Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.


Introduction
The analysis of whole-genome sequences has revealed that most signaling systems consist of multiple ligands that converge on a relatively small set of receptors and pathwayspecific transcription factors. In the case of human transforming growth factor-b (TGFb) signaling, 42 TGFbs converge on seven type I receptors, five type II receptors, and two classes of Smad signal transducers (reviewed in Shi and Massague 2003). This convergence has raised the question of how ligand diversity and signaling specificity among different signals can be achieved. If different TGFbs activate the same receptors, it is unclear how these ligands can vary in their function (diversity) or how a given signal can have a unique role (specificity). Biochemical studies have suggested that ligand diversity can be attained by differential stability and receptor affinity, leading to differences in signaling strength (reviewed in Piek et al. 1999;Shi and Massague 2003). An additional source of ligand variability stems from differential ligand movement through a field of cells, rendering related signals either short-or long-range (Chen and Schier 2001). Finally, specificity and diversity can also be determined by ligand-specific cofactors or inhibitors (Piek et al. 1999;Shi and Massague 2003). A prominent example involves epidermal growth factor-Cripto/FRL-1/Cryptic (EGF-CFC) coreceptors and the TGFbs Activin, Nodal, and Vg1/GDF1 (growth and differentiation factor-1). In this case, differential dependence on a coreceptor leads to ligand diversity and signaling specificity (reviewed in Schier 2003).
Members of the Nodal, Activin, and Vg1/GDF1 subfamilies display similar activities and are potent mesendoderm inducers in vertebrates (reviewed in Schier and Shen 2000). Genetic and biochemical studies have shown that EGF-CFC proteins are essential for signaling by Nodal and Vg1/GDF1 Reissmann et al. 2001;Yeo and Whitman 2001;Bianco et al. 2002;Sakuma et al. 2002;Yan et al. 2002;Cheng et al. 2003). EGF-CFC proteins are extracellular glycosylphosphatidylinositol (GPI)-linked factors and include One-eyed pinhead (Oep) in zebrafish and mammalian Cripto and Cryptic (reviewed in Shen and Schier 2000;Minchiotti et al. 2002;Schier 2003). Genetic studies in zebrafish and mouse have shown that EGF-CFC proteins and Nodal are required for mesoderm and endoderm induction (Conlon et al. 1991(Conlon et al. , 1994Zhou et al. 1993;Ding et al. 1998;Feldman et al. 1998;Gritsman et al. 1999). For example, zebrafish embryos lacking both the maternal and zygotic contribution of Oep (MZoep) lack all endoderm and most mesoderm, similar to the double mutants for the zebrafish Nodal-related genes cyclops and squint (sqt) (Feldman et al. 1998;Gritsman et al. 1999). Moreover, Nodal and Vg1/GDF1 are inactive in MZoep mutants Cheng et al. 2003). During later stages of development, Oep, Cryptic, Nodal, and GDF1 are required for proper left-right axis formation (Gaio et al. 1999;Yan et al. 1999;Bamford et al. 2000;Rankin et al. 2000;Brennan et al. 2002;Long et al. 2003).
Biochemically, EGF-CFC proteins can act as coreceptors for Nodal and Vg1/GDF1 to bind and activate the type I Activin receptor Alk4 and the type II Activin receptor ActRIIB (Reissmann et al. 2001;Yeo and Whitman 2001;Sakuma et al. 2002;Yan et al. 2002;Bianco et al. 2002;Cheng et al. 2003). In the absence of EGF-CFC proteins, these TGFbs cannot form a complex with Activin receptors. Strikingly, Activin utilizes the same receptors as Nodal and Vg1/GDF1, but does not require EGF-CFC coreceptors (Mathews and Vale 1991;Attisano et al. 1992Attisano et al. , 1996Mathews et al. 1992;ten Dijke et al. 1994;Chang et al. 1997). For instance, Activin can signal in MZoep mutants . This ligand diversity between Activin and Nodal or Vg1/GDF1 raises the question of which sequences confer coreceptor dependence or independence. Activin, Nodal, and Vg1/GDF1 are highly related and are thought to acquire very similar folds. Like other TGFb ligands, Activin has four major structural features: a bstranded Finger 1, an a-helical Heel, a b-stranded Finger 2, and three conserved disulfide bonds that form a cysteine knot (Shi and Massague 2003;Thompson et al. 2003). Sequence comparisons indicate that the highest divergence among Activin, Nodal, and Vg1/GDF1 is in the N-terminal segment of Finger 1, the central a-helix, and the loop of Finger 2 with approximately 10%, approximately 15%, and approximately 25% sequence identity, respectively. These regions are potential candidates to determine the specificity of receptor-coreceptor-ligand interactions.
In addition to coreceptors, antagonists represent another class of extracellular factors that control ligand access to receptors (reviewed in Piek et al. 1999;Freeman 2000;De Robertis et al. 2001;Shi and Massague 2003). For example, the divergent TGFb class of Lefty proteins antagonizes Nodal signaling (reviewed in Hamada et al. 2002;Schier 2003). Unlike other TGFbs, Lefty proteins may function as monomers due to the lack of a cysteine residue involved in dimerization (Meno et al. 1996;Thisse and Thisse 1999;Sakuma et al. 2002). Lefty overexpression in zebrafish induces phenotypes identical to cyclops;sqt double mutants and MZoep mutants (Bisgrove et al. 1999;Meno et al. 1999;Thisse and Thisse 1999). Furthermore, the loss of Lefty activity leads to enhanced Nodal signaling during mesoderm induction and left-right axis determination (Meno et al. 1999(Meno et al. , 2001Agathon et al. 2001;Branford and Yost 2002;Chen and Schier 2002;Feldman et al. 2002). Although it has not been determined whether Lefty directly blocks Vg1/GDF1 signaling (Branford et al. 2000), it has been proposed that Lefty inhibits signaling by Activin. Misexpression of Activin or ActRIIB can overcome the inhibitory effects of Lefty (Meno et al. 1999;Thisse and Thisse 1999;Cheng et al. 2000;Tanegashima et al. 2000;Sakuma et al. 2002). Hence, some members of the Lefty family have been called Antivins for their anti-Activin properties (Thisse and Thisse 1999;Cheng et al. 2000;Ishimaru et al. 2000;Tanegashima et al. 2000). However, it has been elusive how Lefty functions at the molecular level.
Here we present genetic and biochemical studies in zebrafish and Xenopus that indicate that Lefty is an in vivo antagonist of EGF-CFC coreceptors. We find that Lefty can antagonize signaling by the coreceptor-dependent ligands Nodal and Vg1/GDF1, but not Activin. Lefty genetically interacts with Cripto and Oep and competes with Nodal for binding to Cripto, representing a novel mechanism for antagonizing TGFb signaling. We identify a short region in Finger 2 of Activin, Nodal, and Vg1 that determines EGF-CFC coreceptor-dependent or coreceptor-independent signaling and governs susceptibility to Lefty. These results indicate that subtle sequence variations in TGFb ligands can dramatically expand signaling diversity by determining interactions with coreceptors and their antagonists.

Results
Lefty Antagonizes Nodal and Vg1 Signaling, but Not Activin Signaling TGFb ligands that activate Activin receptors can be categorized into two classes. The Activin class activates Activin receptors in an EGF-CFC coreceptor-independent manner, whereas the Nodal and Vg1/GDF1 classes require EGF-CFC proteins for receptor activation Cheng et al. 2003). To determine whether these classes are also differentially susceptible to inhibition by the TGFb antagonist Lefty, we coexpressed zebrafish Lefty1 with Xenopus ActivinbB, Xenopus ActivinbA, Sqt (a zebrafish Nodal-related protein), or zebrafish Vg1 (a chimeric molecule containing the Xenopus ActivinbB prodomain fused to the mature domain of zebrafish Vg1) in zebrafish embryos (Smith et al. 1990; Thomsen et al. 1990;van den Eijnden-Van Raaij et al. 1990;Helde and Grunwald 1993;Erter et al. 1998;Feldman et al. 1998;Thisse and Thisse 1999). As a readout for active signaling, we analyzed the ectopic induction of the Nodal downstream gene goosecoid (gsc). ActivinbB, ActivinbA, Sqt, and Vg1-induced ectopic gsc expression in wild-type embryos ( Figure 1D, 1G, 1J, and 1M; Gritsman et al. 1999;Cheng et al. 2003). Coexpression of Lefty1 efficiently inhibited gsc induction by Sqt ( Figure 1K and 1L; Bisgrove et al. 1999;Meno et al. 1999;Thisse et al. 2000) and Vg1 ( Figure 1N and 1O), but not ActivinbB or ActivinbA ( Figure 1E, 1F, 1H, and 1I). To examine whether Lefty1 can antagonize the induction of a gene that responds to very low levels of Activin signaling, we titrated ActivinbB levels so that no tail (ntl; also known as brachyury/T) expression was only weakly induced (see arrowhead in Figure 1A). The coexpression of Lefty1 did not inhibit ntl induction by ActivinbB ( Figure 1B and 1C), but inhibited the dorsal margin expression of ntl that is dependent on endogenous Nodal signaling (see asterisks in Figure 1B and 1C;Feldman et al. 1998). In a more quantitative assay, we overexpressed Lefty1, ActivinbB, Sqt, and Vg1 in zebrafish embryos in the presence of the luciferase reporter A3-luc, which contains FoxH1/P-Smad2 response elements (Chen et al. 1996). Consistent with the gsc and ntl induction assays, Sqt and Vg1 signaling, but not ActivinbB signaling, is inhibited by Lefty1 ( Figure 1P). These results indicate that Lefty1 efficiently antagonizes Nodal and Vg1/GDF1 signaling, but not Activin signaling.

EGF-CFC Proteins Genetically Interact with Lefty
The molecular mechanism of Lefty action has been unresolved. Lefty seems to act upstream of the Activin receptors, as Lefty cannot block signaling from ligandindependent constitutively activated receptors (Thisse and Thisse 1999). Our finding that Lefty blocks Nodal and Vg1 signaling, but not Activin signaling, suggests that Lefty blocks extracellular components specific to the Nodal and Vg1 pathways. The only such factors identified to date are the EGF-CFC coreceptors. We therefore examined whether the EGF-CFC genes zebrafish oep and mouse Cripto genetically interact with Lefty1. Overexpression of Lefty1 in wild-type zebrafish resulted in embryos lacking head and trunk mesendoderm due to inhibition of endogenous Nodal signaling ( Figure 2A1 and 2A9; Bisgrove et al. 1999;Meno et al. 1999;Thisse and Thisse 1999). Coexpression of Cripto or Oep partially suppressed Lefty-induced defects (Figure 2B1-2B3; data not shown), restoring trunk and head mesoderm, including the notochord, and resulting in the separation of the eye field into two eyes. These results indicate an antagonistic relationship between EGF-CFC coreceptors and Leftys.
In reverse experiments, Cripto efficiently coimmunoprecipitated Lefty1 ( Figure 3B). Since Sqt and Lefty1 can both bind to Cripto ( Figure 3C; Reissmann et al. 2001;Yeo and Whitman 2001;Bianco et al. 2002;Sakuma et al. 2002;Yan et al. 2002), these two ligands might compete for binding to Cripto. Indeed, the coexpression of Lefty1 led to decreased interactions between Cripto and Sqt ( Figure 3C). To determine whether Cripto can directly interact with Lefty, we immunoprecipitated purified mouse Lefty1 protein (mLefty1) in the presence of either purified mouse Cripto protein or a control cysteine-rich protein, mouse vascular endothelial growth factor-D (VEGF-D). mLefty1 protein directly interacted with Cripto, but not with VEGF-D. Together, these results suggest that Lefty inhibits Nodal signaling by associating with Cripto and blocking it from interacting with Nodal.

Activin Loop-b8 Region Confers EGF-CFC Coreceptor Independence to Sqt
The finding that TGFb ligands that activate Activin receptors can be grouped into a EGF-CFC coreceptordependent class that is susceptible to inhibition by Lefty (Nodal and Vg1) and a class that is independent of EGF-CFC proteins and resistant to Lefty (Activin) prompted us to examine which sequences underlie this ligand diversity. We therefore generated chimeras between Activins (EGF-CFCindependent) and Sqt or Vg1 (EGF-CFC-dependent) (Figures Note the dorsal expression of ntl (asterisks)-that is, dependent on endogenous Nodal signaling-is inhibited by Lefty1 in these embryos (B and C). Ectopic gsc expression in activin bB and activin bA mRNA-injected embryos was not inhibited by Lefty1 (E and F and H and I,respectively). In contrast, ectopic gsc expression in sqt and Vg1 mRNA-injected embryos was inhibited by both levels of Lefty expression (K and L and N and O, respectively). Wild-type embryos (P) were injected with 10 pg (low) and 20 pg (high) of activin bB/HA, 75 pg of sqt, or 200 pg of Vg1 mRNA. Embryos were further double-injected with 500 pg of LacZ mRNA, 100 pg of lefty1, and 400 pg of LacZ mRNAs, or 500 pg of lefty1 mRNA. Smad2 pathway activation was measured by an Activin response element luciferase reporter, A3-luc. Values are folds over wild-type control injected with 500 pg of LacZ mRNA and A3-luc reporter. An asterisk indicates a significant difference from the level of activation with ligand and LacZ expression alone (Student's t-test, p , 0.05). DOI: 10.1371/journal.pbio.0020030.g001 4 and 5). As a readout for active signaling, we injected mRNAs encoding these chimeric ligands into wild-type and MZoep zebrafish embryos and analyzed the ectopic induction of the downstream genes ntl and gsc. Sqt, Vg1, and Activins induced these genes in wild-type embryos, allowing us to determine which chimeric ligands were active. Activins, but not Sqt or Vg1, were active in MZoep mutants, allowing us to test which sequences conferred EGF-CFC coreceptor dependence or independence.
Initially, swaps of the Finger 1, Heel, or Finger 2 domains of Sqt and ActivinbB were generated. As shown in Figure 4, the Finger 2 region of ActivinbB contains sequence determinants RNAs (1 ng each) encoding ActRIIB(KR)/ Myc, Alk4(KR)/Flag, Cripto/Flag, Lefty1/ HA, or Sqt/HA were injected into Xenopus embryos. After chemical cross-linking, lysates were immunoprecipitated for either Lefty1/HA or Sqt/HA (A) with anti-HA antibody, or ActRIIB(KR)/Myc, Alk4(KR)/HA, Cripto/Flag (B) with, respectively, anti-Myc, anti-HA, or anti-Flag antibodies. Note that Lefty1 specifically interacts with Cripto (A and B), and these Lefty/Cripto complexes do not contain Alk4 (A). Moreover, processed Lefty1 binds much more efficiently to Cripto than full-length Lefty1 precursor (B). In contrast, Sqt can bind to ActRIIB, Alk4, and Cripto (A). The 55 kDa protein marker in (B) is estimated based on molecular weight markers. (C) Lefty1 competes with Nodal for binding to Cripto. RNAs encoding Sqt/HA (1 ng), Cripto/Flag (100 pg), or Lefty1 (2 ng) were injected and anti-Flag antibody was used to immunopreciptate Cripto/Flag. (D) mLefty1 binds directly to Cripto. Purified mouse Lefty1 protein (mLefty1; 10 lg/ml) was mixed with either soluble purified Cripto/His protein (5 lg/ml) or purified control VEGF-D/His protein (5 lg/ml). After chemical cross-linking, mLefty1 was immunoprecipitated with anti-mLefty1 antibody. mLefty1 associates with Cripto, but not with control VEGF-D. Proteins in the coimmunoprecipitates and total extracts were probed in Western blot analysis with the indicated antibodies: ActRIIB(KR)/Myc   that conferred EGF-CFC-independent activity on chimeric ligands. Chimeric SSA, ASA, and SAA that contain the Finger 2 region of ActivinbB were active in both wild-type and MZoep embryos (Figure 4). To further delineate this region, we generated additional chimeras ( Figure 5B). Short stretches of full-length Sqt were replaced by the corresponding region of ActivinbB, including the b6b7, loop, b8, or b9 subregions (Figure 5A and 5B; data not shown). Analysis of these chimeras revealed that the 14 amino acids encoding the loop and b8 region of ActivinbB (Sqt ActbB[loopb8] ; the bracketed region in superscript denotes substituted domains) were sufficient to confer EGF-CFC independence. Further dissection of this region into loop alone (Sqt ActbB [loop] ) or b8 alone (Sqt ActbB[b8] ) yielded no or much weaker activity in MZoep mutants as compared with wild-type embryos ( Figure  5B and 5C). These results suggest that a 14 amino acid region in Activin is sufficient to confer EGF-CFC independence when placed into Sqt.

Activin Loop-b8 Region Confers EGF-CFC Coreceptor Independence to Vg1
To determine whether the loop-b8 region has a wider role in conferring coreceptor independence, we generated additional chimeras using ActivinbA (another EGF-CFC-independent ligand) and Vg1. Sqt ActbA[loopb8] (full-length Sqt with an ActivinbA loop-b8 region) and Vg1 ActbB[loopb8] (Vg1 with an ActivinbB loop-b8 region) both induced gsc expression in MZoep mutants with similar efficiencies as in wild-type embryos ( Figure 5D). These results were also corroborated using the A3-luc reporter assay ( Figure 5E) and suggest that the loop-b8 region has a general role in conferring EGF-CFC coreceptor independence.

Activin Loop-b8 Region Confers Binding to Activin Receptors in the Absence of EGF-CFC Coreceptors
Sqt ActbB[loopb8] can signal in an EGF-CFC-independent manner in vivo, suggesting that this chimeric protein might bind to ActRIIB and Alk4 receptors in the absence of EGF-CFC coreceptors. To test this idea, we coexpressed and immunoprecipitated epitope-tagged ligands (ActivinbB/HA, Sqt/HA, Sqt ActbB[loopb8] /HA), receptors (ActRIIB[KR]/Myc and Alk4[KR]/Flag), and a coreceptor (Cripto/Flag) in Xenopus embryos (Yeo and Whitman 2001;Cheng et al. 2003). We found that Sqt binding to the ActRIIB and Alk4 receptor complex required Cripto ( Figure 5F). In contrast, ActivinbB and Sqt ActbB[loopb8] can bind to Activin receptors in the absence of Cripto. Moreover, Cripto did not significantly increase Sqt ActbB[loopb8] ligand-receptor complex formation. These results indicate that the loop-b8 region is a determinant of TGFb ligand binding to Activin receptors independent of EGF-CFC coreceptors.

Multiple Residues in the Loop-b8 Region Contribute to Coreceptor Independence
An alignment of the loop-b8 region of EGF-CFC-dependent and EGF-CFC-independent TGFbs ( Figure 5A) reveals the Schematic is not drawn to scale. The letters in these three-lettered (XXX) chimeras represent the Finger 1, Heel, and Finger 2, respectively. S denotes Squint; A denotes ActivinbB. Synthetic mRNAs (200 pg) encoding chimeras were injected into wild-type and MZoep embryos. gsc and ntl mRNA expression is shown at shield stage; animal pole views are dorsal to the right. gsc is expressed in the dorsal organizer (shield) in wild-type embryos, but is absent in MZoep mutants. ntl is expressed around the entire margin in wild-type embryos, but the dorsal margin expression is lost in MZoep mutants. The presence of the ActivinbB prodomain and epitope tag does not alter the specificity or functionality of wild-type ActivinbB (AAA) or Sqt (SSS). AAA can induce ectopic gsc and ntl expression in both wild-type and MZoep embryos. In contrast, SSS can induce ectopic gsc and ntl expression in only wild-type embryos. Similar to ActivinbB, chimeras SSA, SAS, ASA, and SAA can induce ectopic gsc and ntl expression in both wild-type and MZoep embryos. Chimeras ASS and AAS are inactive in both wild-type and MZoep embryos. Western blot analysis indicated that all chimeric constructs produce stable ligands (data not shown). DOI: 10.1371/journal.pbio.0020030.g004 presence of several residues unique to Activins. These include (i) a Lys102-X-Asp104 motif (numbering from ActivinbA) that forms a significant binding interface with the type II receptor ActRII (Wuytens et al. 1999;Greenwald et al. 2003;Thompson et al. 2003); (ii) Gln/Pro106 and Asn107, which contribute to the dimerization interface responsible for conformational arrangement (Thompson et al. 2003);and (iii) an Asn insertion at position 99. We therefore mutated the corresponding residues in Sqt, individually or in combination, to the ActivinbB sequence and tested them in the gsc/ntl induction assay ( Figure 6). All constructs were active in wildtype embryos. The Sqt3 and Sqt5 constructs containing the Lys102-X-Asp104 motif and Asn99 insertion showed weak expansion of ntl expression animally and dorsally in MZoep mutants. The incorporation of Pro106-Asn107 (Sqt2, Sqt4, and Sqt5) in Sqt did not enhance activity in MZoep mutants. These results suggest that multiple residues contribute to coreceptor independence, with the type II receptor-binding interface being an essential determinant.

The Loop-b8 Region in Sqt Is Inhibitory
The results described above identified the loop-b8 region of Activin as a region that confers coreceptor-independent signaling to ligands that are normally EGF-CFC-dependent. In a reverse set of experiments, we asked which regions confer dependence on EGF-CFC coreceptors. To identify domains that confer EGF-CFC dependence, chimeras between ActivinbB and Sqt (see Figures 4 and 7) were analyzed for their inability to signal in MZoep mutants. Chimeras containing the Sqt Finger 2 domain (AAS and ASS; see Figure  4) or only the Sqt loop-b8 region (Act Sqt[loopb8] ; Figure 7) were inactive in both wild-type and MZoep embryos. Western blot analysis demonstrated that these chimeras generate stable ligands (data not shown). The addition of Finger 1 in SAS or Act Sqt[Finger1-loopb8] relieved the inhibitory effect of the loop-b8 region of Sqt in wild-type embryos (Figure 7). These chimeras were inactive in MZoep mutants. These results indicate that the loop-b8 region in Sqt acts as an inhibitory domain and that Finger 1 relieves this inhibition by conferring dependence on EGF-CFC coreceptors.

Specificity of Antagonism by Lefty Is Determined by EGF-CFC Coreceptor Dependence
Our genetic and biochemical studies suggested that Lefty blocks Nodal and Vg1 signaling via EGF-CFC coreceptors. In contrast, the coreceptor-independent signaling by Activins cannot be blocked by Lefty. This finding predicts that the EGF-CFC-independent chimeric ligands Sqt ActbB[loopb8] , Sqt ActbA[loopb8] , and Vg1 ActbB[loopb8] should also be resistant to Lefty. Conversely, the coreceptor-dependent chimera Act  should be suspectible to inhibition by Lefty. To test this hypothesis, we coexpressed chimeric ligands and Lefty1 and analyzed gsc expression and A3-luc reporter induction ( Figure 8A-8M). As predicted, Lefty1 did not inhibit signaling by Sqt ActbB[loopb8] ( Figure 8B and 8C), Sqt ActbA[loopb8] ( Figure 8E and 8F), or Vg1 ActbB[loopb8] ( Figure  8H and 8I), but antagonized Act Sqt[Finger1-loopb8] ( Figure 8K and 8L). These results indicate that the incorporation of the Activin loop-b8 region into Nodal and Vg1 can render these ligands EGF-CFC-independent and therefore resistant to Lefty.

Lefty Antagonizes EGF-CFC Coreceptors
Lefty molecules are key regulators of mesendoderm development and left-right axis determination, but the molecular basis of Lefty-mediated antagonism of Activin-like pathways has been elusive (Hamada et al. 2002;Schier 2003). Our genetic and biochemical studies provide three lines of evidence that Lefty blocks EGF-CFC coreceptors. First, Lefty only inhibits EGF-CFC-dependent TGFb ligands such as Nodal and Vg1, but not EGF-CFC-independent ligands such as Activins. A striking example of this coreceptor-specific interaction is the finding that changing only 14 amino acids in Nodal or Vg1 to the corresponding residues in Activins renders the resulting TGFb ligands independent of EGF-CFC coreceptors and resistant to Lefty. Second, the EGF-CFC proteins mouse Cripto and zebrafish Oep can partially suppress the effects of Lefty overexpression in zebrafish. Third, Leftys can bind to EGF-CFC coreceptors and block the coreceptors from interacting with Nodal. Furthermore, Lefty/ EGF-CFC complexes seem to exclude interactions with type I and type II Activin receptors. Taken together, these results indicate that Lefty blocks a subset of TGFb signals by the novel mechanism of blocking pathway-specific coreceptors ( Figure 9A-9D).
The observation that Lefty does not block signaling by Activin seems in apparent contrast to previous studies that led to naming some Lefty family members Antivins, for their anti-Activin properties (Thisse and Thisse 1999;Cheng et al. 2000;Ishimaru et al. 2000;Tanegashima et al. 2000). In particular, it has been found that misexpression of Activin can suppress the defects caused by Lefty misexpression in vivo (Thisse and Thisse 1999). Our results do not undermine this finding, but suggest an alternative explanation. Previous studies have shown that Activin can suppress the loss of EGF-were injected into wild-type and MZoep embryos. Schematic is not drawn to scale. gsc and ntl mRNA expression is at shield stage; animal pole views are dorsal to the right. (B) Sqt ActbB[loopb8b9] and Sqt ActbB[loopb8] can induce gsc and ntl expression in both wild-type and MZoep embryos. (C) Sqt ActbB[b8] can weakly expand ntl expression in MZoep mutants. ntl mRNA expression in MZoep mutants is at shield stage; lateral view. (D) Other TGFbs conform to loop-b8 EGF-CFC-independent determinant. Note that Xenopus ActivinbA can induce ectopic gsc in both wild-type and MZoep embryos. In contrast, Vg1 can only induce gsc in wild-type embryos. Similar to Activins, chimeric Sqt ActbA[loopb8] and Vg1 ActbB[loopb8] can induce ectopic gsc in both wild-type and MZoep embryos. (E) Wild-type and MZoep embryos were injected with 5 pg of activin bB, 100 pg of sqt, 100 pg of Vg1, 125 pg of Sqt ActbB[loopb8] , 250 pg of Sqt ActbA[loopb8] , or 100 pg of Vg1 ActbB[loopb8] mRNA. Smad2 pathway activation was measured by an Activin response element luciferase reporter, A3luc. Luciferase units are relative to wild-type or MZoep control injected with the A3-luc reporter alone. (F) Sqt ActbB[loopb8] can bind to ActRIIB and Alk4 in the absence of EGF-CFC coreceptors. RNAs (1 ng each) encoding ActRIIB ( CFC activity in MZoep mutants Cheng et al. 2003). Analogously, we suggest that the blocking of EGF-CFC activity by Lefty can be bypassed by Activin, because this ligand can activate Activin receptors independent of coreceptors. A similar scenario can also account for the suppression of Lefty gain-of-function phenotypes by misexpression and activation of Activin receptors (Meno et al. 1999;Thisse and Thisse 1999;Sakuma et al. 2002). Hence, Activin and Activin receptors bypass the loss of EGF-CFC coreceptor function that is caused either by mutations in oep or by overexpression of Lefty. Conversely, Lefty cannot block Activin signals and Activin receptors because of their independence from EGF-CFC coreceptors.
Is the block of EGF-CFC coreceptors by Lefty a general and conserved mechanism? Although we have only analyzed a representative subset of these protein families (zebrafish and mouse Lefty1; zebrafish Oep; mouse Cripto; zebrafish Sqt), previous studies have suggested that heterologous Nodal, Lefty, and EGF-CFC proteins have similar activities in zebrafish (Schier 2003). For example, mouse Nodal, mouse Lefty2, and mouse Cripto are active in zebrafish, despite less than 30% overall sequence conservation with their zebrafish counterparts (Toyama et al. 1995;Meno et al. 1999;Gritsman et al. 1999). These studies suggest that the molecular mechanisms described here apply to most, if not all, Nodal/ Lefty/EGF-CFC interactions. This does not exclude the possibility that Lefty has additional means of blocking TGFb signaling. First, Leftys might block the processing of Nodals. However, Sqt is processed normally at levels of Lefty that block Nodal signaling (unpublished data). Second, Leftys might bind Nodal signals. This could result in blocking receptor interactions or antagonizing TGFb dimerization. However, Sqt is not bound by Lefty at Lefty levels that block Nodal signaling and lead to complex formation with Cripto (unpublished data). Moreover, a Sqt protein containing the loop-b8 region of Activin is resistant to Lefty, whereas changing only the dimerization residues in this region does not confer resistance. Third, Leftys might interact with additional extracellular factors. Indeed, the overexpression of the extracellular domain of the type II receptor ActRIIB has been shown to suppress Lefty activity (Meno et al. 1999). Although zebrafish Lefty1 does not appear to bind to ActRIIB, it is conceivable that overexpression of soluble ActRIIB might protect EGF-CFC coreceptors or another yet-to-be identified protein from antagonism by Lefty. In addition, overexpression of EGF-CFC proteins in zebrafish does not induce dominant phenotypes . It is therefore possible that an additional factor would be required to completely block Lefty in these experiments. Alternatively, overexpression levels of EGF-CFC coreceptors might not be high enough to block Lefty at blastula stages. It is also possible that coreceptor overexpression might block Nodal signals, because it has been shown that EGF-CFC proteins and Nodals can directly interact. The complex feedback interactions between Lefty and Nodal might also overcome an initial reduction of Lefty activity by increasing Lefty transcription. These considerations and the data presented here therefore suggest that a major, but perhaps not exclusive, role of Leftys is to block a subset of TGFb signals by interaction with EGF-CFC coreceptors.

Implications for the Role of Lefty during Development
Our finding that Leftys can block Vg1 signaling also has important implications for the developmental control of TGFb signaling. Based on previous studies revealing that Lefty proteins inhibit Nodal signaling, the mouse Lefty mutant phenotypes have been interpreted as a consequence of increased or sustained Nodal signaling (Hamada et al. 2002;Schier 2003). For example, loss of mouse Lefty2 has been thought to increase Nodal signaling, resulting in an enlarged primitive streak (Meno et al. 1999). Similarly, the left-right defects observed in mouse Lefty1 and left-side specific Lefty2 (Lefty2 DASE ) mutants have been thought to be caused by inappropriate spread of Nodal signaling (Meno et al. 1998(Meno et al. , 2001. Our finding that Vg1/GDF1 signaling can also be blocked by Lefty suggests a more complex scenario. In particular, GDF1 (the mouse homologue of Vg1) is required for proper left-right axis determination (Rankin et al. 2000). GDF1 mutants appear to have the opposite phenotypes as Lefty1 and Lefty2 DASE mutants. While GDF1 promotes the expression of left-side-specific genes such as Pitx2 on the left, Leftys appear to block Pitx2 expression on the right (Meno et al. 1998(Meno et al. , 2001Rankin et al. 2000). In light of our findings, we suggest that during left-right axis formation, Leftys act as antagonists of not only Nodal, but also GDF1. In this scenario, loss of Lefty1 or Lefty2 would lead to ectopic and sustained GDF1 signaling. This model is particularly attractive when one considers the expression patterns of Lefty1, Lefty2, the EGF-CFC gene Cryptic, Nodal, and GDF1. Lefty 1 and GDF1 are coexpressed in the developing midline (Meno et al. 1996(Meno et al. , 1997Rankin et al. 2000), whereas Lefty2 and Nodal are coexpressed in left-lateral plate mesoderm (Conlon et al. 1994;Meno et al. 1997). Cryptic is expressed in both the lateral plate and midline (Shen et al. 1997). It is therefore conceivable that GDF1 signaling is restricted by Lefty1mediated inhibition of Cryptic in the midline and its progenitors, whereas Nodal signaling is antagonized by Lefty2-mediated block of Cryptic in the lateral plate.
Our results might also have implications for the role of Cripto in tumorigenesis. Cripto is highly overexpressed in human epithelial cancers, such as breast and colon carcinomas (Salomon et al. 2000), and has been implicated in tumor formation (Ciardiello et al. 1991(Ciardiello et al. , 1994Baldassarre et al. 1996;De Luca et al. 2000;Salomon et al. 2000;Adkins et al. 2003). Although the mechanisms by which Cripto acts in these circumstances are unclear, inhibition of Cripto by antisense or antibody blockade can inhibit tumor cell proliferation (Ciardiello et al. 1994;Baldassarre et al. 1996;De Luca et al. 2000;Adkins et al. 2003;reviewed by Shen 2003). Since Lefty is an in vivo antagonist of EGF-CFC activity, it might also serve as a therapeutic agent to block Cripto.

Subtle Sequence Differences Determine the Interaction with Coreceptors and Inhibitors
The finding that the highly related ligands Activin, Nodal, and Vg1/GDF1 activate the same receptors but differ in their dependence on coreceptors allowed us to determine how ligand diversity and signaling specificity can be achieved. We have identified the loop-b8 region as a 14 amino acid domain, a mere 4% of the entire TGFb signal, that contributes to coreceptor dependence or independence. Sqt and Vg1 incorporating the loop-b8 region of Activin can bind to the Activin receptors in the absence of EGF-CFC proteins. Conversely, Activin incorporating the loop-b8 region of Sqt is inactive, suggesting that the Nodal/Vg1 loop-b8 region might be inhibitory. This inhibition can be relieved by the Finger 1 domain of Sqt, which results in the dependence on EGF-CFC coreceptors ( Figure 9D). These results indicate that rather subtle sequence variations can lead to striking changes in ligand diversity.
Structural considerations suggest that the loop-b8 region determines coreceptor independence or dependence at least in part by its interactions with type II receptors. The In contrast, Lefty1 can inhibit Act  (K and L). (M) Wild-type embryos were injected with 75 pg of either Sqt ActbB[loopb8] , Sqt ActbA[loopb8] , Vg1 ActbB[loopb8] , or 200 pg of Act  mRNA. Embryos were further double-injected with 500 pg of LacZ mRNA, 100 pg of lefty1, and 400 pg of LacZ mRNAs, or 500 pg of lefty1 mRNA. Smad2 pathway activation was measured by an Activin response element luciferase reporter, A3-luc. Values are folds over wild-type control injected with 500 pg of LacZ mRNA and A3-luc reporter. An asterisk indicates a significant difference from the level of activation with ligand and LacZ expression alone (Student's t-test, p , 0.05). DOI: 10.1371/journal.pbio.0020030.g008 Figure 7. Sequence Determinants Conferring EGF-CFC Dependence Synthetic mRNAs (200 pg) encoding ActivinbB with single or double region substitutions from Sqt were injected into wild-type and MZoep embryos. gsc and ntl mRNA expression is shown at shield stage; animal pole views are dorsal to the right. Schematic is not drawn to scale. HA indicates a hemagglutinin epitope tag. Note that Act Sqt[loopb8] containing the loop-b8 region of Sqt is inactive in both wild-type and MZoep embryos. In Act  , the additional substitution of Sqt Finger 1 region relieves the inhibitory presence of the Sqt loop-b8 region. Similar to Sqt, Act  can induce ectopic gsc and ntl in wild-type, but not in MZoep embryos. Western blot analysis indicates that these chimeric constructs produce stable ligands (data not shown). DOI: 10.1371/journal.pbio.0020030.g007 conserved Lys102-X-Asp104 motif in the Activin loop-b8 region has been shown to be important for high-affinity binding to the ActRII receptor (Wuytens et al. 1999;Greenwald et al. 2003;Thompson et al. 2003). In the crystal structure of the ActivinbA-ActRIIB complex, Lys102-X-Asp104 forms an intramolecular salt bridge that interacts with a hydrophobic interface on ActRIIB (Thompson et al. 2003). Mutational analysis has shown that substituting Lys102 with a neutral charge (Ala) significantly reduces receptor binding affinity and signaling (Wuytens et al. 1999). In contrast to Activin, EGF-CFC-dependent ligands such as Nodals and Vg1/GDF1 have the differentially charged residues Met/Leu102 and His104 at the corresponding positions (numbering according to ActivinbA). Similarly, in BMP7 the corresponding residues are Leu102 and Lys104. It has been shown that modeling Lys onto the aligned 102 residue in BMP7 positions it within hydrogen-bonding distance to Glu29 of ActRII and may allow for greater hydrophobic packing at the interface (Greenwald et al. 2003). Analogously, we propose that Sqt ActbA[loopb8] , Sqt ActbB[loopb8] ,Vg1 ActbB[loopb8] , Sqt3, and Sqt5 are coreceptor-independent because of their favorable binding to ActRIIB receptors. Conversely, the corresponding region in Sqt and Vg1 might be inhibitory because of inefficient interaction with ActRIIB receptors. Detailed structural studies should reveal whether EGF-CFC proteins overcome this inhibition by changing the conformation of Nodal and Vg1 or by providing an additional interaction surface that allows the assembly of receptor complexes.
In summary, our results lead to two major conclusions. First, Lefty inhibits a subset of TGFb signals by using the novel mechanism of blocking pathway-specific coreceptors belonging to the EGF-CFC family. Second, subtle sequence changes in TGFbs determine their signaling specificity and dependence on coreceptors. Although Drosophila has an Activin signaling pathway, Nodals, Leftys, and EGF-CFC proteins seem to be restricted to chordates (Brummel et al. 1999;Schier 2003). The evolution of Activin-like signaling pathways therefore represents a remarkable example of how a simple signaling pathway consisting of ligand and receptors can be diversified by subtle sequence changes that modulate the interaction with coreceptors and their inhibitors.

Materials and Methods
Strains and embryos. Adult homozygous fish for oep tz57 were generated as described previously (Zhang et al. 1998;Gritsman et al. 1999). Xenopus embryos were obtained as described in .
Embryo microinjection. Plasmids were linearized and sense strandcapped mRNA was synthesized using the mMESSAGE mMACHINE system (Ambion, Austin, Texas, United States). Zebrafish embryos were dechorinated by pronase treatment and injected between the one-and four-cell stage. Xenopus embryos at the one-to two-cell stage were used for injections into the animal pole.
Phenotypic analysis. Zebrafish embryos at 24 h were mounted in 2% methylcellulose and photographed using a Zeiss (Oberkochen, Germany) M2Bio dissecting microscope. In situ hybrization was Figure 9. Model for EGF-CFC, Activin Receptors, Lefty, and TGFb Interactions (A) In the absence of ligands, the EGF-CFC coreceptor (solid pink) is constitutively bound to the type I receptor Alk4 (solid green). (B) Nodal (solid blue) binds to receptor complexes consisting of EGF-CFC/Alk4 and ActRIIB (solid green). (C) Lefty (solid yellow) sequesters the EGF-CFC coreceptor, thereby preventing Nodal binding to the receptor complexes. (D) Subtle sequence differences determine the interaction with the EGF-CFC coreceptor and the Lefty inhibitor. Nodal and Vg1/GDF1 (solid blue) require the EGF-CFC coreceptor for signaling through ActRIIB and Alk4, while Activin (solid red) does not. Sqt ActbB[loopb8] and Vg1 ActbB[loopb8] (solid blue with red strip) containing the loop-b8 region of ActivinbB can bind to ActRIIB and Alk4 without the EGF-CFC coreceptor and therefore cannot be blocked by Lefty. Act  (solid red with two blue strips) requires the coreceptor for receptor complex binding and can be inhibited by Lefty. DOI: 10.1371/journal.pbio.0020030.g009 performed as described previously (Thisse et al. 1993), using RNA probes to gsc and ntl (Stachel et al. 1993;Schulte-Merker et al. 1994).
Luciferase reporter assay. Luciferase assays were performed with three to six samples and five embryos in each sample. Results are representative of three independent experiments. The injection mixtures were equalized with respect to total mRNA amount with LacZ mRNA. The A3-luc reporter DNA construct (25 pg) (Chen et al. 1996) was also coinjected. Whole zebrafish embryos were harvested at shield stage. Luciferase activity was analyzed using the Luciferase Reporter Assay system (Promega, Madison, Wisconsin, United States) according to the manufacturer's instruction in a Lumat LB9501 (Berthold Technologies, Bad Wildbad, Germany). Owing to the technical aspects of microinjections, in rare circumstances, a single outlier was statistically removed from a population using Grubbs' test/extreme studentized deviate method. Inclusion of outliers into the populations does not change the statistical significance of the p values; that is, p remains ,0.05, where indicated.
Coimmunoprecipitation analysis. Xenopus embryos were harvested at stage 10. For chemical cross-linking of proteins, animal halves were incubated in PBS with 10 mM 3,39-dithiobis(sulfo-succinimidyl propionate) (DTSSP) (Pierce Biotechnology, Rockford, Illinois, United States) and incubated for 2 h on ice. Coimmunoprecipitation was performed as described previously (Yeo and Whitman 2001). Purified processed mouse Lefty1, soluble mouse Cripto, and mouse VEGF-D proteins were obtained from R&D Systems (Minneapolis, Minnesota, United States). Activity assays were performed by R&D Systems. The proteins were incubated in PBS with 1 mM DTSSP for 1 h on ice. Coimmunoprecipitation was performed as described previously (Yeo and Whitman 2001). Samples were treated with 100 mM DTT to cleave DTSSP prior to SDS-PAGE analysis. The following antibodies were used for immunoprecipitation and Western blot analysis: anti-Flag mouse monoclonal antibody (clone M2; Sigma, St. Louis, Missouri, United States), anti-HA mouse monoclonal antibody (clone 16B12; Covance, Princeton, New Jersey, United States), anti-HA rabbit polyclonal antibody (Y-11; Santa Cruz Biotechnology, Santa Cruz, California, United States), anti-c-Myc rabbit polyclonal antibody (A-14; Santa Cruz Biotechnology), anti-c-Myc mouse monoclonal antibody (clone Ab-1; Oncogene Science, Tarrytown, New York, United States), anti-His mouse monoclonal antibody (clone 6-His; Covance), anti-mLefty1 goat polyclonal antibody (R&D Systems), and anti-Glu mouse monoclonal antibody (clone Glu-Glu; Covance). Proteins were visualized using the Super Signal West Pico/ Femto Chemiluminescent Substrate system (Pierce).