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Figure 2. Functional dissection of Cripto. (A) Schematic representation of cripto cDNA derivatives. S.P., signal peptide. (B) Determination of minimal domains required for Cripto activity in cardiomyocyte differentiation. Either wt or deleted cripto mutant derivatives were transfected into Cripto−/− ES cells; empty vector was used as control. The percentage of EBs with rhythmically contracting areas detectable by light microscopy was scored on day 8 to 12. Data are representative of at least two independent experiments. (C) Western blot analysis of conditioned media from 293EBNA cells transfected with cripto cDNA deletion derivatives. Cells were cotransfected with Plgf expression vector as an internal control (see Materials and methods). Lane 1, EGF-CFC; lane 2, EGF long; lane 3, vector. The molecular mass of protein standards is indicated (kD). (D) Expression of cardiac-specific genes MLC2v and αMHC during in vitro differentiation of either wt or Cripto−/− ES cells. RT-PCR was performed on RNA extracted from either undifferentiated ES cells or EBs throughout a differentiation period of 10 d (days 2–10). HPRT gene expression was analyzed as an internal control. (E) RNA expression levels of MLC2v and cardiac αMHC genes during in vitro differentiation of Cripto−/− ES cells overexpressing either wt or cripto deletion mutants. RNA was harvested at days 5, 7, and 10 of the differentiation protocol and subjected to RT-PCR. Empty vector was used as a negative control. HPRT gene expression was analyzed as an internal control. The results are representative of two independent differentiation programs. Image published in: Parisi S et al. (2003) Copyright © 2003, The Rockefeller University Press. 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-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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