Summary Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp 4 activity during forebrain development

Growth factors of the bone morphogenetic protein (Bmp) superfamily are major regulators of cell-cell signaling in animal development (Hogan, 1996; Massague and Chen, 2000). A gradient of Bmp activity establishes dorsoventral patterning in both vertebrate and invertebrate embryos. Studies in vertebrates and Drosophila have shown that Bmp/Dpp activity is regulated in the extracellular space by a network of secreted proteins including Short gastrulation (Sog)/chordin (Chd), Tolloid (Tld)/Xolloid (Xld) and Twisted gastrulation (Tsg) (De Robertis et al., 2000). The Drosophila Tsgmutation was isolated during classical genetic screens for embryonic cuticle defects due to its lack of the amnioserosa, the dorsalmost tissue of the fruit fly embryo (Wieschaus et al., 1984). The Drosophila Tsgproduct is necessary for peak Dpp/Bmp activity in the early Drosophilaembryo, which is required to form the amnioserosa (Mason et al., 1994). Tsg contains two conserved cysteine-rich domains and was shown to bind both Bmp and Chd (Oelgeschläger et al., 2000). The Tsg N-terminal domain binds to Bmp and shares similarity with the cysteinerich (CR) Bmp-binding modules of Chd, whereas the Cterminal domain is conserved only among the Tsg homologues (Oelgeschläger et al., 2000; Vilmos et al., 2001; Oelgeschläger et al., 2003a). In addition, Drosophila Tsgis highly diffusible and can act at a long distance from its site of expression (Mason et al., 1997). Tsg has multiple biochemical activities. First, it promotes the formation of stable ternary Bmp/Chd/Tsg complexes (Oelgeschläger et al., 2000; Chang et al., 2001; Larrain et al., 2001; Scott et al., 2001). As this ternary complex prevents binding of Bmp to its cell surface receptors, in this aspect of its function Tsg behaves as a Bmp antagonist. Second, the stability of these inhibitory ternary complexes is controlled by the Tld metalloprotease, which cleaves Chd/Sog at specific sites (Marques et al., 1997; Piccolo et al., 1997). In the presence of Tsg, Chd/Sog is a better substrate for cleavage by the Tld enzyme (Larrain et al., 2001; Scott et al., 2001; Shimmi and O’Connor, 2003). By promoting Chd degradation in the presence of Tld, Tsg releases Bmp that is now able to signal through Bmp receptors. In this second aspect of its activity, Tsg functions to increase Bmp activity (Piccolo et al., 1997; Larrain et al., 2001; Shimmi and O’Connor, 2003). The opposing functions of the Tsg protein may explain why conflicting results have been reported in various microinjection assays. In Xenopus, overexpression of Tsg mRNA results in Bmp-promoting effects (Oelgeschläger et al., 2000; Oelgeschläger et al., 2003a; Oelgeschläger et al., 2003b). The opposite observation was made in zebrafish embryos, in which microinjection of zebrafish Tsgor Xenopus Tsgcaused dorsalization (Ross et al., 2001; Oelgeschläger et al., 2003b). These different outcomes have been attributed to the different levels of the Tld protease in the two model embryos (Larrain et al., 2001). In zebrafish, endogenous levels of the Tolloid protease are low, as indicated by the weak phenotype of tolloid/mini-fin mutant embryos which are viable and lack the ventral tail fin (Connors et al., 1999). At these low Xolloid concentrations, microinjection of Tsg mRNA favors the formation of inhibitory Bmp/Chd/Tsg complexes. In the Xenopusembryo, high levels of Tld activity are present, Tsg promotes the cleavage of Chd, and ventralization is observed. However, when the endogenous Tld protease is inhibited by co-injection of dominant-negative Xld mRNA the opposite result, dorsalization, is seen (Larrain et al., 2001). These experiments suggest that the proteolytic cleavage of Chd 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. Tsghomozygous 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 Tsgfor 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.