XB-ART-3978PLoS Biol. February 1, 2004; 2 (2): E30.
Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta 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 TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.
PubMed ID: 14966532
PMC ID: PMC340941
Article link: PLoS Biol.
Grant support: T32HD07520 NICHD NIH HHS
Genes referenced: acvr1b acvr2b ass1 egf fgd1 gdf1 gsc igf2bp3 kcnj2 lefty myc nodal nodal1 ptpn11 smad2 t tbx2 tdgf1.3 tgfb1 tspan31 vegfa vegfd
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|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 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).|