Srt1-GFP expressing Arabidopsis protoplasts. Although both proteins were found exclusively in CCs, only SUC1 complemented the developmental defects of suc2-4 and suc2-5 mutants. As SUC1 and Srt1 are well characterized, this result provides an insight into the properties that are essential for sucrose transporters to load the phloem successfully.     

Wingless gradient formation in the Drosophila wing

BACKGROUND: Secreted signaling proteins of the Wingless (Wg)/Wnt, Hedgehog and bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) families function as morphogens to control growth and pattern formation during development. Although these proteins have been shown to act directly on distant cells in the developing limbs of the fruit fly Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. RESULTS: A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement did not require internalization by dynamin-mediated endocytosis. Dynamin activity was, however, required for Wg secretion. By reversibly blocking Wg secretion, we found that Wg moves rapidly to form a long-range extracellular gradient. CONCLUSIONS: The Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. We propose that the extracellular Wg gradient forms by diffusion.     

Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.