ZFIN Image: Cheng et al., 2004, Figure 5

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Figure Caption

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) Sqt^Act^β^B[loop^β⁸^β^9] and Sqt^Act^β^B[loop^β^8] can induce gsc and ntl expression in both wild-type and MZoep embryos.

(C) Sqt^Act^β^B[^β^8] 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 Sqt^Act^β^A[loop^β^8] and Vg1^Act^β^B[loop^β^8] 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 Sqt^{ActβB[loopβ8]}, 250 pg of Sqt^{ActβA[loopβ8]}, or 100 pg of Vg1^{ActβB[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) Sqt^Act^β^B[loop^β^8] 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 Sqt^Act^β^B[loop^β^8]/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 Sqt^Act^β^B[loop^β^8]/HA (mature ligand, approximately 22 kDa; anti-HA).

Acknowledgments

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