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. This study shows 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 fly embryos were constructed lacking all early Tsg activity (tsg;;srw double mutants); they were shown to still establish a BMP signalling gradient. Thus, these results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution (Nunes da Fonseca, 2010).
Twisted gastrulation gene products have been identified from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG ortholog, chordin. Tsg binds both the vertebrate Decapentaplegic ortholog BMP4 and chordin, and these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. It is concluded that Tsg acts as a cofactor in chordin's antagonism of BMP signaling (Scott, 2001).
Tsg is coexpressed with chordin and various BMPs in vertebrate development. In Xenopus, maternal Tsg RNA was detected in eggs by RT (reverse transcription)-PCR, while whole-mount in situ hybridization showed uniform Tsg expression across the entire animal hemisphere and marginal zone of the early gastrula. At tailbud stage, Tsg, chordin and BMP4 expression domains partially overlap in the developing tail, anterior brain, eye and heart. In mouse, Tsg is broadly expressed throughout the 7.5-days-post-coitus (7.5-d.p.c.) gastrula and in extraembryonic tissues. Chordin, Tsg and BMPs 2, 4 and 7 are highly expressed in the digital rays of 15.5- and 17.5-d.p.c. embryo hindlimbs. Strong chordin expression in the interzone of the joint cavity is juxtaposed with strong Tsg expression at the joint articular surfaces and the interzone. Thus, Tsg is properly situated for potential interactions with chordin and BMPs during various stages of vertebrate embryogenesis (Scott, 2001).
In Drosophila, TSG influences cleavage of the chordin ortholog Short gastrulation (SOG) by Tolloid, altering the pattern of SOG cleavage products. There are four mammalian tolloid-related proteases. Two of these, BMP1 and mammalian tolloid-like 1 (mTll1), each cleave chordin at two specific sites, yielding fragments of relative molecular mass (Mr) 15K, 13K and 83K, corresponding to the amino-terminal, carboxy-terminal, and internal portions of chordin, respectively. Murine Tsg appears to enhance cleavage of mouse chordin and to influence the relative abundance of cleavage products, such that fragments of Mr 65K and 29K, minor forms in the absence of Tsg, become major products in the presence of Tsg. A third related protease, mammalian tolloid (mTld), which has little detectable chordin-cleaving activity, has significant activity in the presence of Tsg, also producing the 65K and 29K fragments as major forms. The fourth mammalian tolloid-like protease, mTll2, lacks chordin-processing activity in the presence or absence of Tsg (Scott, 2001).
The 65K and 29K chordin cleavage products preferentially produced in the presence of Tsg are subfragments of the 83K internal chordin fragment, as established by N-terminal amino-acid sequencing, and result from cleavage at a previously unmapped site between Ala 670 and Thr 671. Thus, the 29K form contains chordin cysteine-rich repeats (CRs) 2 and 3, whereas the 65K form contains no CR domains (Scott, 2001).
To determine how Tsg affects chordin cleavage, Tsg's ability to physically interact with tolloid-like proteases was examined. Co-immunoprecipitation of Tsg with BMP1 or mTll1 fails to detect physical interactions. However, co-immunoprecipitations show that Tsg binds chordin. Whether Tsg/chordin interactions might influence chordin's ability to bind BMP4 was examined. Co-immunoprecipitation of chordin and BMP4 is greatly enhanced in the presence of Tsg. It was also found that Tsg binds BMP4. In summary, Tsg's interactions with chordin and/or BMP4 enhance chordin/BMP4 complex formation, suggesting that Tsg might enhance chordin's antagonism of BMP signaling (Scott, 2001).
Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). The molecular mechanism by which Tsg regulates BMP signaling has been analyzed. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, since they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay has revealed that Tsg has two distinct and sequential activities on BMP signaling. (1) Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. (2) After cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated (Larraín, 2001).
The opposing activities of Tsg on BMP binding to its receptor suggest a sequential molecular mechanism that may help reconcile disparate observations in the literature. (1) Tsg forms a ternary complex with Chordin and BMP, which is a potent inhibitor of BMP signaling. This antagonist function must be the predominant one in zebrafish, because loss-of-function of Tsg and Chordin using antisense morpholinos ventralizes the embryo. (2) After cleavage of Chordin by Xolloid, Tsg competes the residual activity of Chordin fragments, providing a permissive signal that promotes BMP binding to its cognate receptor. This function is consistent with injection experiments in Xenopus embryos, in which reduction of endogenous Xenopus Tsg activity enhances the anti-BMP activity of CR1 fragments. (3) Overexpression of Tsg facilitates the degradation of endogenous Chordin in Xenopus. This activity may help explain why Tsg can ventralize the embryo and inhibit axis duplication by Chordin in a Xolloid-dependent manner. It is proposed that in overexpression experiments, an excess of Tsg protein displaces the equilibrium in the reaction, so that after cleavage of Chordin by Xolloid, Tsg dislodges BMP from the proteolytic products and facilitates their degradation in vivo. The Tsg/BMP binary complex acts as a permissive signal, because at physiological concentrations Tsg does not interfere with BMP binding to its receptor. Finally, at high concentrations, Tsg can also act as a BMP antagonist in the absence of Chordin, inducing in animal cap explants the cement gland marker XAG-1, but not the neural marker NCAM, by partially inhibiting BMP activity (Larraín, 2001).
The present results provide mechanistic insights into how sharp borders may be generated in embryos. In Drosophila, Tsg is required for the peak BMP signaling that induces a sharp band of Mad phosphorylation in the dorsal-most tissue. In lateral regions of the Xenopus embryo, where free full-length Chordin is still present, Tsg/BMP binary complexes released by Xolloid will have a higher affinity for Chordin than for the BMP receptor promoting the re-formation of inhibitory ternary complexes that can diffuse further. However, once all Chd is proteolytically cleaved by Xolloid, the function of Tsg switches from an inhibitory to a permissive signal that increases binding of BMPs to their cognate receptors. This switch in activity would facilitate the formation of sharp boundary differences. In lateral regions, where ternary complexes are constantly re-formed and re-cleaved as diffusion takes place, the situation is conceptually analogous to that occurring in an organic chemistry fractional distillation column. Although much remains to be learned about this interesting patterning system, the opposing functions of Tsg suggest a novel molecular mechanism for the establishment of cell differentiation territories in the embryo (Larraín, 2001).
BMP activity is controlled by several secreted factors including the antagonists chordin and Short gastrulation (Sog). A second secreted protein, Twisted gastrulation (Tsg), enhances the antagonistic activity of Sog/chordin. In Drosophila, visualization of BMP signaling using anti-phospho-Smad staining shows that the tsg and sog loss-of-function phenotypes are very similar. In S2 cells and imaginal discs, Tsg and Sog together make a more effective inhibitor of BMP signaling than either of them alone. Blocking Tsg function in zebrafish with morpholino oligonucleotides causes ventralization similar to that produced by chordin mutants. Co-injection of sub-inhibitory levels of morpholines directed against both Tsg and chordin synergistically enhances the penetrance of the ventralized phenotype. Tsgs from different species are functionally equivalent, and it has been concluded that Tsg is a conserved protein that functions with SOG/chordin to antagonize BMP signaling (Ross, 2001).
Since the phenotypes of tsg and sog mutants are similar, attempts were made to determine whether Tsg can enhance the binding of Sog to ligand. Co-immunoprecipitation of DPP by Sog is greatly enhanced when these two factors are coexpressed in S2 cells along with Tsg. To test whether the combination of Sog and Tsg blocks Dpp signaling better than Sog alone, an S2 cell-culture assay was developed for Dpp signaling. At high concentration Tsg alone can block Dpp signaling; however, at lower concentration, the combination of Tsg and Sog together dramatically reduces the Dpp-dependent accumulation of P-MAD much more efficiently than either can alone. In vivo overexpression of sog and tsg together can completely reverse the phenotype of ectopic dpp expression in the wing, whereas the expression of either alone has no effect. It is concluded that a complex of Tsg and Sog is an efficient antagonist of Dpp signaling (Ross, 2001).
To determine whether Tsg is conserved among other species, genes in the database related to Drosophila Tsg were sought and found in human, mouse, zebrafish and Xenopus. In addition, a second tsg-related sequence was found in Drosophila (tsg2) and a second zebrafish tsg (tsg1) was obtained using degenerate polymerase chain reaction (PCR) methods. The protein products show extensive similarity with about 50% of 202 amino-acid residues matching in all four species. The pairs of tsg genes in fly and fish are closer to each other than to tsg in any other species, suggesting independent gene-duplication events in these two species. The human, mouse and zebrafish (tsg1) genes were mapped by a combination of fluorescence in situ hybridization (FISH) or radiation hybrid mapping. The mouse gene maps to 17E1.3–E2, a region that is syntenic to 18p11.2–3 where the human homologue resides. In zebrafish, tsg1 is located at linkage group 24-74.5, which is syntenic to the human locus and indicates that all three genes are probably functional orthologues (Ross, 2001).
The zebrafish tsg1 gene is expressed uniformly in early embryos, whereas zebrafish tsg2 is only expressed at later stages. Hence, the analysis was focused on zebrafish tsg1 and morpholino oligonucleotides were used to reduce the function of this gene in early zebrafish development. Injection of a tsg1 morpholino oligonucleotide (ztsg-MO: see Taylor, 1996 for a description of the morpholino modification) produces a phenotype characteristic of expanded BMP signaling. Using morphological criteria and fluorescent red blood cells, it was found that embryos develop expansions of the ventral fin region that correspond to ectopic blood islands, a tissue derived from ventral mesoderm. Injected embryos also show an expansion of GATA2, loss of paraxial mesoderm (visualized with the marker myoD), and a mild reduction of anterior ectodermal tissues (detected by staining for krox20). Caudal expression of bmp4 is also expanded in these embryos, while the anterior ectodermal marker otx2 is reduced. Treated embryos also exhibit an expansion in apoptotic cells ventral to the yolk extension, similar to dino and mercedes mutants. Overall, this phenotype is very similar to that of ogon/mercedes mutants and moderate chordin loss-of-function mutants, and represents a modest ventralized phenotype (Ross, 2001).
Since Drosophila data suggest that one function of Tsg is to cooperate with Sog to inhibit BMP signaling, it was asked whether the same relationship is true in vertebrates by determining whether a modest reduction of zebrafish chordin activity can enhance the effect of a moderate reduction in tsg1 activity. Sub-inhibitory levels of a zebrafish chordin morpholino oligonucleotide and tsg1-MO were injected into wild-type embryos, and the effect on ectopic blood island development was scored. These two morpholino oligonucleotides synergistically enhance blood island expansion, supporting the view that both of these gene products co-operatively inhibit BMP signaling. As with the Drosophila components, it was found that the combination of purified mouse chordin and Tsg is better able to inhibit mouse BMP-stimulated phosphorylation of Mad in S2 cells than either can alone (Ross, 2001).
A test was performed for synergy between Tsg and chordin mRNA in Xenopus embryos by co-injecting their mRNAs and scoring for enhancement of secondary axis formation. Co-injection of Xenopus Tsg and chordin reveals a dose-response optimum. When a sub-inhibitory dose of chordin mRNA is supplemented with increasing levels of Tsg mRNA, the fraction of embryos exhibiting a secondary axis increases up to 4.5-fold over chordin alone at a 1/5 ratio of Tsg/chordin mRNA. However, if the Tsg/chordin ratio is increased to 1:1 or higher, the number of secondary axes is reduced to basal levels and the resulting tadpoles have normal morphology. Injection of 150 pg Tsg alone (the highest concentration of Tsg mRNA used in these experiments) has no effect on embryonic development. Notably, if the level of Tsg relative to chordin is increased in the S2 experiments, no reversal of the inhibition phenotype is seen, suggesting that additional factors probably modulate the in vivo response. Taken together, it is concluded that, like Drosophila Tsg, vertebrate Tsg can co-operate with chordin to inhibit BMP signaling (Ross, 2001).
As a final test of the functional equivalence of the vertebrate and invertebrate tsg genes, the human and mouse genes were expressed under the control of the UAS promoter in flies, and Drosophila Tsg mRNA was injected into zebrafish embryos. The phenotype of animals expressing human Tsg and Drosophila sog in wing discs resembles that of dpp shortvein alleles and is very similar to that produced by coexpression of the Drosophila tsg and sog genes. When injected into zebrafish, Drosophila tsg produces a dorsalized phenotype equivalent to that produced by zebrafish tsg1, which includes reduced axial length and expansion of krox20 (Ross, 2001).
These experiments suggest that Tsg has three molecular functions. (1) It can synergistically inhibit Dpp/BMP action in both Drosophila and vertebrates by forming a tripartite complex between itself, Sog/chordin and a BMP ligand. (2) Tsg seems to enhance the Tld/BMP-1-mediated cleavage rate of Sog/chordin and may change the preference of site utilization. (3) Tsg can promote the dissociation of chordin cysteine-rich (CR)-containing fragments from the ligand. Different organisms may exploit each of these properties to different degrees during development depending on the relative in vivo concentrations of each molecule. It is proposed that in Drosophila and zebrafish the primary function of Tsg is to form a tripartite complex between itself, Sog/chordin and a BMP ligand. In Drosophila, this complex acts to redistribute a limiting amount of Dpp, such that activity is elevated dorsally at the expense of being lowered laterally. The net driving force for this redistribution is likely to be diffusion of Sog from its ventral source of synthesis. This is consistent with the finding that Sog diffusion is essential for activation of genes such as race that require high levels of Dpp/SCW signaling. In this model Tld would serve to modulate both the net movement of Dpp and its release from the inhibitory complex by cleaving Sog. The ability of Tsg to enhance the rate of Sog cleavage may also be an important aspect of this model in that it helps ensure the proper timing of these rapid developmental events. It seems unlikely that Tsg is needed to remove an inhibitory CR-containing fragment from Dpp, since the affinity of full-length Sog for Dpp in the absence of Tsg seems to be low. Likewise, in zebrafish the phenotype of reduced Tsg function is ventralized and not dorsalized as would be predicted if Tsg were primarily needed to release inhibitory CR fragments from ligand. In Xenopus, however, perhaps the endogenous levels of full-length chordin and CR fragments are higher than in zebrafish, thereby making the CR displacement activity of Tsg the more important biological function. Determination of the in vivo levels of these proteins, along with a more careful analysis of the concentration optima for each type of reaction involving Tsg function, will be required before all of its in vivo activities can be understood (Ross, 2001).
Human Twisted gastrulation (TSG) was isolated in a screen for secreted factors, and mouse and Xenopus Tsg were isolated by low-stringency hybridization using human TSG as the probe. These vertebrate Tsgs have a high sequence homology to one another (more than 80% identical) and are about 30% identical to Drosophila Tsg at the amino-acid level. Tsg is expressed maternally and in all developmental stages in Xenopus, and at least from gastrula stages onward in mouse. Expression of Tsg is also detected in a variety of adult tissues in both mouse and human (Chang, 2001).
To study the function of Tsgs, their activities were examined in Xenopus ectodermal explants (animal caps). Human, mouse and Xenopus Tsg induce the cement gland and the neural markers XAG-1, OtxA and NRP-1 with comparable efficiency, suggesting that these vertebrate Tsgs function similarly in Xenopus. The induction of cement gland and neural markers in animal caps in the absence of mesoderm is normally associated with inhibition of the BMP signaling, so whether Tsg could directly block the activity of BMP was addressed. The effect of Tsg on ventralization of the ectodermal cells by BMPs was examined. Intact animal caps express high levels of epidermal keratin. This expression is suppressed when caps from blastula stages are dissociated for 4 h. Cement gland and neural markers are turned on in these dissociated samples. While Bmp2 restores the transcription of epidermal keratin and inhibits the expression of neural markers, it cannot do so in the presence of Tsg. These results indicate that Tsg directly antagonizes the neural inhibition and epidermal induction activity of BMPs in dissociated animal caps (Chang, 2001).
The effect of Tsg on ventralization of the mesodermal explants by BMPs was examined. Bmp4 inhibits dorsal and induces ventral gene expression in dorsal marginal zone (DMZ) explants. Coexpression of Xenopus Tsg with Bmp4 re-establishes dorsal marker expression and reduces the ventral gene induction in these explants, indicating that it also inhibits BMP activity in the mesodermal cells. Notably, a reduction in the level of OtxA (a marker for anterior neural tissue at tailbud stages) was detected when Tsg alone was expressed in the DMZ. This phenotype has not been observed with other BMP antagonists, and suggests that Tsg may be involved in processes other than inhibition of BMP signaling. To determine whether Tsg also blocks other signal transduction pathways, the effect of Xenopus Tsg on marker induction by activin, basic fibroblast growth factor (FGF) and Wnt8 was examined. Tsg specifically inhibits BMP-dependent mesoderm induction, but does not interfere with the expression of the markers induced by the other signaling molecules. These results demonstrate that Tsg is specific for a BMP pathway and does not block activin, FGF or Wnt signaling (Chang, 2001).
The in vivo function of Tsg was examined by gain-of-function studies. Injection of Tsg RNAs from different vertebrates in early embryos induces a similar phenotype, indicating that they have similar activities in vivo. The embryos injected with Tsg RNA exhibit several developmental defects at tadpole stages, such as an enlargement of the dorsal fin, malformation of the proctodeum, and a reduction of the head. Lineage tracing with nuclear ß-gal shows that the effect induced by Tsg is non-cell autonomous. The phenotype of Tsg-expressing embryos is unique and does not resemble the phenotypes induced by overexpression of other BMP antagonists. Therefore, whether Tsg affects the cells and tissues that rely on the BMP signaling was addressed. The formation of the blood cells, which are derived from ventral tissues and require active BMP signals, was examined. In control embryos, the blood cells stained with benzidine were localized at the ventral side of the embryos; however, this staining was not observed in embryos injected with Xenopus Tsg RNA. Furthermore, in situ hybridization with a blood-specific anti-T1-globin probe reveals that blood cells are absent in the Tsg-expressing embryos. The formation of the heart, which has been reported in both the chick and the frog to require BMP signaling, was examined. In situ hybridization with a heart marker, Nkx2.5, shows that the Tsg-expressing embryos have reduced transcription of Nkx2.5. In contrast, the expression of a dorsal mesoderm marker in muscle is not decreased by Tsg. These data show that Tsg interferes with the development of several tissues that require BMP signaling (Chang, 2001).
To further analyse the effects of Tsg, expression of both dorsal and ventral genes was assayed in Tsg-injected embryos at different developmental stages. In gastrula embryos, several ventral markers, such as Xhox3, Vent1 and Msx1, are downregulated by Xenopus Tsg, whereas dorsal markers, including Sox2, goosecoid and Hex, are not much affected. The expression of the dorsal gene chordin also remains the same in most cases, although a slight reduction of its level was occasionally seen. The pattern of gene expression changes during development, so that at later stages, Xhox3 and Msx1 are also expressed in the neural tissue and neural crest cells. The reduction of their expression is less profound, and at tailbud stages Xhox3 expression is normal. In agreement with whole-mount staining results, the transcripts of both globin and Nkx2.5 are reduced by Tsg, whereas the muscle actin remains intact at tailbud stages. Unexpectedly, the reduction of several anterior genes, such as Hex (a marker for anterior endoderm) and goosecoid (a prechordal mesoderm gene), by Xenopus Tsg is seen from neurula stages onward. Sox2, whose expression domain includes the anterior neural region, is also slightly downregulated. These data demonstrate that Tsg reduces BMP signaling at early developmental stages; furthermore, Tsg may have one or more additional functions in anterior embryonic development at a stage after the onset of gastrulation (Chang, 2001).
Although Tsg can inhibit BMP function, it does not induce a partial secondary axis when injected into the ventral side of early Xenopus embryos. One possible explanation for this is that Tsg may be a highly diffusible molecule, as has been observed in Drosophila, and does not accumulate to high concentrations at the ventral side to establish an organizer-like activity. To test this hypothesis, a membrane-tethered form of Tsg was constructed by fusing the full-length Xenopus Tsg in frame with an integral membrane protein CD2. The chimaeric protein, Tsg-CD2, induces cement gland and neural markers in animal caps to the same extent as wild-type Xenopus Tsg, suggesting that the two proteins have similar activities. Injection of the CD2 RNA into early frog embryos does not induce any phenotype, but ventral injection of Tsg-CD2 RNA leads to embryos with partial secondary axes. These results indicate that localized Tsg behaves like other BMP antagonists and can induce ectopic dorsal structures on the ventral side of frog embryos (Chang, 2001).
To investigate whether Tsg modifies the BMP inhibitory activity of chordin, the effect of coexpression of chordin and Xenopus Tsg in both ectodermal explants and whole embryos was examined. In animal caps, a low dose of either chordin or Tsg does not efficiently block Brachyury (Bra) induction by Bmp4; coexpression of both genes leads to a more effective inhibition of the BMP activity. In whole embryos, chordin induces a partial secondary axis when expressed at the ventral side; coexpression with Xenopus Tsg at a ratio of Tsg/chordin below 1/1 does not prevent the ectopic axis-induction by chordin. Instead, many embryos show a dorsalized phenotype with reduced trunk and tail. Notably, with an increasing Tsg/chordin ratio (2/1 or higher), the secondary axis disappears in an increasing number of embryos, and these embryos resume a typical Tsg phenotype. Tsg and chordin still inhibit Bra induction by Bmp4 in animal caps at these high ratios. The data suggest that although Tsg and chordin expression together lead to a more efficient BMP inhibition in ectodermal explants regardless of the amounts of RNA injected, the in vivo phenotypes depend on the relative abundance of the two genes. The exact mechanism underlying this observation remains unclear. There are at least two possibilities that are not mutually exclusive. The highly diffusible Tsg may help to diffuse the chordin protein through complex formation, which may result in dilution of the chordin function at the ventral side and inhibition of the secondary axis induction by chordin. Alternatively, other endogenous factors may interact with chordin and/or Tsg and modify their activities and the phenotypes of coexpression of chordin and Tsg (Chang, 2001).
Tsg, first identified in Drosophila and shown to have a function in development of the dorsal midline, was originally proposed to potentiate DPP activity. This hypothesis, however, has recently been challenged by the observation that a processed SOG product, which has a broader inhibitory spectrum towards BMP members, rescues the tsg mutant phenotype, whereas Dpp does not. This result indicates that Tsg may participate in alternative processing of Sog to generate a 'supersog' to block the DPP signaling. Tsg may therefore be involved in inhibiting, rather than enhancing, the Dpp activity. A similar mechanism suggesting that Tsg may participate in inhibition of the BMP signaling has now been proposed to also work in vertebrates. Tsg stimulates chordin cleavage at a unique site, and Tsg enhances chordin activity in both Xenopus and zebrafish. This study shows that Tsg can function as a BMP antagonist in embryonic explants and interfere with BMP-dependent tissue formation in frog embryos. The activity of Tsg, however, is different from that of other BMP antagonists, since Tsg also induces defects in the anterior tissues, such as the anterior endoderm expressing the Hex gene and the prechordal mesoderm that expresses goosecoid. This phenotype may underlie the recent interpretation that vertebrate Tsg acts to promote BMP signaling, since it has been reported that elevated BMP expression leads to reduction of dorsal markers at the gastrula stages, which results in truncation of anterior and dorsal tissues at late stages. It is currently unclear, however, whether the defects induced by TSG truly reflect an enhancement of BMP signaling. Although the possibility cannot be ruled out that Tsg can function both as a BMP agonist and antagonist depending on the presence or absence of other factors in specific regions of the embryos at particular developmental stages, it is also possible that the late effects are still mediated by inhibition of a BMP pathway. Several BMPs are expressed in the anterior endoderm and/or prechordal mesoderm in chick and fish, and in chick, BMPs may be involved in specification of prechordal mesoderm. It is possible that Tsg inhibits a different spectrum of BMPs from other BMP antagonists, either alone or by alteration of chordin specificity, thereby leading to the unique phenotype in the anterior tissues. Another possibility is that Tsg may have one or more additional functions independent of BMP signaling. Further investigation is required to understand the in vivo function of Tsg (Chang, 2001).
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, morpholino (MO)-based knockdown studies of Tsg1 were performed in zebrafish. 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) enhance dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppresses the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). These results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning (Little, 2004).
The characterization of vertebrate Tsg homologs is reported. Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. These data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis (Chang, 2001).
BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. The protein twisted gastrulation (TSG) is a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. The in vivo function of TSG in early Xenopus development has been examined using a loss-of-function approach. Reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, it is noted that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos (Blitz, 2003).
The twisted gastrulation gene encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. Tsg homologs from human, mouse, zebrafish, and Xenopus share 72%-98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with dpp. The vertebrate orthologs of Dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and this analysis places the gene within 5 Mbp of TG-interacting factor (Graf, 2001).
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. The function of Tsg in embryonic patterning was investigated by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain are unable to bind BMP, yet ventralize the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity requires an intact C-terminal domain and can block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity is specific for CR-containing proteins since it does not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants are 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 (Oelgeschläger, 2003).
Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. A TSG cDNA was isolated as a secreted molecule from the mouse aorta-gonad-mesonephros region. TSG-deficient mice are born healthy, but more than half of the neonatal pups show severe growth retardation shortly after birth and display dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus is atrophic, and phosphorylation of SMAD1 is augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, these findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, these findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency (Nosaka, 2003).
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. Tsg loss-of-function was examined in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. Evidence is provided 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 (HPE), first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development (Zakin, 2004).
The human Tsg gene maps close to the HPE locus 4 on chromosome 18p11.3. However no mutations in the human Tsg gene could be detected in familial cases of HPE at locus 4. It has been proposed in the multi-hit hypothesis that sporadic HPE may result from mutations in more than one gene. This is what was observed in the current study, in which HPE requires mutations in two genes, Tsg and Bmp4, to become manifest. However, sporadic cases of HPE have been recently observed in Tsg-/- embryos in mice that had been bred for six generations into B6SJL/F1 background. Importantly, these Tsg-/- embryos with HPE still developed eyes, whereas Tsg-/-;Bmp4+/- had HPE with anophthalmia. In earlier crosses, the ones reported in this study, HPE was observed in Tsg-/- embryos only when one copy of Bmp4 was removed. It is likely that a genetic modifier of unknown nature was changed by breeding in the laboratory (Zakin, 2004). Tsg and Bmp4 are expressed in adjacent or overlapping regions. The holoprosencephalic phenotype of the Tsg-/-;Bmp4+/- mutants is not of early onset, for the anterior visceral endoderm and the anterior neural ridge are normally formed. At E8.5 the expression of two crucial signaling factors, Fgf8 and Shh, was defective in Tsg-/-;Bmp4+/- embryos and this can explain the phenotypes observed. Shh is required for the growth of the ventral forebrain and when mutated causes a more severe HPE than the one described in this study since it includes cyclopia. In addition, Shh-/- mice fail to express Fgf8 in the ventral forebrain. Fgf8 null embryos die at gastrulation, but the study of Fgf8 hypomorphic alleles has demonstrated its requirement for forebrain formation and first branchial arch development (Zakin, 2004).
Multiple experiments in mouse and chick embryos have shown that signals from Bmps, Shh and Fgf8 regulate the growth of the telencephalon through proliferation and cell death. For example, the implantation of Bmp4-coated beads causes HPE. Ectopic expression of Bmp4 and Bmp5 in the chick forebrain causes cyclopia and HPE. Chd;Nog double mutants, which should have increased Bmp signaling, also show HPE. However, ectopic expression of the Bmp antagonist Nog also inhibits telencephalic growth. Therefore, it appears that the growth of the forebrain requires a fine balance of Bmp, Fgf8 and Shh signaling and that both excessive and insufficient signaling can result in similar phenotypes (Zakin, 2004).
Is the HPE in Tsg-/-;Bmp4+/- embryos indicative of a pro-Bmp4 or an anti-Bmp4 effect? This question can be answered by a simple genetic argument. In the absence of Tsg, two copies of Bmp4 are compatible with normal head development. However, when in addition one copy of Bmp4 is removed, development of the ventral forebrain and first branchial arch are impaired. It is concluded from these dose-dependent genetic interactions that during head development Tsg is required to promote Bmp4 signaling (Zakin, 2004).
Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. The loss of Bmp7 in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 binds to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm (Zakin, 2005).
Sirens were discovered in the mouse (Gluecksohn-Schoenheimer, 1945) among the progeny of parents carrying various combinations of the Short-tail (T locus), anury (t0), Fused and ur mutations. The siren pups obtained had no tail, various degrees of reduction and fusion of elements of the hindlimbs, abnormalities of the spine, and fusion of ribs. Even though Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- sirenomelic pups do form tails (albeit shorter), the limb bud phenotypes observed are very similar to those seen in the Gluecksohn-Schoenheimer study. Could the old and new mutations be linked in any way? It is noted that the T locus (including brachyury), Fused (corresponding to Axin) and Tsg are all located on chromosome 17. The us mutation (urogenital syndrome), which is phenotypically identical to the now extinct ur (urogenital) mutant, and Bmp7 are both located on chromosome 2. Although the respective locations of these genes on these chromosomes are distant from each other, mutations at the T locus correspond to important chromosomal rearrangements, often leading to duplications and deficiencies of chromosome segments upon cell division (Gluecksohn-Schoenheimer, 1945). Thus, it is conceivable, although perhaps unlikely, that the occurrence of sirens in the initial description was associated with disruptions of the Tsg and/or Bmp7 genes. Unfortunately, some of the original mutations have been lost, so this is not a testable proposition (Zakin, 2005 and references therein).
In subsequent work, Hoornbeek found sirenomelic neonates in crosses between SM/J and BUA strains studied for the incidence of the 'careener' phenotype (Hoornbeek, 1970). These sirens have the same phenotype as in this study (fused hindlimbs, a tail, an abnormal umbilical artery). The genes affected in these crosses are not known, but the carriers of the 'siren' mutation (Hoornbeek, 1970) had tightly twisted tails; this is relevant because Tsg-/- or Bmp7-/- mutants also have kinked tails (Zakin, 2005 and references therein).
In conclusion, in the absence of Bmp7, two copies of Tsg are required for the proper differentiation of ventral and posterior structures. In the mouse, when Tsg and Bmp7 are mutated, the siren phenotype results from the fusion of the limb buds in the ventroposterior midline owing to a paucity of posterior ventral mesoderm. In Xenopus, knockdown of Tsg and Bmp7 results in an analogous phenotype: loss of posteroventral cell fates associated with decreased Bmp activity. These results demonstrate a common mechanism, mediated by Bmp signaling, in mouse and frog in the patterning of the dorsoventral axis (Zakin, 2005).
Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicate a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. This study shows by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore, evidence is provided that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impairs the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibits BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation (Schmidl, 2006).
Bone morphogenetic protein (BMP) signaling controls various aspects of organogenesis, including skeletal development. The pro-BMP function of Crossveinless 2 (Cv2) has been shown to be required for axial and non-axial skeletal development in mice. Skeletal defects in the Cv2-null mutant ae reversed by the additional deletion of Twisted gastrulation (Tsg). Whereas the Cv2-/- mutant lacks a substantial portion of the lumbar vertebral arches, Cv2-/-;Tsg-/- mice have almost normal arches. Suppression of Cv2-/- phenotypes is also seen in the non-axial skeleton, including the ribs, humerus, skull, and laryngeal and tracheal cartilages. In contrast, the Tsg-/- phenotype in the head is not significantly affected by the Cv2 mutation. These findings demonstrate that Tsg mutation is epistatic to Cv2 mutation in the major skeletal phenotypes, suggesting that the pro-BMP activity of Cv2 is, at least in part, dependent on Tsg. Genetic evidence is presented for the context-dependent functional relationship between Tsg and Cv2 during mouse development (Ikeya, 2008).
Developing tissues form spatial patterns by establishing concentration gradients of diffusible signaling proteins called morphogens. The bone morphogenetic protein (BMP) morphogen pathway uses a family of extracellular modulators to reshape signaling gradients by actively 'shuttling' ligands to different locations. It has remained unclear what circuits are sufficient to enable shuttling, what other patterns they can generate, and whether shuttling is evolutionarily conserved. Using a synthetic, bottom-up approach, this study compared the spatiotemporal dynamics of different extracellular circuits. Three proteins-Chordin (Drosophila Sog), Twsg (Drosophila Tsg), and the BMP-1 protease (Drosophila Tolloid)-successfully displaced gradients by shuttling ligands away from the site of production. A mathematical model explained the different spatial dynamics of this and other circuits. Last, combining mammalian and Drosophila components in the same system suggests that shuttling is a conserved capability. Together, these results reveal principles through which extracellular circuits control the spatiotemporal dynamics of morphogen signaling (Zhu, 2023).
BMP, Chordin, Twsg1, and BMP-1 have been shown to enable ligand shuttling in Drosophila and Xenopus dorsal-ventral patterning. On the other hand, two recent studies in zebrafish embryos showed that BMP ligands were not shuttled despite the presence of all four circuit components, provoking the question of whether shuttling can occur in mammals and what minimal set of components is sufficient to generate it. By systematically reconstituting the shuttling circuit one component at a time, this study was able to identify four distinct behaviors enabled by different combinations of components: BMP alone can form simple monotonically decreasing gradients; Chordin can delay and extend those gradients; Twsg1 with Chordin can suppress gradients; and BMP-1 with the other components can generate shuttling. In this last case, time-lapse movies revealed that gradients form at a distance from the source rather than propagating outward from it (Zhu, 2023].
A simple mathematical model shows how the four-component circuit is sufficient to enable ligand shuttling, notably predicts all of the four behaviors observed in these experiments, and highlights how the qualitative properties of the shuttling gradient depend on BMP-1 expression level. To do so, the model relies only on known interactions and previously measured parameters. Looking ahead, it will be important to identify more complex and developmentally relevant conditions in which the model fails, as additional components are included (Zhu, 2023].
Shuttling appears to be conserved during evolution. Biochemical and genetics studies have shown that interactions among BMP, Chordin, Twsg1, and BMP-1 are largely conserved across metazoans. These observations raise the provocative question of whether hybrid systems with components from distantly related organisms can still function. The reconstituted system allows one to directly examine this question. By substituting Drosophila Sog for mouse Chordin, it was found that some features of the resulting circuit are preserved, including gradient inhibition and lengthening. This substitution degraded the shuttling behavior, eliminating gradient displacement. This could be due to quantitative differences in BMP-Sog interactions, such as weaker binding. Another difference between Drosophila Sog and mammalian Chordin is that Tolloid cleavage of Sog is dependent on BMP binding, while BMP-1 can cleave vertebrate Chordin both in its free form and within a BMP-Chordin complex. While BMP-dependent cleavage is not necessary for shuttling, this mechanism has been suggested to increase the robustness of shuttling. It would be interesting to introduce Drosophila Tolloid into the reconstituted system and compare the robustness of shuttling enabled by Drosophila Sog and Tolloid, and mammalian Chordin and BMP-1. Analysis of other hybrid interspecies circuits across a broader range of expression levels could help to understand how evolutionary changes in molecular interactions cause changes in patterning (Zhu, 2023].
These results show that shuttling can occur with mammalian components but do not imply that all BMP-Chordin dependent behaviors involve shuttling. The BMP-Chordin system appears capable of multiple qualitatively distinct patterning behaviors, depending on factors such as the expression level of circuit components or the expression configurations. For example, the configuration demonstrated here, where BMP and Chordin are expressed at the same region, resembles certain developmental contexts such as mouse vertebral field, and ectopic expression systems in embryos. However, it differs from the configuration in zebrafish early embryos, where BMP and Chordin are expressed from opposing poles, and a source-sink, rather than a shuttling, mechanism has been demonstrated. It is possible that the more linear gradient generated by the source-sink mechanism is more desired in this developmental context. Future studies with the reconstituted system should allow investigation of other developmentally relevant configurations (Zhu, 2023).
While this study focused on shuttling, BMP pathway components generate a much broader range of behaviors, including scaling with embryo size and spatially oscillatory patterning of complex tissues, such as digits. By extending the platform described in this study to incorporate additional components [e.g., other ligands, modulators, and receptors], feedback loops in which signaling regulates pathway components, and other geometric configurations, one could, in principle, reconstitute other gradient behaviors and quantitatively explore these phenomena in a simplified setting. One could also analyze the role of co-occurring combinations of multiple BMP ligands such as BMP2, BMP4, and BMP7 in space and time. In addition, one could investigate the proposed ability of shuttling to enhance the robustness of gradient formation and, within a larger expander-repressor system, to enable gradient scaling. By exploring a wide range of circuits, these experiments could also provide a foundation for the development of synthetic circuits to program multicellular pattern formation within the nascent field of synthetic developmental biology (Zhu, 2023).
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