XB-IMG-82774
Xenbase Image ID: 82774
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Figure 2. Emilin1 Inhibits Nodal/TGF-β Signaling through the EMI Domain(A) Schematic representation of the structure of Emilin1 protein. The EMI domain at the N terminus is a cysteine-rich region distinctive of the EDEN protein family.(B�E) Overexpression of Emilin1 attenuates mesoderm development in Xenopus embryos through the EMI-domain. Eight cell-stage embryos were injected in the marginal zone with mRNAs encoding for full-length Emilin1 (2 ng) (C), EMI-domain alone (400 pg) (D), or a deleted version of Emilin1 (2 ng, Emilin1δEMI) lacking the EMI-domain (E). The embryos were processed by in situ hybridization for the paraxial mesoderm marker MyoD. Similar results were obtained by staining gastrula stage embryos with the pan-mesodermal marker VegT (Figure S2).(F and G) Emilin1 inhibits nodal/TGF-β signaling, and this requires the EMI-domain. Two cell-stage embryos were injected in the animal pole with the indicated mRNAs. Animal caps were explanted at stage 9 and harvested at stage 11 for RT-PCR analysis. (F) Activation of the mesoderm markers Eomes and Mixer by Xnr-1 mRNA (60 pg) is antagonized by Emilin1 mRNA but not by Emilin1δEMI mRNA. (G) Overexpression of EMI-domain mRNA is sufficient to inhibit mesoderm induction by coinjected Xnr-1. As shown in Figure S2, induction of ectopic bottle cells or axial structures, typical effects of TGF-β/Nodal/Smad signaling, is also antagonized by Emilin1.(H) RT-PCR of animal caps explanted from embryos injected as in (F) with constitutive-active (ca)-TGF-β type I receptor (Alk5) mRNA (100 pg) alone or in combination with EMI-domain mRNA (800 pg). Emilin1 acts upstream of TGF-β receptor activity.(I�K) Embryos were injected as in (F) with EMI-domain mRNA (800 pg) in combination with BMP2, eFGF, or Xwnt8 mRNAs (100 pg, 2 pg, and 20 pg, respectively). Explanted animal caps were analyzed by RT-PCR for the indicated markers. EF1α serves as loading control. Image published in: Zacchigna L et al. (2006) Copyright © 2006. Image reproduced with permission of the Publisher, Elsevier B. V.
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