The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.     

On the role of glypicans in the process of morphogen gradient formation

Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding.     

Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and wingless transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. In this study, we demonstrate that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. We also demonstrate that Wg is secreted apically in the disc epithelium and that apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action.     

Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity

The glypican family of heparan sulfate proteoglycans has been implicated in formation of morphogen gradients. Here, we examine the role of the glypican Dally-like protein (Dlp) in shaping the Wingless gradient in the Drosophila wing disc. Surprisingly, we find that Dlp has opposite effects at high and low levels of Wingless. Dlp promotes low-level Wingless activity but reduces high-level Wingless activity. We present evidence that the Wg antagonist Notum acts to induce cleavage of the Dlp glypican at the level of its GPI anchor, which leads to shedding of Dlp. Thus, spatially regulated modification of Dlp by Notum employs the ligand binding activity of Dlp to promote or inhibit signaling in a context-dependent manner. Notum-induced shedding of Dlp could convert Dlp from a membrane-tethered coreceptor to a secreted antagonist.     

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.     

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.     

Carrier of Wingless (Cow), a Secreted Heparan Sulfate Proteoglycan,Promotes Extracellular Transport of Wingless

Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. We identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen.     

Formation and maintenance of morphogen gradients: an essential role for the endomembrane system in Drosophila melanogaster wing development

As early as 1964 it was suggested that simple diffusion of morphogens away from their secretion source did not provide an adequate explanation for the formation and maintenance of morphogen gradients. Involvement of the endosome in morphogen distribution models provides an explanation for the slow, directional movement of morphogens, as well as their ability to form intracellular and extracellular gradients independent of morphogen production rates. Drosophila melanogaster morphogens Wg and Dpp form stable, steep, long-range gradients that specify the polarity of the wing disc. The process of endocytosis is imperative to the two central themes in gradient formation: active transport facilitating long-range signaling and degradation of morphogen to sustain gradient shape. This review investigates the endomembrane-mediated processes of re-secretion, degradation and argosome transport of Wg and Dpp in the hope that a better understanding of the endomembrane system will contribute to a more accurate and comprehensive model for morphogen gradient formation and maintenance.     

Expression of a secreted form of Dally, a Drosophila glypican, induces overgrowth phenotype by affecting action range of Hedgehog

Glypicans, a family of heparan sulfate proteoglycans attached to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor, play essential roles in morphogen signaling and distributions. A Drosophila glypican, Dally, regulates the gradient formation of Decapentaplegic (Dpp) in the developing wing. To gain insights into the function of glypicans in morphogen signaling, we examined the activities of two mutant forms of Dally: a transmembrane form (TM-Dally) and a secreted form (Sec-Dally). Misexpression of tm-dally in the wing disc had a similar yet weaker effect in enhancing Dpp signaling compared to that of wild-type dally. In contrast, Sec-Dally shows a weak dominant negative activity on Dpp signal transduction. Furthermore, sec-dally expression led to patterning defects as well as a substantial overgrowth of tissues and animals through the expansion of the action range of Hh. These findings support the recently proposed model that secreted glypicans have opposing and/or distinct effects on morphogen signaling from the membrane-tethered forms.     

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.     

Fast-tracking morphogen diffusion

The readout of morphogen concentrations has been proposed to be an essential mechanism allowing embryos to specify cell identities [Wolpert Trends Genet 12 (1996) 359], but theoretical and experimental results have led to conflicting ideas as to how useful concentration gradients can be established. In particular, it has been pointed out that some models of passive extracellular diffusion exhibit traveling waves of receptor saturation, inadequate for the establishment of positional information. Two alternative (but not mutually exclusive) models are proposed here, which are based on recent experimental results highlighting the roles of extracellular glycoproteins and morphogen oligomerization. In the first model, inspired from the interactions of Dally and Dally-like with Wingless and Decapentaplegic in the third-instar Drosophila wing disc, two morphogen populations are considered: one in a cell-membrane phase, and another one in an extracellular matrix phase, which does not interact with receptors; in the second model, inspired from biochemical studies of Sonic Hedgehog, morphogen oligomers are considered to diffuse freely without interacting with receptors. The existence of a dynamic sub-population of freely diffusing morphogen allows the system to establish a gradient of bound receptor that is suitable for the specification of positional information. Recent experimental results are discussed within the framework of these models, as well as further possible experiments. The role of Notum in the setup of the Wg gradient is also shown to be likely not to involve a gradient in Notum distribution, even though Notum is only expressed close to the source of Wg synthesis.     

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp^ΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp^ΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.     

Refinement of wingless expression by a wingless- and notch-responsive homeodomain protein,defective proventriculus

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.     

Dally regulates Dpp morphogen gradient formation in the Drosophila wing

Decapentaplegic (Dpp), a Drosophila TGF beta/bone morphogenetic protein homolog, functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. We show evidence that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thick veins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. We propose that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient.     

Formation and maintenance of morphogen gradients: An essential role for the endomembrane system in Drosophila melanogaster wing development

As early as 1964 it was suggested that simple diffusion of morphogens away from their secretion source did not provide an adequate explanation for the formation and maintenance of morphogen gradients. Involvement of the endosome in morphogen distribution models provides an explanation for the slow, directional movement of morphogens, as well as their abilty to form intracellular and extracellular gradients independent of morphogen production rates. Drosophila melanogaster morphogens Wg and Dpp form stable, steep, long-range gradients that specify the polarity of the wing disc. The process of endocytosis is imparative to the two central themes in gradient formation; active transport facilitating long-range signalling, and degradation of morphogen to sustain gradient shape. This review investigates the endomembrane mediated processes of re-secretion, degradation, and argosome transport of Wg and Dpp in the hope that a better understanding of the endomembrane system will contribute to a more accurate and comprehensive model for morphogen gradient formation and maintenance.     

Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila.

Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins which have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here we describe the isolation of an amber mutation of fz2 which truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, we show that Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos as well as in the wing imaginal disc. We also show that Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila.     

Regulation of BMP activity and range in Drosophila wing development

Bone morphogenetic protein (BMP) signaling controls development and maintenance of many tissues. Genetic and quantitative approaches in Drosophila reveal that ligand isoforms show distinct function in wing development. Spatiotemporal control of BMP patterning depends on a network of extracellular proteins Pent, Ltl and Dally that regulate BMP signaling strength and morphogen range. BMP-mediated feedback regulation of Pent, Ltl, and Dally expression provides a system where cells actively respond to, and modify, the extracellular morphogen landscape to form a gradient that exhibits remarkable properties, including proportional scaling of BMP patterning with tissue size and the modulation of uniform tissue growth. This system provides valuable insights into mechanisms that mitigate the influence of variability to regulate cell-cell interactions and maintain organ function.     

Drosophila heparan sulfate 6-O endosulfatase regulates Wingless morphogen gradient formation

Heparan sulfate proteoglycans (HSPGs) play critical roles in the distribution and signaling of growth factors, but the molecular mechanisms regulating HSPG function are poorly understood. Here, we characterized Sulf1, which is a Drosophila member of the HS 6-O endosulfatase class of HS modifying enzymes. Our genetic and biochemical analyses show that Sulf1 acts as a novel regulator of the Wg morphogen gradient by modulating the sulfation status of HS on the cell surface in the developing wing. Sulf1 affects gradient formation by influencing the stability and distribution of Wg. We also demonstrate that expression of Sulf1 is induced by Wg signaling itself. Thus, Sulf1 participates in a feedback loop, potentially stabilizing the shape of the Wg gradient. Our study shows that the modification of HS fine structure provides a novel mechanism for the regulation of morphogen gradients.     

Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans

The Drosophila transforming growth factor beta (TGF-beta) homolog Decapentaplegic (Dpp) acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, we found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. We demonstrate that a blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, we show that Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). Our results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly.     

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.