Zacchigna L , Vecchione C , Notte A , Cordenonsi M , Dupont S , Maretto S , Cifelli G , Ferrari A , Maffei A , Fabbro C , Braghetta P , Marino G , Selvetella G , Aretini A , Colonnese C , Bettarini U , Russo G , Soligo S , Adorno M , Bonaldo P , Volpin D , Piccolo S , Lembo G , Bressan GM .

TI_GGP04030 Telethon, GGP04030 Telethon

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.

Figure S2. Emilin1 Inhibits Nodal/TGF-β Signaling through Its EMI Domain(A) Control embryo. (B) Long-term effects of Emilin1 overexpression in Xenopus embryos. (C�F) Emilin1 antagonizes endogenous TGF-β ligands in Xenopus embryos, through its EMI-domain. Eight-cell stage embryos were injected in the marginal zone with mRNAs (800 pg) encoding full-length Emilin1 (D), EMI-domain alone (E), or a deleted version of Emilin1 (EmilinδEMI) lacking the EMI-domain (F). At gastrula stage, the embryos were harvested for in situ hybridization with the mesoderm marker VegT. Emilin full-length or the EMI domain alone, but not EmilinδEMI, are sufficient to block the expression of VegT. (G) Western blotting of Xenopus extracts shows expression of the proteins from injected mRNAs. (H) EMI-domain inhibits Xbra expression in Xenopus embryos. Eight-cell stage embryos were injected and processed as in (C�F). (I�O) Emilin1 is able to antagonize the phenotypic effects of Nodal/TGF-β signaling. Two-cell stage embryos were injected in the animal cap with Xnr-1 mRNA (40pg) or TGF-β3 mRNA (300 pg) alone or in combination with 1 ng of Emilin1, EMI-domain or Emilin1δEMI. Overexpression of Xnr-1 mRNA in ectoderm cells induces their differentiation into pigment bottle-cells (J, M, and N�arrowhead). (P and Q) Overexpression of TGF-β3 induces secondary trunk/tail structures that are inhibited by coinjected Emilin1. (R and S) Two-cell stage embryos were injected in the animal pole with BMP2 mRNA (400 pg) alone or in combination with EMI domain (1 ng). BMP overexpression produces strong ventralized embryos lacking the anterior dorsal structures. EMI-domain does not reduce this ventralized phenotype.

Raman, TGF-beta regulation by Emilin1: new links in the etiology of hypertension. 2006, Pubmed

Raman, TGF-beta regulation by Emilin1: new links in the etiology of hypertension. 2006, Pubmed