Cheng SK , Olale F , Brivanlou AH , Schier AF .

T32HD07520 NICHD NIH HHS

	Figure 1. Lefty Antagonizes Nodal and Vg1 Signaling, but Not Activin Signaling, in Zebrafishntl mRNA expression (A–C) and gsc mRNA expression (D–O) in wild-type zebrafish embryos at shield stage, animal pole view. Embryos were injected with low levels (1 pg) of activin βB mRNA (A–C), high levels (10 pg) of activin βB mRNA (D–F), 200 pg of activin βA mRNA (G–I), 75 pg of sqt mRNA (J–L), or 200 pg of Vg1 (M–O). Embryos were further double-injected with either 500 pg of LacZ mRNA (A, D, G, J, and M), 100 pg of lefty1 and 400 pg of LacZ mRNAs (B, E, H, K, and N), or 500 pg of lefty1 mRNA (C, F, I, L, and O). Ectopic ntl expression (arrowheads) in activin βB mRNA-injected embryos was not inhibited by Lefty1 (B and C) when compared with LacZ mRNA-coinjected controls (A). 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 βB and activin βA 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 βB/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).
	Figure 2. EGF-CFC Coreceptors Genetically Interact with LeftyLive wild-type zebrafish embryos at 30 h postfertilization (hpf).(A1, B1, and C1) Ventral views of the head.(A1′, B2, B3, and C1′) Lateral views, with anterior to the left, dorsal up.(A1, A1′, B1, B2, and B3) Wild-type embryos were injected with 20 pg of lefty1 mRNA. Embryos were further double-injected with either 200 pg of LacZ mRNA (A1 and A1′) or 200 pg of Cripto mRNA (B1, B2, and B3).(C1 and C1′) Wild-type embryos injected with 200 pg of Cripto mRNA and 20 pg of LacZ mRNA.Misexpression of Lefty1 results in cyclopia and other head and trunk mesoderm defects ([A1 and A1′] 32 of 32 embryos had the phenotype shown; arrow shows cyclopia). Coexpression of Cripto with Lefty in embryos leads to rescue of two eyes ([B1] four of 50; arrows show two eyes), notochord ([B2] 20 of 50; inset shows trunk somites and notochord, red bar delineates notochord), and trunk somites ([B3] 50 of 50). Embryos injected with Cripto mRNA only show normal wild-type phenotype ([C1 and C1′] 30 of 30; arrow in [C1] shows two normal eyes, and inset in [C1′] shows normal notochord and trunk somites, red bar delineates notochord).
	Figure 3. Lefty Binds to Cripto, but Not to the Activin Receptors ActRIIB and Alk4(A and B) Lefty1 interacts with Cripto. 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 μg/ml) was mixed with either soluble purified Cripto/His protein (5 μg/ml) or purified control VEGF-D/His protein (5 μg/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 (kinase-defective receptor, approximately 120 kDa; anti-Myc), Alk4(KR)/Flag (kinase-defective receptor, approximately 70 kDa; anti-Flag), Cripto/Flag (approximately 30 kDa; anti-Flag), Lefty1/HA (mature ligand, approximately 36–40 kDa; anti-HA; Sakuma et al. 2002), Sqt/HA (unprocessed precursor, approximately 55 kDa; mature ligand, approximately 22 kDa; anti-HA), Lefty1/Glu (unprocessed precursor, approximately 55 kDa; mature ligand, approximately 38 kDa; anti-Glu; Sakuma et al. 2002), mLefty1 (mature ligand, approximately 36 kDa, anti-mLefty1; Sakuma et al. 2002), Cripto/His (soluble form, approximately 22–25 kDa; anti-His), and VEGF-D/His (mature ligand, approximately 15–20 kDa; anti-His).
	Figure 4. Chimera Analysis to Identify TGFβ Sequence Determinants Conferring EGF-CFC Coreceptor Dependence or IndependenceSchematic depiction of chimeras of mature ligand domains, Finger 1 (F1), Heel (H), and Finger 2 (F2), between Xenopus ActivinβB and zebrafish Sqt. HA indicates an hemagglutinin epitope tag. 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 ActivinβB. 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 ActivinβB prodomain and epitope tag does not alter the specificity or functionality of wild-type ActivinβB (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 ActivinβB, 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).
	Figure 5. Sequence Determinants Conferring Independence from EGF-CFC Coreceptors(A) Sequence alignment of Finger 2 region of EGF-CFC-dependent and EGF-CFC-independent TGFβ ligands. Location of secondary structure elements, β-sheets (β6–β9) and loop, are shown (Kirsch et al. 2000). Residue numbering is from mouse ActivinβA.(B–E) Synthetic mRNAs (200 pg) encoding chimeras of Finger 2 subregions between Xenopus ActivinβB or ActivinβA and zebrafish Sqt or Vg1 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) SqtActβB[loopβ8β9] and SqtActβB[loopβ8] can induce gsc and ntl expression in both wild-type and MZoep embryos.(C) SqtActβB[β8] can weakly expand ntl expression in MZoep mutants. ntl mRNA expression in MZoep mutants is at shield stage; lateral view.(D) Other TGFβs conform to loop-β8 EGF-CFC-independent determinant. Note that Xenopus ActivinβA 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 SqtActβA[loopβ8] and Vg1ActβB[loopβ8] can induce ectopic gsc in both wild-type and MZoep embryos.(E) Wild-type and MZoep embryos were injected with 5 pg of activin βB, 100 pg of sqt, 100 pg of Vg1, 125 pg of SqtActβB[loopβ8], 250 pg of SqtActβA[loopβ8], or 100 pg of Vg1ActβB[loopβ8] mRNA. Smad2 pathway activation was measured by an Activin response element luciferase reporter, A3-luc. Luciferase units are relative to wild-type or MZoep control injected with the A3-luc reporter alone.(F) SqtActβB[loopβ8] can bind to ActRIIB and Alk4 in the absence of EGF-CFC coreceptors. RNAs (1 ng each) encoding ActRIIB(KR)/Myc, Alk4(KR)/Flag, Cripto/Flag, ActivinβB/HA, Sqt/HA, or SqtActβB[loopβ8]/HA were injected into Xenopus embryos. Proteins in the coimmunoprecipitates and total extracts were probed in Western blot analysis with the indicated antibodies: ActRIIB(KR)/Myc (approximately 120 kDa; anti-Myc), Alk4(KR)/Flag (approximately 70 kDa; anti-Flag), Cripto/Flag (approximately 30 kDa; anti-Flag), ActivinβB/HA (mature ligand, approximately 16 kDa; anti-HA), Sqt/HA (mature ligand, approximately 22 kDa; anti-HA), and SqtActβB[loopβ8]/HA (mature ligand, approximately 22 kDa; anti-HA).
	Figure 6. Conserved Residues in Activin Loop-β8 Region Confer Independence from EGF-CFC CoreceptorsSynthetic mRNAs (200 pg) encoding Sqt harboring multiple mutations from ActivinβB (shown in red) 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. Note that the Sqt3 and Sqt5 constructs containing the Lys102–X–Asp104 motif and Asn99 insertion show weak expansion of ntl expression animally and dorsally in MZoep mutants.
	Figure 7. Sequence Determinants Conferring EGF-CFC DependenceSynthetic mRNAs (200 pg) encoding ActivinβB 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 ActSqt[loopβ8] containing the loop-β8 region of Sqt is inactive in both wild-type and MZoep embryos. In ActSqt[Finger1-loopβ8], the additional substitution of Sqt Finger 1 region relieves the inhibitory presence of the Sqt loop-β8 region. Similar to Sqt, ActSqt[Finger1-loopβ8] 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).
	Figure 8. EGF-CFC Coreceptor Depen-dence Determines Susceptibility to Antagonism by Lefty(A–L) Embryos were injected with 75 pg of SqtActβB[loopβ8] mRNA (A–C), 75 pg of SqtActβA[loopβ8] mRNA (D–F), 200 pg of Vg1ActβB[loopβ8] mRNA (G–I), or 200 pg ActSqt[Finger1-loopβ8] mRNA (J–L). Embryos were further double-injected with either 500 pg of LacZ mRNA (A, D, G, and J), 100 pg of lefty1 and 400 pg LacZ mRNAs (B, E, H, and K), or 500 pg of lefty1 mRNA (C, F, I, and L). gsc mRNA expression in wild-type zebrafish embryos is shown at shield stage, animal pole view. Note that both levels of Lefty1 cannot inhibit the ectopic gsc expression induced by SqtActβB[loopβ8] (B and C), SqtActβA[loopβ8] (E and F), and Vg1ActβB[loopβ8] (H and I). In contrast, Lefty1 can inhibit ActSqt[Finger1-loopβ8] (K and L).(M) Wild-type embryos were injected with 75 pg of either SqtActβB[loopβ8], SqtActβA[loopβ8], Vg1ActβB[loopβ8], or 200 pg of ActSqt[Finger1-loopβ8] 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).
	Figure 9. Model for EGF-CFC, Activin Receptors, Lefty, and TGFβ 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. SqtActβB[loopβ8] and Vg1ActβB[loopβ8] (solid blue with red strip) containing the loop-β8 region of ActivinβB can bind to ActRIIB and Alk4 without the EGF-CFC coreceptor and therefore cannot be blocked by Lefty. ActSqt[Finger1-loopβ8] (solid red with two blue strips) requires the coreceptor for receptor complex binding and can be inhibited by Lefty.

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