Matching catalytic activity to developmental function: tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing

The Drosophila tolloid (tld) and tolloid related (tlr) gene products belong to a family of developmentally important proteases that includes Bone Morphogenetic Protein 1 (Bmp1). Tld is required early in Drosophila development for proper patterning of dorsal embryonic structures, whereas Tlr is required later during larval and pupal stages of development. The major function of Tld is to augment the activity of Decapentaplegic (Dpp) and Screw (Scw), two members of the Bmp subgroup of the Tgf beta superfamily, by cleaving the Bmp inhibitor Short gastrulation (Sog). In this study, we provide evidence that Tlr also contributes to Sog processing. Tlr cleaves Sog in vitro in a Bmp-dependent manner at the same three major sites as does Tld. However, Tlr shows different site selection preferences and cleaves Sog with slower kinetics. To test whether these differences are important in vivo, we investigated the role of Tlr and Tld during development of the posterior crossvein (PCV) in the pupal wing. We show that tlr mutants lack the PCV as a result of too little Bmp signaling. This is probably caused by excess Sog activity, as the phenotype can be suppressed by lowering Sog levels. However, Tld cannot substitute for Tlr in the PCV; in fact, misexpressed Tld can cause loss of the PCV. Reducing levels of Sog can also cause loss of the PCV, indicating that Sog has not only an inhibitory but also a positive effect on signaling in the PCV. We propose that the specific catalytic properties of Tlr and Tld have evolved to achieve the proper balance between the inhibitory and positive activities of Sog in the PCV and early embryo, respectively. We further suggest that, as in the embryo, the positive effect of Sog upon Bmp signaling probably stems from its role in a ligand transport process.     

Shaping BMP morphogen gradients through enzyme-substrate interactions

Bone morphogenetic proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. We identified Sog characteristics responsible for making its cleavage dependent on BMP binding. "Chordin-like" variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning.     

Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), we followed signaling directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp.     

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.     

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.     

Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. We have analyzed Tsg loss-of-function in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. We provide evidence that Tsg interacts genetically with Bmp4. When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg-/-;Bmp4+/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development.     

Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. We demonstrate that Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, we provide biochemical and genetic evidence that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. We propose that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, we demonstrate mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens.     

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.     

Presence and roles of calcium gradients along the dorsal-ventral axis in Drosophila embryos

Dorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways which have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, we found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. We further show that this calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic (dpp) or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. We conclude that calcium plays a role in specification of the dorsal embryonic region.     

Integrins modulate Sog activity in the Drosophila wing

Morphogenesis of the Drosophila wing depends on a series of cell-cell and cell-extracellular matrix interactions. During pupal wing development, two secreted proteins, encoded by the short gastrulation (sog) and decapentaplegic (dpp) genes, vie to position wing veins in the center of broad provein territories. Expression of the Bmp4 homolog dpp in vein cells is counteracted by expression of the Bmp antagonist sog in intervein cells, which results in the formation of straight veins of precise width. We screened for genetic interactions between sog and genes encoding a variety of extracellular components and uncovered interactions between sog and myospheroid (mys), multiple edematous wing (mew) and scab (scb), which encode betaPS, alphaPS1 and alphaPS3 integrin subunits, respectively. Clonal analysis reveals that integrin mutations affect the trajectory of veins inside the provein domain and/or their width and that misexpression of sog can alter the behavior of cells in such clones. In addition, we show that a low molecular weight form of Sog protein binds to alphaPS1betaPS. We find that Sog can diffuse from its intervein site of production into adjacent provein domains, but only on the dorsal surface of the wing, where Sog interacts functionally with integrins. Finally, we show that Sog diffusion into provein regions and the reticular pattern of extracellular Sog distribution in wild-type wings requires mys and mew function. We propose that integrins act by binding and possibly regulating the activity/availability of different forms of Sog during pupal development through an adhesion independent mechanism.     

Twisted gastrulation enhances BMP signaling through chordin dependent and independent mechanisms

BMP signaling is modulated by a number of extracellular proteins, including the inhibitor Chordin, Tolloid-related enzymes (Tld), and the interacting protein Twisted Gastrulation (Tsg). Although in vitro studies have demonstrated Chordin cleavage by Tld enzymes, its significance as a regulatory mechanism in vivo has not been established in vertebrates. In addition, Tsg has been reported in different contexts to either enhance or inhibit BMP signaling through its interactions with Chordin. We have used the zebrafish gastrula to carry out structure/function studies on Chordin, by making versions of Chordin partially or wholly resistant to Tld cleavage and introducing them into chordin-deficient embryos. We examined the cleavage products generated in vivo from wild-type and altered Chordins, and tested their efficacy as BMP inhibitors in the embryo. We demonstrate that Tld cleavage is crucial in restricting Chordin function in vivo, and is carried out by redundant enzymes in the zebrafish gastrula. We also present evidence that partially cleaved Chordin is a stronger BMP inhibitor than the full-length protein, suggesting a positive role for Tld in regulating Chordin. We find that depletion of the embryo for Tsg leads to decreased BMP signaling, and to increased levels of Chordin. Finally, we show that Tsg also enhances BMP signaling in the absence of Chordin, and its depletion can partially rescue the chordin mutant phenotype, demonstrating that important components of the BMP signaling pathway remain unidentified.     

sog and dpp exert opposing maternal functions to modify toll signaling and pattern the dorsoventral axis of the Drosophila embryo

The short gastrulation (sog) and decapentaplegic (dpp) genes function antagonistically in the early Drosophila zygote to pattern the dorsoventral (DV) axis of the embryo. This interplay between sog and dpp determines the extent of the neuroectoderm and subdivides the dorsal ectoderm into two territories. Here, we present evidence that sog and dpp also play opposing roles during oogenesis in patterning the DV axis of the embryo. We show that maternally produced Dpp increases levels of the I(kappa)B-related protein Cactus and reduces the magnitude of the nuclear concentration gradient of the NF(kappa)B-related Dorsal protein, and that Sog limits this effect. We present evidence suggesting that Dpp signaling increases Cactus levels by reducing a signal-independent component of Cactus degradation. Epistasis experiments reveal that sog and dpp act downstream of, or in parallel to, the Toll receptor to reduce translocation of Dorsal protein into the nucleus. These results broaden the role previously defined for sog and dpp in establishing the embryonic DV axis and reveal a novel form of crossregulation between the NF(kappa)B and TGF(beta) signaling pathways in pattern formation.     

Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is homologous to the vertebrate proto-oncogene c-rel. In wild-type embryos, the dorsal protein is found in the cytoplasm during cleavage. After the nuclei migrate to the periphery of the embryo, a ventral-to-dorsal gradient of nuclear dorsal protein is established. The formation of the nuclear gradient is disrupted in mutant embryos from other maternally active dorsal-ventral polarity genes: in dorsalized embryos only cytoplasmic protein is observed, while in ventralized embryos the nuclear gradient is shifted dorsally. My findings suggest that nuclear localization is critical for dorsal to function as a morphogen and that the distribution of the dorsal protein determines cell fate along the dorsal-ventral axis.     

Robust stability of the embryonic axial pattern requires a secreted scaffold for chordin degradation

Dorsal axial formation during vertebrate embryogenesis exhibits robust resistance to perturbations in patterning signals. However, how such stability is supported at the molecular level remains largely elusive. Here we show that Xenopus ONT1, an Olfactomedin-class secreted protein, stabilizes axial formation by restricting Chordin activity on the dorsal side. When ONT1 function is attenuated, the embryo becomes hyperdorsalized by a normally subeffective dose of Chordin. ONT1 binds Chordin and BMP1/Tolloid-class proteinases (B1TP) via distinct domains and acts as a secreted scaffold that enhances B1TP-mediated Chordin degradation by facilitating enzyme-substrate association. ONT1 is indispensable for fine-tuning BMP signaling in the axial tissue, and a similar role has been suggested for dorsally expressed BMPs such as ADMP. Simultaneous inhibition of ONT1 and dorsally expressed BMPs (ADMP and BMP2) synergistically caused drastic dorsalization. These results indicate that stable axial formation depends on two compensatory regulatory pathways involving ONT1/B1TP and dorsally expressed BMPs.     

Cysteine repeat domains and adjacent sequences determine distinct bone morphogenetic protein modulatory activities of the Drosophila Sog protein

The Drosophila short gastrulation gene (sog) encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. Here we present a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, our analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities.