The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.     

Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans

Glycosaminoglycans such as heparan sulphate and chondroitin sulphate are extracellular sugar chains involved in intercellular signalling. Disruptions of genes encoding enzymes that mediate glycosaminoglycan biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, which encodes a UDP-glucose dehydrogenase, impairs developmental signalling through the Wnt family member Wingless, and signalling by the fibroblast growth factor and Hedgehog pathways. Heparan sulphate is involved in these pathways, but little is known about the involvement of chondroitin. Undersulphated and oversulphated chondroitin sulphate chains have been implicated in other biological processes, however, including adhesion of erythrocytes infected with malaria parasite to human placenta and regulation of neural development. To investigate chondroitin functions, we cloned a chondroitin synthase homologue of Caenorhabditis elegans and depleted expression of its product by RNA-mediated interference and deletion mutagenesis. Here we report that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis. Reversion of cytokinesis is often observed in chondroitin-depleted embryos, and cell division eventually stops, resulting in early embryonic death. Our findings show that chondroitin is required for embryonic cytokinesis and cell division.     

Specificities of heparan sulphate proteoglycans in developmental processes

Heparan sulphate proteoglycans are abundant cell-surface molecules that consist of a protein core to which heparan sulphate glycosaminoglycan chains are attached. The functions of these molecules have remained mostly underappreciated by developmental biologists; however, the actions of important signalling molecules, for example Wnt and Hedgehog, depend on them. To understand both the mechanisms by which ligands involved in development interact with their receptors and how morphogens pattern tissues, biologists need to consider the functions of heparan sulphate proteoglycans in signalling and developmental patterning.     

Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells

In the Drosophila midgut, multipotent intestinal stem cells (ISCs) that are scattered along the epithelial basement membrane maintain tissue homeostasis by their ability to steadily produce daughters that differentiate into either enterocytes or enteroendocrine cells, depending on the levels of Notch activity. However, the mechanisms controlling ISC self-renewal remain elusive. Here we show that a canonical Wnt signalling pathway controls ISC self-renewal. The ligand Wingless (Wg) is specifically expressed in the circular muscles next to ISCs, separated by a thin layer of basement membrane. Reduced function of wg causes ISC quiescence and differentiation, whereas wg overexpression produces excessive ISC-like cells that express high levels of the Notch ligand, Delta. Clonal analysis shows that the main downstream components of the Wg pathway, including Frizzled, Dishevelled and Armadillo, are autonomously required for ISC self-renewal. Furthermore, epistatic analysis suggests that Notch acts downstream of the Wg pathway and a hierarchy of Wg/Notch signalling pathways controls the balance between self-renewal and differentiation of ISCs. These data suggest that the underlying circular muscle constitutes the ISC niche, which produce Wg signals that act directly on ISCs to promote ISC self-renewal. This study demonstrates markedly conserved mechanisms regulating ISCs from Drosophila to mammals. The identification of the Drosophila ISC niche and the principal self-renewal signal will facilitate further understanding of intestinal homeostasis control and tumorigenesis.     

Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing.

Distinct and evolutionarily conserved signal-transduction cascades mediate the survival or death of cells during development. The c-Jun amino-terminal kinases (JNKs) of the mitogen-activated protein kinase superfamily are involved in apoptotic signalling in various cultured cells. However, the role of the JNK pathway in development is less well understood. In Drosophila, Decapentaplegic (Dpp; a homologue of transforming growth factor-beta) and Wingless (Wg; a Wnt homologue) proteins are secretory morphogens that act cooperatively to induce formation of the proximodistal axis of appendages. Here we show that either decreased Dpp signalling in the distal wing cells or increased Dpp signalling in the proximal wing cells causes apoptosis. Inappropriate levels of Dpp signalling lead to aberrant morphogenesis in the respective wing zones, and these apoptotic zones are also determined by the strength of the Wg signal. Our results indicate that distortion of the positional information determined by Dpp and Wg signalling gradients leads to activation of the JNK apoptotic pathway, and the consequent induction of cell death thereby maintains normal morphogenesis.     

Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis

Defects in glycosaminoglycan biosynthesis disrupt animal development and can cause human disease. So far much of the focus on glycosaminoglycans has been on heparan sulphate. Mutations in eight squashed vulva (sqv) genes in Caenorhabditis elegans cause defects in cytokinesis during embryogenesis and in vulval morphogenesis during postembryonic development. Seven of the eight sqv genes have been shown to control the biosynthesis of the glycosaminoglycans chondroitin and heparan sulphate. Here we present the molecular identification and characterization of the eighth gene, sqv-5. This gene encodes a bifunctional glycosyltransferase that is probably localized to the Golgi apparatus and is responsible for the biosynthesis of chondroitin but not heparan sulphate. Our findings show that chondroitin is crucial for both cytokinesis and morphogenesis during C. elegans development.     

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.     

Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus

The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate orthologue glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Here we show that Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form (Ci75), and by stimulating the activity of accumulated Ci155 (refs 2-6). Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes including decapentaplegic (dpp) and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signalling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3-phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. We propose that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets it for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation.     

Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation.

Cell-fate specification of R7 photoreceptors in the developing Drosophila eye depends on an inductive signal from neighbouring R8 cells. Mutations in three genes, sevenless (sev), bride-of-sevenless (boss) and seven-in-absentia (sina) cause the R7 precursor to become a non-neural cone cell. The sev gene encodes a receptor protein tyrosine kinase (Sev) localized on the R7 surface, activated by a boss-encoded ligand presented by R8. The sina gene encodes a nuclear factor required in R7. Reduction in the dosage of the Ras1 gene impairs Sev-mediated signalling, suggesting that activation of Ras1 may be an important consequence of Sev activation. We report here that Ras1 activation may account for all of the signalling action of Sev; an activated Ras1Va112 protein rescues the normal R7 precursor from transformation into a cone cell in sev and boss null mutants and induces the formation of supernumerary R7 cells. Similar activation of the Drosophila Ras2 protein does not produce these effects, demonstrating Ras protein specificity.     

arrow encodes an LDL-receptor-related protein essential for Wingless signalling

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.     

Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin

Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF-heparin and FGF-FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1-FGFR2-heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.     

The evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling

Dorsal-ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Here we describe Xenopus Twisted gastrulation (xTsg), another member of this signalling pathway. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signalling agonist. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signalling in the embryo. Drosophila Tsg also binds BMPs and is expressed dorsally, supporting the proposal that the dorsal-ventral axis was inverted in the course of animal evolution.     

Three loci related to the src oncogene and tyrosine-specific protein kinase activity in Drosophila

Rous sarcoma virus (RSV) is an acutely oncogenic avian retrovirus which induces sarcomas in animals and transforms fibroblasts in cell culture. Genetic analysis indicates that the viral src gene (v-src) mediates neoplastic transformation. The product of v-src is a 60,000 molecular weight (MW) phosphoprotein (pp60v-src) possessing the enzymatic activity of a tyrosine-specific protein kinase. The viral src gene is derived from a cellular gene (c-src) which also encodes a 60,000 MW phosphoprotein (pp60c-src) with tyrosine-specific protein kinase activity. Both birds and mammals are known to possess c-src. Shilo and Weinberg have reported that the genome of the fruit fly, Drosophila melanogaster, contains nucleotide sequences that are homologous to v-src. We report here the molecular cloning and chromosomal mapping of three loci from the Drosophila genome that contain such sequences. We also show that Drosophila contain both phosphotyrosine and a tyrosine-specific protein kinase activity immunoprecipitated by antisera directed against pp60v-src. It should now be possible to identify the precise locus that encodes a src-specific protein kinase in Drosophila, and to explore the role of c-src in the growth and development of D. melanogaster.     

Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers

The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. Here we show that the Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as org-1, a Drosophila homologue of mammalian TBX1, in these cells.     

The Mg-chelatase H subunit is an abscisic acid receptor

Abscisic acid (ABA) is a vital phytohormone that regulates mainly stomatal aperture and seed development, but ABA receptors involved in these processes have yet to be determined. We previously identified from broad bean an ABA-binding protein (ABAR) potentially involved in stomatal signalling, the gene for which encodes the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signalling. Here we show that Arabidopsis ABAR/CHLH specifically binds ABA, and mediates ABA signalling as a positive regulator in seed germination, post-germination growth and stomatal movement, showing that ABAR/CHLH is an ABA receptor. We show also that ABAR/CHLH is a ubiquitous protein expressed in both green and non-green tissues, indicating that it might be able to perceive the ABA signal at the whole-plant level.     

Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic

The anterior/posterior (A/P) and dorsal/ventral (D/V) compartment borders that subdivide the wing imaginal discs of Drosophila third instar larvae are each associated with a developmental organizer. Decapentaplegic (Dpp), a member of the transforming growth factor-beta (TGF-beta) superfamily, embodies the activity of the A/P organizer. It is produced at the A/P organizer and distributes in a gradient of decreasing concentration to regulate target genes, functioning non-autonomously to regulate growth and patterning of both the anterior and posterior compartments. Wingless (Wg) is produced at the D/V organizer and embodies its activity. The mechanisms that distribute Dpp and Wg are not known, but proposed mechanisms include extracellular diffusion, successive transfers between neighbouring cells, vesicle-mediated movement, and direct transfer via cytonemes. Cytonemes are actin-based filopodial extensions that have been found to orient towards the A/P organizer from outlying cells. Here we show that in the wing disc, cytonemes orient towards both the A/P and D/V organizers, and that their presence and orientation correlates with Dpp signalling. We also show that the Dpp receptor, Thickveins (Tkv), is present in punctae that move along cytonemes. These observations are consistent with a role for cytonemes in signal transduction.     

Distalization of the Drosophila leg by graded EGF-receptor activity

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-beta (TGF-beta) and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. Here I revise our understanding of limb development in flies and show that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals.