XB-IMG-116370
Xenbase Image ID: 116370
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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). Image published in: Cheng SK et al. (2004) Copyright: ©2004 Cheng et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |