Lee HX , Mendes FA , Plouhinec JL , De Robertis EM .

HD21502-23 NICHD NIH HHS , Howard Hughes Medical Institute , R01 HD021502-23 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 1. BMP1 CUB domains dorsalize ventral half-embryos independently of Chordin. (A) Diagram of the generic Tolloid family metalloproteinase primary structure, BMP1, and the BMP1 CUB1/2/3 construct. (B) Xenopus laevis embryos were bisected into dorsal and ventral halves at late blastula/early gastrula stage using a surgical blade. (C,C′) Bisected wild-type embryos result in the dorsal half-embryo that self-regulates into a relatively normal embryo and a ventral belly piece lacking all neural structures marked by the pan-neural marker Sox2. (D,D′) BMP1 CUB1/2/3 mRNA (1 ng per embryo injected at the two-cell stage) dorsalizes the ventral half-embryo (n = 86, 96% positive for Sox2). (E,E′) Microinjection of morpholino oligos against Chordin (ChdMO) reduced neural structures in the dorsal half. (F,F′) The dorsalizing effect of BMP1 CUB1/2/3 mRNA in the ventral half-embryo is not affected by Chordin depletion (n = 82, 95% positive for Sox2).
	Figure 2. A fluorometric assay for the Chordinase activity of Tolloids shows that BMP4 is a specific inhibitor of the reaction. (A) A new fluorogenic peptide substrate for Tolloids containing the sequence of the Xenopus Chordin cleavage site. (B) The Chd peptide substrate is efficiently digested by BMP1 enzyme, a reaction that is competitively inhibited by 60 nM full-length Chordin proteins. The Ki (17 nM) is similar to the Km of tolloids for full-length chordin (Lee et al. 2006). (C) BMP4, but not other regulators of D–V patterning, inhibits BMP1 metalloproteinase activity. All proteins were added at 120 nM final concentration.
	Figure 3. BMP4 is a noncompetitive inhibitor of the BMP1 enzyme. (A) BMP4 inhibits BMP1 activity in a dose-dependent manner. (B) BMP4 inhibition cannot be competed by increasing amounts of substrate, as shown by the difference in maximal velocity with or without BMP4. This result is consistent with that expected of a noncompetitive enzyme inhibitor. (C) BMP4 affects the maximal velocity (Vmax), but not the Michaelis constant (Km) of the BMP1 enzyme. (D) Effects of different concentrations of inhibitor at two different substrate concentrations. The Dixon plot shows that BMP4 inhibited BMP1 with an inhibition constant (Ki) of 40 nM. (E) BMP4 does not exhibit cooperativity when inhibiting BMP1, indicating one-to-one binding is required for inhibition of catalytic activity.
	Figure 4. BMP1 CUB domains bind BMP4 growth factor. (A) Diagram of BMP1 and BMP1 CUB1/2 constructs and their mutations. (B) Mimicking an antimorph revertant mutant in BMP1 (DN-BMP1WR) or BMP1 CUB1/2 impairs protein secretion. (C–E) Overexpression of DN-BMP1 dorsalized embryos (note the enlarged head marked by Otx2), but the second site revertant mutation (DN-BMP1WR) had no effect. (F) BMP4 binds to full-length BMP1 on a BIAcore sensor chip. (G) Purified BMP1 CUB1 and CUB2 domains are sufficient for BMP4 binding. (H,I) Embryos injected with BMP1 CUB1/2 RNA were dorsalized, as indicated by the expanded Otx-2 expression domain (n = 83, 81% dorsalized). (J) Dorsalization (anti-BMP) effect was also observed after injection of BMP1 CUB1/2 purified protein into the blastula cavity (n = 85, 82% dorsalized).
	Figure 5. dTld binds BMP4 and BMP7. (A) Diagram of dTld and the dTld CUB1/2 and CUB4/5 constructs. (B) dTld CUB1/2δL mutant is not secreted, and CUB4/5SR mutant has impaired secretion in transfected 293T cells. Multiple bands are presumably due to glycosylation. (C) dTld CUB1/2 binds BMP4 within the physiological range (KD ≈ 17 nM). (D) When BMP7 is placed on the sensor chip, dTld CUB1/2 protein is able to bind to it (KD ≈ 26 nM). (E,F) Both dTld CUB4/5 and CUB4/5SR bind BMP4 with similar KD. (G–J) dTld CUB1/2 (n = 81, 78% dorsalized) and CUB4/5 (n = 79, 77% dorsalized) have anti-BMP (dorsalizing) activity in a Xenopus assay, while dTld CUB4/5SR did not show any anti-BMP activity (n = 52).
	Figure 6. Mathematical modeling of the effects of the BMP negative feedback loop on BMP1/Tolloid (Tld) activity. (A) Partial differential equation describing the temporal evolution of free Tld concentration. The terms accounting for the inhibition of Tld by BMP (reaction 10 in Supplemental Fig. 1) are boxed. The reactions involving Tld and their corresponding parameters are described in Supplemental Figure 2. (B) Schematic diagram of the negative feedback loop between Tld, BMP, and Chd, which regulates the D–V gradient of pSmad1 activity. (C,D) Effect of BMP negative feedback on the temporal evolution of free Tld concentration profile. D and V indicate the position of the dorsal and ventral sides. Each concentration profile is labeled from t0 (0 h) to t4 (2 h) to indicate their evolution over time. The arrows indicate the final level of free Tld on the ventral side without (C) or with (D) BMP4 feedback. Note that the model predicts that free (active) Tld is low in the dorsal midline due to the large amounts of Chd substrate secreted by the dorsal side (Supplemental Fig. 3).
	Figure 7. Enzymatic regulation of Xenopus D–V patterning. This diagram depicts the extracellular network of biochemical protein–protein interactions that establishes the embryonic D–V axis through protein–protein interactions (black arrows), BMP-dependent transcription (blue arrows), and the flux of Chordin/BMP complexes from more dorsal regions to the ventral side, where it is bound by CV2 (red arrows). Tolloid and CV2 act as sinks for Chordin in the ventral side, where BMPs made in more dorsal regions can be released by Tolloid to reach peak signaling levels. The system is self-regulating because transcription of dorsal genes is repressed by BMP signals, while ventral genes are under the opposite transcriptional regulation (Reversade and De Robertis 2005; Lee et al. 2006; Ambrosio et al. 2008; Ben-Zvi et al. 2008). The two new reactions reported in this study are the inhibitory black arrows from BMP4/7 to Tolloid in the ventral side (enzyme activity inhibition) and from Tolloid to BMP4/7 (inhibition through binding and sequestration of the growth factor). Other important regulators of D–V patterning, such as Noggin and Follistatin (Khokha et al. 2005), are not included in this simplified model.
	Lee et al. Supplemental Figure 1. Alignment of CUB domain protein sequences. CUB domains 1 and 2 from Xenopus laevis Xolloid-related, Drosophila melanogaster Tolloid, and human BMP1 were aligned using the Multalin algorithm (bioinfo.genotoul.fr/multalin/multalin.html). Red denotes high-consensus amino acids. Drosophila second-site mutations that suppress tolloid antimorphs are shown.
	Lee et al. Supplemental Figure 2. The 14 Biochemical Reactions Included in the Mathematical Model. For each reaction the name and value of its biochemical parameters are shown. We estimated the D-V circumference of the Xenopus gastrula embryo to be 4400 μm and Chordin to be expressed in the dorsal 30% of the embryo (700 μm). BMP4 and Tld/BMP1 were assumed to be expressed uniformly in the early embryo. The diffusion constant for Chd (DChd), BMP (DBMP), and ChdBMP (DChdBMP) was set to D=102 μm2.s-1, and to 0 μm2.s-1 for the other molecular species; relevant references for these parameters are listed in the figure. The initial concentrations of the different components were set between 0.3 and 1.0 nM for Tld, between 5 and 10 nM for the BMP receptor complex (BMPR), and at 0 nM for the rest. The concentrations of each molecular species were allowed to evolve for 2 hours, which represents the time taken by the Xenopus embryo to develop from late blastula to early gastrula.
	Lee et al. Supplemental Figure 3. Free Tld Levels Decrease on the Dorsal Side as the Concentration of its Substrate Chordin Increases. Free Tld decreases as free Chd increases, because Chordin binds to the enzyme that normally degrades it. Our mathematical modeling unexpectedly revealed that a gradient of Tld activity is formed by the action of free Chordin, which limits the availability of free Tld for other substrates such as Chordin/BMP4 complexes. Vertebrate BMP1/tolloids digest free Chordin protein (Piccolo et al. 1997; Wardle et al. 1999), although cleavage is more efficient in the presence of BMP4 (our unpublished observations). Drosophila Tolloid enzyme cleaves Sog complexed with Dpp efficiently, but uncomplexed Sog very inefficiently or not at all (Marqués et al. 1997). It is not known at present whether Tolloid would bind to free Sog in Drosophila embryos, but if it did it would act as an even stronger competitive inhibitor than its vertebrate counterpart because it would not be cleaved efficiently. The curves labeled from t0 to t4 indicate the evolution over time of the free Tld and free Chd gradients. Note that the final level of free Tld on the ventral side (arrows) differs without or with BMP4 feedback inhibition, but that the levels of free Tld or Chd on the dorsal midline are not affected.

Ambrosio, Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. 2008, Pubmed, Xenbase

Ambrosio, Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. 2008, Pubmed , Xenbase
Ball, Forging patterns and making waves from biology to geology: a commentary on Turing (1952) 'The chemical basis of morphogenesis'. 2015, Pubmed
Ben-Zvi, Scaling of the BMP activation gradient in Xenopus embryos. 2008, Pubmed , Xenbase
Blitz, Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation. 2000, Pubmed , Xenbase
Bork, The CUB domain. A widespread module in developmentally regulated proteins. 1993, Pubmed
Canty, A complete domain structure of Drosophila tolloid is required for cleavage of short gastrulation. 2006, Pubmed
Celeste, Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. 1990, Pubmed
Childs, Two domains of the tolloid protein contribute to its unusual genetic interaction with decapentaplegic. 1994, Pubmed
Dale, Xolloid-related: a novel BMP1/Tolloid-related metalloprotease is expressed during early Xenopus development. 2002, Pubmed , Xenbase
De Robertis, Evo-devo: variations on ancestral themes. 2008, Pubmed
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
Eldar, Robustness of the BMP morphogen gradient in Drosophila embryonic patterning. 2002, Pubmed
Ferguson, Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. 1992, Pubmed
Finelli, Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homolog. 1994, Pubmed
Geach, Molecular determinants of Xolloid action in vivo. 2008, Pubmed , Xenbase
Hartigan, Bone morphogenetic protein-1 (BMP-1). Identification of the minimal domain structure for procollagen C-proteinase activity. 2003, Pubmed
Hojima, Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization. 1985, Pubmed
Hopkins, The bone morphogenetic protein 1/Tolloid-like metalloproteinases. 2007, Pubmed
Inomata, Robust stability of the embryonic axial pattern requires a secreted scaffold for chordin degradation. 2008, Pubmed , Xenbase
Jasuja, bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis. 2006, Pubmed
Jasuja, Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4. 2007, Pubmed
Jürgens, Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : II. Zygotic loci on the third chromosome. 1984, Pubmed
Khokha, Depletion of three BMP antagonists from Spemann's organizer leads to a catastrophic loss of dorsal structures. 2005, Pubmed , Xenbase
Larraín, BMP-binding modules in chordin: a model for signalling regulation in the extracellular space. 2000, Pubmed , Xenbase
Lee, Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. 2006, Pubmed , Xenbase
Little, Extracellular modulation of BMP activity in patterning the dorsoventral axis. 2006, Pubmed , Xenbase
Mao, Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. 2002, Pubmed
Marqués, Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. 1997, Pubmed , Xenbase
Meinhardt, Pattern formation by local self-activation and lateral inhibition. 2000, Pubmed
Mizutani, Formation of the BMP activity gradient in the Drosophila embryo. 2005, Pubmed
Muraoka, Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein. 2006, Pubmed
O'Connor, Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing. 2006, Pubmed
Oelgeschläger, Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos. 2003, Pubmed , Xenbase
Petropoulou, Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1). 2005, Pubmed
Piccolo, The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. 1999, Pubmed , Xenbase
Piccolo, Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. 1997, Pubmed , Xenbase
Plouhinec, Systems biology of the self-regulating morphogenetic gradient of the Xenopus gastrula. 2009, Pubmed , Xenbase
Reversade, Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field. 2005, Pubmed , Xenbase
Sampath, Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. 1981, Pubmed
Serpe, The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila. 2008, Pubmed
Shimmi, Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. 2005, Pubmed
Siva, Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model. 2008, Pubmed
Sundin, Extreme hyperopia is the result of null mutations in MFRP, which encodes a Frizzled-related protein. 2005, Pubmed
Takahara, Type I procollagen COOH-terminal proteinase enhancer protein: identification, primary structure, and chromosomal localization of the cognate human gene (PCOLCE). 1994, Pubmed
Tucker, The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis. 2008, Pubmed
Umulis, Robust, bistable patterning of the dorsal surface of the Drosophila embryo. 2006, Pubmed
Urist, Bone: formation by autoinduction. 1965, Pubmed
Wang, Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning. 2005, Pubmed
Wardle, Bone morphogenetic protein 1 regulates dorsal-ventral patterning in early Xenopus embryos by degrading chordin, a BMP4 antagonist. 1999, Pubmed , Xenbase
Wozney, Novel regulators of bone formation: molecular clones and activities. 1988, Pubmed
Zhang, von Willebrand factor type C domain-containing proteins regulate bone morphogenetic protein signaling through different recognition mechanisms. 2007, Pubmed