Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

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.     

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.     

Drosophila heparan sulfate, a novel design

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.     

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.     

Biosynthesis of heparan sulfate

Microsomal preparations from Englebreth-Holm-Swarm mouse sarcoma were incubated with UDP-N-acetyl[³H] glucosamine and UDP-[¹⁴C]glucuronic acid to form proteoglycan containing [³H,¹⁴C]glycosaminoglycan with equimolar amounts of [³H]glucosamine and [¹⁴C]glucuronic acid. The labelled glycosaminoglycan was totally resistant to degradation by testicular hyaluronidase, but could be degraded readily by a crudeFlavobacter heparinum enzyme preparation which is capable of degrading heparin and heparan sulfate. Chromatography of the [³H,¹⁴C]glycosaminoglycan on DEAE-cellulose provided a pattern with three peaks: the first appearing before hyaluronic acid, the second and largest appearing at the site of hyaluronic acid, and a third appearing slightly beyond hyaluronic acid but before a standard of chondroitin sulfate. When 3-phosphoadenosine 5-phosphosulfate was also included in the reaction mixture, a change appeared in the [³H,¹⁴C]glycosaminoglycan so that chromatography on DEAE-cellulose presented a pattern with a significant amount of material which cochromatographed in the area where heparan sulfate would be found. There was no material that co-chromatographed with the more highly sulfated substance, heparin. This indicates that the microsomal preparation from the Englebreth-Holm-Swarm sarcoma is capable of producing a heparan sulfate-like molecule and is controlled in its sulfation of precursors so that heparin is not formed.     

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.     

Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS‐CoV‐2. Notably, neutralizing antibodies against SARS‐CoV‐2 isolated from COVID‐19 patients interfered with SARS‐CoV‐2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS‐CoV‐2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS‐CoV‐2, both DC subsets efficiently captured SARS‐CoV‐2 via heparan sulfate proteoglycans and transmitted the virus to ACE2‐positive cells. Notably, human primary nasal cells were infected by SARS‐CoV‐2, and infection was blocked by pre‐treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS‐CoV‐2 infection.     

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.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

Preparation and characterization of N-enriched, size-defined heparan sulfate precursor oligosaccharides

We report the preparation of size-defined [¹⁵N]N-acetylheparosan oligosaccharides from Escherichia coli-derived ¹⁵N-enriched N-acetylheparosan. Optimized growth conditions of E. coli in minimal media containing ¹⁵NH₄Cl yielded [¹⁵N]N-acetylheparosan on a preparative scale. Depolymerization of [¹⁵N]N-acetylheparosan by heparitinase I yielded resolvable, even-numbered oligosaccharides ranging from disaccharide to icosaccharide. Anion-exchange chromatography-assisted fractionation afforded size-defined [¹⁵N]N-acetylheparosan oligosaccharides identifiable by ESI-TOFMS. These isotopically labeled oligosaccharides will prove to be valuable research tools for the chemoenzymatic synthesis of heparin and heparan sulfate oligosaccharides and for the study of their structural biology.     

Single-minded and the evolution of the ventral midline in arthropods

Glypicans are multifunctional proteoglycans with regulatory roles in several intercellular signaling pathways. Here, we examine the functional requirements for glypican regulation of bone morphogenetic protein (BMP)-mediated body length in C. elegans. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit BMP signaling in vivo: the N-terminal furin protease product and the C-terminal region containing heparan sulfate attachment sequences. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. Cleavage of LON-2 into two parts at the conserved furin protease site is not required for LON-2 to inhibit BMP-like signaling. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 activity. Finally, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity, suggesting that LON-2 inhibits BMP signaling by acting as a scaffold for BMP and an RGD-binding protein.     

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.     

Egg production and fertility in Drosophila depend upon the number of yolk-protein gene copies

Summary The yolk proteins of Drosophila melanogaster comprise a family of three related yolk polypeptides each encoded by a single-copy gene. We show by genetic crosses that each gene makes an equivalent contribution to the fecundity and fertility of the female and they do not individually provide unique functions to the embryo. We show that the number of eggs laid by a female depends upon the number of genes encoding yolk polypeptides present in the genome and furthermore that the probability of an egg hatching into an adult also critically depends upon the number of yolk protein genes present in the mother. This suggests that the three yolk protein-encoding genes in Drosophila melanogaster may have arisen by duplication, then been maintained for quantitative reasons because they increased egg production and fertility, rather than each protein evolving a different function as is the case with most small gene families, such as tubulins and collagen genes.     

Spatial differences in patterns of modification: selection on hairy in Drosophila melanogaster wings

Artificial selection was carried out for over 45 generations to enhance and suppress expression of the mutation hairy on the Drosophila melanogaster wing. Whole chromosome mapping of X‐linked and autosomal modifiers of sense organ number displayed regional differences in magnitude and direction of their effects. Regional specificity of modifier effects was also seen in some interchromosomal interactions. Scanning electron microscopy allowed precise measurement of sense organ size and position along the L3 longitudinal wing vein. Sense organ size varied in a predictable fashion along the proximal–distal axis, and the dorsal pattern differed from the ventral pattern. The high and low selection lines differed most in the proximal portion of the L3 vein. Extra sense organs in the High line were often associated with vein fragments at locations predicted from ancestral vein patterns. Thus, regional specificity of polygenic or quantitative trait locus modifier effects was identified in several different parts of the wing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative study of structure and function of blood cells from two Drosophila species

Summary Hemocytes of Drosophila melanogaster and Drosophila yakuba larvae have been defined in terms of their ultrastructure and functions in coagulation, wound healing, encapsulation, phenol-oxydase activity, and phagocytosis. The position of these cells among the classical hemocyte types of insects is determined. We distinguish two plasmatocyte types (macrophage plasmatocytes and lamellocytes) which do not seem to belong to the same lineage, and oenocytoids which are the crystal cells of the literature.I should like to thank Dr. N. Plus for her help in this study     

The function of type IV collagen during Drosophila embryogenesis

A collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a peptide related to vertebrate basement membrane type IV collagen chains. To study the function of type IV collagen during Drosophila development, we transformed flies with a partially truncated Dcg1 gene under the control of a heat-shock promotor. This construct induced synthesis of shortened pro- chains which associated with normal ones and thereby caused degradation of the shortened and normal pro- chains through a process called pro-collagen suicide. A large proportion of embryos expressing the transgene developed a phenotype exhibiting absence or partial retraction of the germ band with defects in nerve cord condensation and dorsal closure. Together these results indicated that, during embryogenesis, type IV collagen was an essential guiding factor for cell-matrix interactions in morphogenetic events.