Biglycan is a new extracellular component of the Chordin-BMP4 signaling pathway

The BMP4 signaling pathway plays key roles during early embryonic development and for maintenance of adult homeostasis. In the extracellular space, BMP4 activity is regulated by a group of interacting molecules including the BMP antagonist Chordin, the metalloproteinase Tolloid and Twisted gastrulation (Tsg). In this study, we identified Biglycan (Bgn), a member of the small leucine-rich proteoglycan family, as a new extracellular modulator of BMP4 signaling. Xenopus Bgn (xBgn) is expressed uniformly in the ectoderm and mesoderm and their derivatives during development. Microinjection of Bgn mRNA induced secondary axes, dorsalized the mesoderm and inhibited BMP4 activity in Xenopus embryos. Biochemical experiments showed that Bgn binds BMP4 and Chordin, interaction that increased binding of BMP4 to Chordin. Bgn was also able to improve the efficiency of Chordin-Tsg complexes to block BMP4 activity. Using antisense morpholinos, we demonstrated that Bgn required Chordin to induce double axes in Xenopus. This work unveiled a new function for Bgn, its ability to regulate BMP4 signaling through modulation of Chordin anti-BMP4 activity.     

The pro-BMP activity of Twisted gastrulation is independent of BMP binding

The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.     

Molecular determinants of Xolloid action in vivo

Xld (Xolloid) is a member of the Tolloid family of metalloproteases found in embryos of the frog Xenopus laevis. It cleaves Chordin, an inhibitory binding protein for BMP2/4, releasing fragments with reduced affinity for these important ventralizing signals. As a consequence, increasing Xld activity ventralizes Xenopus embryos. We have used this phenotype as an assay to determine the requirement for the C-terminal, nonprotease component of Xld for in vivo activity. This part of the protein is composed of five complement C1r/C1s-sea urchin epidermal growth factor-BMP1 (CUB) and two epidermal growth factor domains, which are thought to be involved in protein-protein interactions and may confer substrate specificity. Our results show that the protease coupled to CUB1 and CUB2 is the minimum domain structure required to ventralize Xenopus embryos and to block the dorsal axis-inducing activity of Chordin. Xld-CUB1-CUB2 cleaves Chordin, and a protease-inactive version co-precipitates Chordin. Our results indicate that the first and second CUB domains bind Chordin and present it to the protease domain. Protease-inactive Xld blocks the cleavage of Chordin by wild-type Xld and dorsalizes injected Xenopus embryos. We find that protease-inactive Xld-CUB1-CUB2 does not share this activity and that all of the C-terminal domains are required to generate the dorsalized phenotype.     

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.     

BMP-binding modules in chordin: a model for signalling regulation in the extracellular space

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

The binding of von Willebrand factor type C domains of Chordin family proteins to BMP-2 and Tsg is mediated by their SD1 subdomain

The VWC domain of Chordin family proteins consists of subdomains SD1 and SD2. In previous experiments with VWC1 from CV-2 SD-1 was shown to be crucial for BMP interaction. Now the SD1 from VWC1 and VWC3 of Chordin and CHL2 were established to confer BMP affinity and specificity to these proteins also. In addition, these SD1 subdomains are mediating binding to Tsg. Mutational analysis revealed similar binding epitopes of the various SD1 proteins for BMP-2 and Tsg. Inhibitory activity of CHL2 in C2C12 cells is reduced by mutations in SD1 of VWC1 and even more of VWC3. These results together provide strong evidence that the SD1 subdomain module of about 40 residues represents the crucial binding partner for BMPs and Tsg in these Chordin family proteins and likely in other BMP-binding VWC domains also.     

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.     

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.     

Identification of a second Xenopus twisted gastrulation gene

Twisted Gastrulation (Tsg) is a secreted molecule which regulates BMP signalling in the extracellular space as part of an evolutionarily conserved network of interacting proteins. In Xenopus, maternal xTsg mRNA can be found throughout the early embryo. After gastrulation, xTsg is expressed as part of the BMP4 synexpression group until late tadpole stages. Here we report the identification of a second Xenopus Tsg gene (xTsg-2). Xenopus Tsg-2 is highly homologousto xTsg. In particular, amino acid residues which have been shown to be required for the binding of xTsg to BMP and to Chordin are conserved. The expression of Xenopus Tsg-2 mRNA was restricted to late stages of embryonic development; it was detected at tadpole stages in lateral plate mesoderm, neural crest, branchial arches and head mesenchyme. In microinjection experiments, the activity of xTsg-2 mRNA was similar to that of xTsg. We conclude that two Tsg genes act in distinct temporal and spatial territories in the course of Xenopus embryonic development.     

A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic (DPP) and Screw (SCW). short gastrulation (sog), which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Here we demonstrate that SOG has a second function, which is to promote BMP signaling on the dorsal side of the embryo. We show that a weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and that the lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of SOG is directed against SCW, we present data that suggests that the positive activity of SOG is directed towards DPP. We demonstrate that Chordin shares the same apparent ligand specificity as does SOG, preferentially inhibiting SCW but not DPP activity. However, in Drosophila assays Chordin does not have the same capacity to elevate BMP signaling as does SOG, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates.     

Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.     

Cell-surface heparan sulfate proteoglycans potentiate chordin antagonism of bone morphogenetic protein signaling and are necessary for cellular uptake of chordin

Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed.     

Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.     

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted Gastrulation

Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2; Tsg and Cv2; Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.     

Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning

The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.     

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.