Processing of the Drosophila Sog protein creates a novel BMP inhibitory activity

Structurally unrelated neural inducers in vertebrate and invertebrate embryos have been proposed to function by binding to BMP4 or Dpp, respectively, and preventing these homologous signals from activating their receptor(s). In this study, we investigate the functions of various forms of the Drosophila Sog protein using the discriminating assay of Drosophila wing development. We find that misexpression of Drosophila Sog, or its vertebrate counterpart Chordin, generates a very limited vein-loss phenotype. This sog misexpression phenotype is very similar to that of viable mutants of glass-bottom boat (gbb), which encodes a BMP family member. Consistent with Sog selectively interfering with Gbb signaling, Sog can block the effect of misexpressing Gbb, but not Dpp in the wing. In contrast to the limited BMP inhibitory activity of Sog, we have identified carboxy-truncated forms of Sog, referred to as Supersog, which when misexpressed cause a broad range of dpp(-) mutant phenotypes. In line with its phenotypic effects, Supersog can block the effects of both misexpressing Dpp and Gbb in the wing. Vertebrate Noggin, on the other hand, acts as a general inhibitor of Dpp signaling, which can interfere with the effect of overexpressing Dpp, but not Gbb. We present evidence that Sog processing occurs in vivo and is biologically relevant. Overexpression of intact Sog in embryos and adult wing primordia leads to the developmentally regulated processing of Sog. This in vivo processing of Sog can be duplicated in vitro by treating Sog with a combination of the metalloprotease Tolloid (Tld) plus Twisted Gastrulation (Tsg), another extracellular factor involved in Dpp signaling. In accord with this result, coexpression of intact Sog and Tsg in developing wings generates a phenotype very similar to that of Supersog. Finally, we provide evidence that tsg functions in the embryo to generate a Supersog-like activity, since Supersog can partially rescue tsg(-) mutants. Consistent with this finding, sog(- )and tsg(-) mutants exhibit similar dorsal patterning defects during early gastrulation. These results indicate that differential processing of Sog generates a novel BMP inhibitory activity during development and, more generally, that BMP antagonists play distinct roles in regulating the quality as well as the magnitude of BMP signaling.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.     

The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing

In the early Drosophila embryo, Bone morphogenetic protein (BMP) activity is positively and negatively regulated by the BMP-binding proteins Short gastrulation (Sog) and Twisted gastrulation (Tsg). We show here that a similar mechanism operates during crossvein formation, utilizing Sog and a new member of the tsg gene family, encoded by the crossveinless (cv) locus. The initial specification of crossvein fate in the Drosophila wing requires signaling mediated by Dpp and Gbb, two members of the BMP family. cv is required for the promotion of BMP signaling in the crossveins. Large sog clones disrupt posterior crossvein formation, suggesting that Sog and Cv act together in this context. We demonstrate that sog and cv can have both positive and negative effects on BMP signaling in the wing. Moreover, Cv is functionally equivalent to Tsg, since Tsg and Cv can substitute for each other's activity. We also confirm that Tsg and Cv have similar biochemical activities: Sog/Cv complex binds a Dpp/Gbb heterodimer with high affinity. Taken together, these studies suggest that Sog and Cv promote BMP signaling by transporting a BMP heterodimer from the longitudinal veins into the crossvein regions.     

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.     

Dpp/BMP transport mechanism is required for wing venation in the sawfly Athalia rosae

The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.     

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range.     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.     

The function of a Drosophila glypican does not depend entirely on heparan sulfate modification

Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core.     

A juxtamembrane basolateral targeting motif regulates signaling through a TGF-β pathway receptor in Drosophila

In polarized epithelial cells, receptor–ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

Receptor-ligand interactions can be restricted by different spatial distributions of the two interacting components, giving rise to an underappreciated layer of regulatory complexity. This study reveals that an evolutionarily conserved mechanism for polarized delivery of a transmembrane receptor (the Dpp Type II receptor Punt) supports robust morphogen signaling in a model epithelial tissue, the Drosophila wing disc.     

Dpp gradient formation in the Drosophila wing imaginal disc

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. Here we make use of a biologically active form of Dpp tagged with GFP to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusted to compartment size when this was experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation.     

Long-range Dpp signaling is regulated to restrict BMP signaling to a crossvein competent zone

The sensitivity of the crossveins of the Drosophila wing to reductions in BMP signaling provides a valuable system for characterizing members of this signaling pathway. We demonstrate here two reasons for that sensitivity. First, the initial stage of posterior crossvein development depends on BMP signaling but is independent of EGF signaling. This is the opposite of the longitudinal veins, which rely of EGF signaling for their initial specification. Second, BMP signaling in the posterior crossvein depends on Decapentaplegic (Dpp) at a stage when it is being produced in the longitudinal veins. Thus, the posterior crossvein will be especially vulnerable to reductions in the levels or range of Dpp signaling. We investigated the roles of the BMP receptor Thickveins (Tkv) and the BMP inhibitor Short gastrulation (Sog) in allowing this long-range signaling. Expression of both is downregulated in the developing posterior crossvein. The Tkv downregulation depends on BMP signaling and may provide a positive feedback by allowing the spread of Dpp. The Sog downregulation is independent of BMP signaling; Sog misexpression experiments indicate that this prepattern is essential for posterior crossvein development. However, this requirement can be overridden by co-misexpression of the BMP agonist Cv-2, indicating the presence of as yet unknown cues; we discuss possible candidates.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.