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Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling

Juan Larraín^*, Michael Oelgeschläger^*, Nan I. Ketpura, Bruno Reversade, Lise Zakin and E. M. De Robertis

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA*These authors contributed equally to this work

View larger version (69K): [in a new window]   Fig. 1. Tsg and Xolloid ventralize the Xenopus embryo. (A) Uninjected stage 18 control embryos stained for krox20 and otx2. (B) Embryos microinjected at the four-cell stage four times at the animal pole with 100 pg Xolloid, (C) 250 pg mouse Tsg or (D) both mRNAs. Same results were obtained using Xenopus Tsg mRNA (data not shown). For each mRNA combination at least 25 embryos were analyzed. (E-H) LiCl-treated embryos. (E) Radially dorsalized LiCl-treated embryo (n=40; dorsoanterior index, DAI=9.5); (F) embryo microinjected into a single blastomere of the marginal zone at the 16-cell stage with 200 pg Xolloid (26% with trunk/tail structures, n=23, DAI=8); (G) 500 pg Xenopus Tsg (32%, n=33, DAI=8.1); or (H) both mRNAs (51%, n=27, DAI=7). Lineage tracing with lacZ and Red-Gal shows that the cells injected with Xenopus Tsg or Xolloid mRNA contributed mostly to ventroposterior mesoderm in the rescued tail region.

View larger version (70K): [in a new window]   Fig. 2. Tsg promotes the degradation of endogenous Chordin fragments. (A) Schematic representation of the cleavage sites of Xolloid in Chordin (arrows). The regions of Chordin protein used to generate the anti-N-Chd and anti-I-Chd antibodies are indicated. (B-D) Embryos were injected marginally into each blastomere at the four-cell stage with Xenopus Tsg (2 ng total, lane 2), Xolloid (0.8 ng total, lane 4) or both mRNAs (lane 3). Dorsal marginal zones (DMZ, lanes 1-4) or ventral marginal zones (VMZ, lane 5) were explanted at early gastrula (stage 10), cells were dissociated and the Chordin protein secreted during 3 hours analyzed in western blots using anti-I-Chd (B) or anti-N-Chd (C) antibodies. (D) Loading control showing a protein that crossreacts with the secondary antibody. (E) DMZs isolated at stage 11 and incubated for 12 hours at room temperature show the canonical Chordin degradation fragments but no additional products. (F) Proteins secreted by animal caps from uninjected (lane 1) and chordin-injected embryos (lane 2) were detected by anti-N-Chd immunoblot. Xenopus embryos were injected into the animal pole with 200 pg of chordin mRNA, ectodermal explants isolated at blastula stage, and dissociated cells incubated for 12 hours at room temperature. (G) Western blot analysis of Xenopus Chordin protein probed with anti-N-Chd or anti-I-Chd after digestion for 10 hours at room temperature with control medium (lane 1), Xolloid (lane 2), and Xolloid and Xenopus Tsg (lane 3). Note that the pattern obtained for Chordin digestion in vivo (E,F) is the same one as obtained after in vitro digestion of Chordin (G), and that additional proteolytic fragments were not observed in the embryo.

View larger version (86K): [in a new window]   Fig. 3. The ventralizing activity of Tsg is dependent on endogenous Xolloid. (A) Albino embryos were microinjected once into an animal cell with 500 pg Xenopus Tsg and lacZ mRNA at the 32-cell stage and krox20 in situ hybridization performed. Note that krox20 expression is reduced on the injected side. (B) krox20 in situ hybridization of uninjected embryos at neural plate stage. (C) Embryos injected with 250 pg Xenopus Tsg mRNA, (D) 250 pg dominant negative (dn) Xld mRNA or, (E) co-injected with Xenopus Tsg and dnXld mRNAs (n=25 or more for each mRNA combination). All embryos were injected 4 times in the animal pole at the four-cell stage. Similar results were obtained using mouse Tsg mRNA (data not shown). (F) Ventral injection of 5 pg mouse Chd mRNA induces secondary axes; (G) injection of 5 pg mouse Chd and 500 pg mouse Tsg mRNA; (H) injection of 5 pg mouse Chd and 500 pg dnXld mRNA, and (I) 5 pg mouse Chd, 500 pg mouse Tsg and 500 pg dnXld mRNAs. (J,K) Injection of 500 pg of dnXld mRNA(J) and uninjected controls (K). Similar results were obtained using Xenopus Tsg and Xenopus chd mRNA (data not shown). Injection of 5 pg of chd mRNA induced strong secondary axes in 47% of the cases (F); these axes were not seen after chd and Tsg co-injection (G), but in 14-50% of the embryos injected with chd, Tsg and dnXld mRNA, strong secondary axes were rescued (I). Note in J that dnXld was unable to induce secondary axes on its own. (L) Ventral injection of 5 pg Xenopus Chd mRNA induces secondary axes (44%). (M) Co-injection of 500 pg dnTsg mRNA reduced the axis-forming activity of Xenopus Chd (14%). (N) 20 pg Xenopus CR1 mRNA induced weak secondary axes. (O) Co-injection of dnTsg mRNA enhanced the secondary axis phenotype caused by Xenopus CR1. For all injections, at least 35 embryos were analyzed. (P) RT-PCR analysis of animal cap explants injected with the indicated combinations of mRNAs and analyzed at stage 25; total amounts of mRNA injected per embryo were 800 pg Xenopus Tsg, 40 pg Xenopus chd and 1 ng dnXld. NCAM is a pan-neural marker, -Glo (-globin), a ventral mesoderm marker, and EF1 was used as a loading control.

View larger version (36K): [in a new window]   Fig. 4. Binding of Xenopus Tsg to Chordin requires an uncleaved C-terminal Xolloid cleavage site. (A) Schematic representation of the cleavage sites of Xolloid in Chordin (arrows). Fragments of chordin mimicking the products of Xolloid digestion were prepared in 293T cells and designated as Chd-A, Chd-B, Chd-C, Chd-A+B and Chd-B+C. Each of these constructs contains the Xenopus Chordin signal peptide and an N-terminal Flag tag (except for Chd-A+B, which lacks the flag tag). (B) Western blot analysis of Xenopus Tsg-HA (5 nM) bound to the different Chordin fragments (5 nM each) after immunoprecipitation of Chordin in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of 5 nM BMP4. As a loading control, membranes were stripped and probed with anti-Flag (lanes 1 to 8) or anti-I-Chd (lanes 9 to 11). (C) Anti-BMP4 immunoblot analysis of Xenopus Tsg-BMP-Chd ternary complexes after crosslinking with DSS (disuccinimidyl suberate). (D) BMP4 is dislodged from preformed Chd-A/BMP4 complexes by Xenopus Tsg. Chd-A and BMP4 were incubated for 1 hour at room temperature, followed by another hour of incubation in the presence of increasing amounts of Xenopus Tsg, and after that the crosslinker DSS was added. The complexes formed were analyzed by anti-BMP4 immunoblot. Note that Xenopus Tsg dislodges most the BMP4 from Chd-A at equimolar concentrations (lane 5) and that no Chd-A/BMP4/Xenopus Tsg ternary complexes were formed at any concentration.

View larger version (60K): [in a new window]   Fig. 5. Xenopus Tsg functions as a BMP antagonist in the presence of full-length Chordin, but promotes binding of BMP to its receptor in the presence of Chordin fragments. BMP4 was incubated for 1 hour at room temperature with Chordin, CR1, and affinity purified Xenopus Tsg-HA. Subsequently, type I BMP-receptor-Fc fusion protein (R&D Systems) was added, precipitated using protein-A, and analyzed by anti-BMP immunoblot. (A) Xenopus Tsg makes Chordin a better BMP antagonist; the concentration of each component in nM is indicated. (B) Xenopus Tsg restores binding of BMP4 to its cognate receptor in the presence of 20 nM CR1 (lane 3). Note that at high concentrations (100 nM, lane 6) Xenopus Tsg by itself can function as a BMP antagonist in this biochemical assay. (C) The Chd/BMP/Xenopus Tsg ternary complex was digested for 10 hours at room temperature with Xolloid. Lane 3 shows that BMP4 is reactivated and binds to its receptor. (D) Xenopus Tsg does not bind to BMPR-IA. Xenopus Tsg, BMP4 and the BMPRIA-Fc were incubated 1 hour at room temperature before DSS crosslinker was added. The complexes formed were analyzed by anti-BMP4 western blot after protein A immunoprecipitation. The arrow indicates the BMPR-Fc-BMP4 complex. The band at 75 kDa is unspecific as it is also observed in the absence of BMP4 in the reaction (lane 3).

View larger version (19K): [in a new window]   Fig. 6. Model for a biochemical pathway that regulates BMP signaling in the extracellular space. Tsg-BMP complexes can be bound by full-length Chordin to form a ternary complex that is a potent BMP antagonist. Xolloid cleaves Chordin releasing Tsg-BMP binary complexes and Chordin fragments. In the presence of full-length Chordin, the binary complex will re-bind to Chordin, re-forming the ternary complex. After all full-length Chordin is cleaved by Xolloid, however, Tsg is able to dislodge BMP from Chordin and to destabilize the Chordin proteolytic products, displacing the equilibrium. This model explains why Tsg has the dual ability to increase BMP antagonism by full-length Chordin and to promote BMP signaling after Xolloid cleavage.