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Cell, Vol. 104, 801–804, March 23, 2001, Copyright 2001 by Cell Press Twisted Perspective: New Insights into Extracellular Modulation of BMP Signaling during Development Robert P. Ray and Kristi A. Wharton* Department of Molecular Biology, Cell Biology and Biochemistry Division of Biology and Medicine Box G-J160 Brown University Providence, Rhode Island 02912 During the past several decades, considerable research efforts have elucidated the central components of many signal transduction pathways in animal systems. More recently attention has shifted to the importance of modulating intercellular signaling processes during development, as it is clear that fine-tuned regulation of signaling systems in vivo results in subtle distinctions at the cellular level. Extracellular modulators have been a particular focus of investigation, including proteins that affect receptor stability, ligand function, or ligand availability. In these latter types of regulation, proteins such as proteoglycans, heparan sulfate modifying enzymes, and proteases have been recognized as key players, as have a diverse class of diffusible modulators that influence ligand activity by inhibiting or facilitating its function (see references contained within reviews by Capdevila and Belmonte, 1999; Christian, 2000). The topic of this review focuses on the function of one such diffusible modulator that influences the availability of BMP ligands, a family of the TGF- superfamily of signaling molecules. It appears that for TGF- signaling in general a major means of signal regulation is the modulation of ligand availability and as yet this system provides one of the best studied paradigms for this kind of regulation in animal systems. In recent years, our understanding of the mechanism of signaling by TGF- superfamily ligands has increased tremendously. A little over a decade ago, the few TGF-like proteins that had been characterized were “orphan” ligands for which neither receptors nor cellular targets had been identified. The discovery of the TGF- receptors, followed shortly thereafter by the characterization of the founding member of the Smad family of transcriptional regulators, the Drosophila gene Mothers against dpp (Mad), initiated an intensive period of research focused on the downstream components in the pathway. These studies revealed a relatively simple signal transduction system in which the ligand dimer binds to a heteromeric complex of Type I and Type II transmembrane serine/threonine kinase receptors that phosphorylate cellular transcription factors of the Smad family that directly move into the nucleus and regulate target gene expression (Massagué, 1998). More recently, studies on a number of model systems have demonstrated that an important and evolutionarily conserved mechanism of regulation for one family of * To whom correspondence [email protected]). should be addressed (e-mail: Minireview TGF--like ligands, the Bone Morphogenetic Proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (reviewed in De Robertis and Sasai, 1996; Figure 1). In amphibians, opposing activities of BMP4 and its antagonist Chordin (Chd) are responsible for subdivision of the embryonic dorsal–ventral axis. A similar role for BMP4 and Chd orthologs has been implicated in dorsal–ventral patterning of the zebrafish embryo (Mullins, 1998). Thus, in the vertebrates, BMP activity specifies ventral fates in the early embryo, and is antagonized by localized expression of Chd orthologs dorsally. In Drosophila, the same relationship exists between the BMP homologs decapentaplegic (dpp) and screw (scw) and the Chd ortholog short gastrulation (sog), but the axis is inverted: dpp and scw specify dorsal fates in the embryo and are antagonized by sog, which is expressed ventrally. In both vertebrates and invertebrates, the activity of Chd orthologs is antagonized by a family of metalloproteases including Drosophila Tolloid (Tld), Xenopus Xolloid (Xol), Human BMP-1, and their orthologs in other vertebrates. Biochemical studies on the Xenopus, Drosophila, and mouse proteins have demonstrated that Tld/Xol/BMP1 cleavage inactivates Sog/Chd and promotes BMP signaling (Marqués et al., 1997; Piccolo et al., 1997; Scott et al., 1999). Thus, Tld/ Xol proteins function as BMP agonists. In three papers appearing this month in Nature (Chang et al., 2001; Ross et al., 2001; Scott et al., 2001), studies on the Drosophila gene twisted gastrulation (tsg) and its vertebrate orthologs reveal another level of complexity in the extracellular modulation of BMP signaling during development. tsg was first identified in Drosophila as a zygotic lethal gene that, based on its mild ventralized phenotype and its localized requirement to dorsal blastomeres, was allied with dpp, scw, tld, and other zygotic ventralizing genes (Ray et al., 1991 and references therein). Subsequent molecular characterization revealed tsg encodes a secreted, cysteine-rich protein that was proposed to act in combination with dpp to specify the dorsal-most pattern elements in the embryo (Mason et al., 1994). This role for tsg as a dpp agonist was supported by a study on the Xenopus ortholog, xTsg, in which overexpression of xTsg was shown to result in a loss of neural and head structures, and a reduction in dorsal expressed molecular markers. These results led to the conclusion that xTsg promotes BMP signaling (Oelgeschläger et al., 2000). In apparent contradiction to this earlier work, the current studies instead show that Tsg orthologs can function in combination with Chd/ Sog to antagonize BMP signaling. Tsg orthologs have now been isolated from Drosophila, Xenopus, zebrafish, chick, mouse, and humans. Drosophila and zebrafish each have two Tsg homologs, and based on sequence comparisons, the pairs appear to have arisen by independent gene duplication events in these two species (Ross et al., 2001; Scott et al., 2001). Studies on both Drosophila and Xenopus indicate that Tsg acts nonautonomously and is highly diffusible (Mason et al., 1997; Chang et al., 2001). Tsg does not appear to play a role in establishing asymmetries in the early Cell 802 Figure 1. A Conserved Mechanism of Extracellular Signals Establishes Dorsal–Ventral Pattern in Invertebrates and Vertebrates (for references, see De Robertis and Sasai, 1996) (Top) Schematic frontal section of a Drosophila blastoderm embryo showing dorsal–ventral fates. Sog, expressed in the ventral ectoderm (gold) diffuses dorsally and antagonizes BMP signaling by Dpp and Scw in the dorsal ectoderm (blue). Tld, also expressed dorsally, antagonizes Sog’s antagonism and thus promotes BMP signaling. (Bottom) Schematic fate map of a Xenopus embryo showing molecular markers used in the current studies. The Sog ortholog Chd is expressed dorsally in the organizer and antagonizes BMP signaling, confining it ventrally. As in Drosophila, Chd’s action is antagonized by the Tld ortholog Xol. The same fate map and relationship between BMP2b and zchordin applies to the zebrafish embryo. While the double negative regulation and the proteins involved are conserved, the axis is inverted: Sog is expressed ventrally in Drosophila and antagonizes BMP signaling dorsally, and Chd is expressed dorsally and antagonizes BMP signaling ventrally. embryo (i.e., like Chordin) as in most organisms the tsg RNA is not specifically localized or does not require specific localization. Rather, Tsg acts generally or depends on the localized activities of other components in the pathway. The recent loss-of-function and gain-of-function analyses support the notion that Tsg acts as an antagonist of BMP signaling in early embryogenesis. Loss-of-function studies in Drosophila and zebrafish indicate that Tsg inactivation produces a phenotype resembling that of mutations in the BMP antagonists sog and chordin, respectively, rather than that expected from a loss of BMP function. In Drosophila, the sog and tsg mutant phenotypes exhibit the same effects on the expression patterns of a number of genes that respond to the BMP gradient, and these phenotypes are distinct from those produced by dpp, tld, or scw mutants (Ross et al., 2001). Thus, given that studies on sog have demonstrated unequivocally that it functions as a dpp antagonist, it follows that tsg, which produces the same phenotype, might also act in this capacity. In zebrafish, a similar result is seen. Using morpholino oligonucleotides, it has been shown that the phenotype associated with the loss of ztsg1 activity consists of an expansion of ventral mesodermal tissues, a loss of paraxial mesoderm, and a mild reduction of anterior ectoderm, i.e., a ventralized phenotype, consistent with a role for ztsg1 in antagonizing BMP signaling (Ross et al., 2001). Gain-of-function studies in Xenopus, zebrafish, and Drosophila are consistent with the loss-of-function data and also support the notion that Tsg functions as a BMP antagonist. Microinjection of Tsg RNA into Xenopus embryos results in a dorsalization of the embryo as evidenced by loss of ventral cell fate markers such as Xhox3, Msx1, and Vent1 in gastrula and loss of blood and the heart marker Nkx2.5 in the tadpole (Chang et al., 2001). In ectodermal explants (animal caps), overexpression of Tsg results in suppression of epidermal keratin and induces the expression of cement gland and neural markers, responses that are normally associated with an inhibition of BMP signaling (Chang et al., 2001). Similarly, in zebrafish, injection of ztsg1 RNA results in a phenotype resembling that of a mutant for the BMP7 homolog, snailhouse, consistent with a dorsalization of the embryo (Ross et al., 2001). Thus, in the vertebrate embryo, loss of Tsg results in ventralization of the embryo, while overexpression leads to a dorsalization. Significantly, overexpression of Tsg in the Drosophila embryo has no effect on embryonic development, and misexpression of Tsg in the wing disk produces either no phenotype or only a mild venation defect (Ross et al., 2001; Yu et al., 2000). Thus, the vertebrate embryo either shows a sensitivity to Tsg that is not apparent in Drosophila, or microinjection experiments in vertebrate embryos may reveal aspects of Tsg function that cannot be uncovered using the genetic tools of Drosophila. The conclusion, based on mutant phenotypes, that Chd/Sog and Tsg both act as BMP antagonists is supported by biochemical studies. Previous studies have shown that Chd/Sog acts to antagonize BMP signaling by binding BMP4/Scw and preventing the ligand from interacting with its receptor (as reviewed in Podos and Ferguson, 1999). Coimmunoprecipitations of tagged Tsg, Chd, and BMP proteins indicate that Chd and Tsg are both able to bind directly to BMP, and, more importantly, that Tsg promotes the binding of Chd to BMP (Oelgeschläger et al., 2000; Chang et al., 2001; Ross et al., 2001; Scott et al., 2001). This effect is particularly clear in Drosophila where it has been shown that on its own Sog can antagonize Scw signaling but not Dpp signaling, while, in the presence of Tsg, Sog can antagonize both Dpp and Scw (Ross et al., 2001). Furthermore, Tsg has been shown to bind Chd/Sog and the data from both systems support the hypothesis that Tsg, Chd, and BMP form a ternary complex that inactivates BMP signaling more effectively than Chd alone (Figure 2). A number of in vivo experiments indicate that Tsg and Chd act synergistically to antagonize BMP signaling. Loss-of-function studies on zebrafish show that inactivation of both Tsg and Chd results in a higher penetrance of the mutant phenotype than with either alone (Ross et al., 2001). Similarly in Xenopus, coinjection of both Tsg and Chd is more potent in the induction of secondary axes than either alone (Scott et al., 2001), and the combination is able to repress expression of Xbra (Chang et al., 2001). In Drosophila, overexpression of either Tsg or Sog alone is unable to suppress wing Minireview 803 Figure 2. Summary of Binding and Functional Studies on Tsg Demonstrated in vitro binding relationships are indicated in red, demonstrated functional relationships in black, and potential functional relationships in gray. The central triangle illustrates the “ternary complex” of Tsg/Chd/BMP (Chang et al., 2001; Ross et al., 2001; Scott et al., 2001). Potential functions of Tsg include direct binding and inactivation of BMP (Ross et al., 2001), cooperative binding and inactivation of BMP in combination with Sog/Chd (Chang et al., 2001; Ross et al., 2001; Scott et al., 2001), and enhancement of Sog/Chd cleavage by Tld (Scott et al., 2001). In the latter case, it is not clear if the new Tsg-dependent cleavages activate Sog/Chd to produce “supersog-like” molecules, or inactivate it as suggested by the function of Tld in vivo. phenotypes associated with ectopic expression of Dpp, while the combination of the two is able to suppress these phenotypes. Similarly, overexpression of either Sog or Tsg alone in the wing disk results in only minor venation defects, but the two together repress the endogenous dpp functions in the disk resulting in a phenocopy of dpp mutant wing phenotypes (Yu et al., 2000; Ross et al., 2001). Notably, this result is consistent with the biochemical data indicating that Sog is only able to inactivate Dpp in the presence of Tsg. Interactions between Chd/Sog and BMP are mediated by two of the four cysteine-rich domains in the Chd protein, CR1 and CR3, though, in isolation, these domains bind BMP with a 10-fold lower affinity than fulllength Chd (Oelgeschläger et al., 2000, and references therein). These domains have weak homology with the cysteine-rich domains in Tsg, and in Xenopus have been shown to be responsible for the interaction with BMP4 (Oelgeschläger et al., 2000). Significantly, the CR1 and CR3 domains of Chd also mediate interactions with Tsg. Both CR1 and CR2/CR3 fragments of mouse Chd coimmunopreciptate with Tsg, and Tsg enhances the coimmunoprecipitation of CR1 and BMP4 (Scott et al., 2001). Taken together, these results indicate that the cysteinerich domains represent protein–protein interaction motifs that appear to mediate the associations between Tsg, Chd, and BMP. In contrast to the results with mouse CR1, Xenopus CR1 failed to form a ternary complex with BMP and Tsg in crosslinking experiments (Oelgeschläger et al., 2000). Thus, while the CR domains appear to mediate interaction between Tsg, Chd, and BMP, the specifics of their role in ternary complex formation has yet to be resolved. Biochemical studies on mouse and Drosophila proteins suggest that one function of Tsg in the Tsg/Chd/ BMP complex may be to promote cleavage of Chd by Tld. Cleavage of Chd by Tld has been documented in a number of different species, and the data support the model that Tld activity inactivates Chd and thus releases the active BMP ligand dimer (Marqués et al., 1997; Piccolo et al., 1997; Scott et al., 1999). Tld cleavage sites have been mapped roughly in Drosophila and Xenopus and precisely in mouse, and despite the conservation between these systems, the sites do not all fall in the same location of the Chd/Sog proteins (Figure 3). In mouse, two cleavage sites have been documented, one just after the CR1 domain, and one just after the CR3 domain, producing fragments of 15, 83, and 13 kDa (Scott et al., 1999), similar fragments have been documented in Xenopus (Piccolo et al., 1997). In Drosophila, there is an analogous cleavage site just after CR1, but two other sites are documented, one before the CR2 domain and one just after, producing fragments of 20, 55, 20, and 25 kDa (Marqués et al., 1997). Assuming that the mobility of these fragments is not altered by modifications such as glycosylation, neither of the C-terminal sites in Drosophila corresponds to the second vertebrate site. The addition of Tsg alters the pattern of Chd/ Sog cleavage in both mouse and Drosophila. In the mouse, four Tld homologs have been identified, and these differentially affect Chd cleavage in the presence and absence of Tsg (Scott et al., 2001). BMP-1 and mTLL-1 cleave Chd at the documented sites in the absence of Tsg, while in the presence of Tsg an additional site becomes prominent that subdivides the 84 kDa fragment into two fragments of 29 and 65 kDa. Notably, this Tsg-dependent site corresponds to the second Tld cleavage site in Drosophila Sog. A third Tld-like protein, mTLD does not cleave Chd at all in the absence of Tsg, but if Tsg is present, Chd is cleaved in the same way as with BMP-1 and mTLL-1. The fourth Tld-like protease, mTLL-2, is incapable of cleaving Chd either in the presence or absense of Tsg. In Drosophila, Sog cleavage is also altered in the presence of Tsg but the precise location of the altered cleavages is not known (Yu et al., 2000). The mouse Tsg-dependent Tld cleavage site of Chd just before the CR2 domain coincides with one of the reported Tld cleavage sites in Drosophila Sog Figure 3. Cleavage Patterns of Mouse Chd and Drosophila Sog The N-terminal signal sequence is shown as a black box, and the four cysteine-rich (CR) repeats are shown as white boxes. Tld cleavage sites in the absence of Tsg are shown in blue, Tsg-dependent sites are shown in red. According to the current data, vertebrates and invertebrates share common sites just after the CR1 (Piccolo et al., 1997; Scott et al., 1999; Marqués et al., 1997) domain and a Tsg-dependent site just before the CR2 domain (Scott et al., 2001). The final C-terminal site is different between the two: the vertebrate site falls just after the CR3 domain, while the invertebrate site falls just after the CR2 domain (Scott et al., 1999; Marqués et al., 1997). Cell 804 (Marqués et al., 1997). The mapping of the Drosophila Tld cleavage sites was done in tissue culture cells and thus, it is possible that cleavage at the site before CR2 may have been due to endogenous Tsg expression in this cell line. Given the in vivo data indicating that Tsg can act as an antagonist of BMP signaling during development, the fact that the Tsg/Chd/BMP complex promotes cleavage by Tld is somewhat paradoxical. How is it that Tsg can act as a BMP antagonist, and yet do so by promoting the activity of Tld, a known BMP agonist? While a precise answer to this question cannot be made based on the available data, it must be kept in mind that while the biochemical data indicates that Tsg can form complexes with BMP and with Chd/BMP, as yet, these complexes have not been ascribed to a particular Tsg function. Is BMP inactive when complexed with Tsg, indicating that Tsg can act as an antagonist independent of Chd? Does Tsg binding to Chd/BMP act in some cases to lock Chd onto BMP while in other cases to promote Tld cleavage and thus release BMP? Do the novel Chd cleavage fragments produced in the presence of Tsg actually serve to inactivate Chd, or do they act as superrepressors, i.e., “superchordins” by analogy with the “supersogs” (Yu et al., 2000) that have been generated in vitro in Drosophila? Finally, is it possible that these different complexes and fragments have distinct and perhaps even opposing functions depending on the context in which they were generated? The studies on Xenopus indicate that Tsg may be multifunctional, as it has been proposed to be both a BMP agonist and antagonist (Oelgeschläger et al., 2000; Chang et al., 2001; Scott et al., 2001). In this case, similar types of experiments done in different laboratories under slightly different conditions produced results that suggest Tsg may exhibit opposing functions in development. While the resolution of the different results awaits further analyses, one potentially important criterion that arises from these data is that the relative levels of Tsg and Chd may dictate the nature of Tsg’s behavior—at least in Xenopus. For example, it has been shown that the ability for Chd to induce secondary axes when injected in ventral blastomeres can be enhanced by addition of Tsg, but only if the ratio of Tsg:Chd is less than 1:1. If the ratio of Tsg:Chd is 2:1 or greater, the enhancement of secondary axis formation is inhibited (Chang et al., 2001; Ross et al., 2001). This result seems to indicate that the types of interactions that Tsg may be involved in depend to a large degree on the relative levels of Tsg and Chd and perhaps a variety of other proteins involved in extracellular modulation of BMP signaling (e.g., Tld). In Drosophila, issues such as relative concentrations of Sog and Tsg or other extracellular factors may also account for the paradox of how both sog and tsg act as antagonists yet promote peak levels of BMP signaling in the dorsal cells of embryo. More detailed studies are required to resolve these issues. These recent studies have highlighted the importance of extracellular modulation of ligand availability, a once underappreciated means of regulating cell fate specification. The current work clearly demonstrates that Tsg can act as a BMP antagonist, yet the full extent of activities that Tsg possesses in the context of the developing organism remains to be clarified. Considering the cur- rent models for patterning events mediated by BMP signaling, there does not appear to be a requirement for an additional antagonist, in particular, since Tsg does not function simply as a cofactor for Chd. Indeed this fact alone indicates that the complete function or functions of Tsg are likely more complex. Added information on the importance of the relative levels of Tsg and Chd/ Sog, as well as on the functions of the different Chd/ Sog cleavage products, will be required to bring us to the end of this long and twisted road. Selected Reading Capdevila, J., and Belmonte, J.C.I. (1999). Curr. Opin. Genet. Dev. 9, 427–433. Chang, C., Holtzman, D.A., Chau, S., Chickering, T., Woolf, E.A., Holmgren, L.M., Bodorova, J., Gearing, D.P., Holmes, W.E., and Brivanlou, A.H. (2001). Nature 410, 483–486. Christian, J.L. (2000). Curr. Opin. Cell Biol. 12, 244–249. De Robertis, E.M., and Sasai, Y. (1996). Nature 380, 37–40. Marqués, G., Masacchio, M., Shimell, M.J., Wünnenberg-Stapleton, K., Cho, K.W.Y., and O’Connor, M.B. (1997). Cell 91, 417–426. Mason, E.D., Konrad, K.D., Webb, C.D., and Marsh, J.L. (1994). Genes Dev. 8, 1489–1501. Mason, E.D., Williams, S., Grotendorst, G.R., and Marsh, J.L. (1997). Mech. Dev. 64, 61–75. Massagué, J. (1998). Annu. Rev. Biochem. 67, 753–791. Mullins, M.C. (1998). Trends Genet. 14, 127–129. Oelgeschläger, M., Larrain, J., Geissert, D., and De Robertis, E.M. (2000). Nature 405, 757–763. Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L., and De Robertis, E.M. (1997). Cell 91, 407–416. Podos, S.D., and Ferguson, E.L. (1999). Trend Genet. 15, 396–402. Ray, R.P., Arora, K., Nüsslein-Volhard, C., and Gelbart, W.M. (1991). Development 113, 35–54. Ross, J.J., Shimmi, O., Vilmos, P., Petryk, A., Kim, H., Gaudenz, K., Hermanson, S., Ekker, S.C., O’Connor, M.B., and Marsh, J.L. (2001). Nature 410, 479–482. Scott, I.C., Blitz, I.L., Pappano, W.N., Imamura, Y., Clark, T.C., Steiglitz, B.M., Thomas, C.L., Maas, S.A., Cho, K.W.Y., and Greenspan, D.S. (1999). Dev. Biol. 213, 283–300. Scott, I.C., Blitz, I.L., Pappano, W.N., Maas, S.A., Cho, K.W.Y., and Greenspan, D.S. (2001). Nature 410, 475–478. Yu, K., Srinivasan, S., Shimmi, O., Biehs, B., Rashka, K.E., Kimelman, D., O’Connor, M.B., and Bier, E. (2000). Development 127, 2143– 2154.