Parisi S , D'Andrea D , Lago CT , Adamson ED , Persico MG , Minchiotti G .

	Figure 1. Schematic representation of the experimental protocol used for ES cell differentiation into cardiomyocytes (adapted from Maltsev et al., 1993).
	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.
	Figure 3. Cripto expression profile during the in vitro differentiation of ES cells. Total lysates of either undifferentiated ES cells or EBs at different days of differentiation (2–7 d), derived from either RI (wt) or DE7 (Cripto−/−) ES cells, were collected in lysis buffer and analyzed by Western blot using a polyclonal anti-Cripto antiserum (Minchiotti et al., 2000). Data were normalized to the expression level of Porin.
	Figure 4. Dynamics of Cripto signaling in cardiomyocyte differentiation. (A) Definition of the temporal activity of Cripto. Percentage of Cripto−/− EBs containing beating areas after addition of recombinant Cripto protein. 10 μg/ml of soluble Cripto protein was added to EBs at 24-h intervals starting from time 0 of the in vitro differentiation assay (scheme in Fig. 1). The number of EBs containing beating areas was scored from day 8 to 12 of in vitro differentiation. (B) Dose-dependent activity of Cripto protein. 2-d-old Cripto−/− EBs were treated with increasing amounts of recombinant soluble Cripto protein for 24 h and then cultured for the remaining days. Appearance of beating areas was scored from day 8 to 12 of the in vitro differentiation. (C) Duration of Cripto signaling. 2-d-old Cripto−/− EBs were treated with 10 μg/ml of recombinant soluble Cripto protein for different lengths of time, 1, 12, 24, and 72 h. EBs were then washed to remove the protein and cultured for the remaining days. Cells were examined for cardiac differentiation as described above. In all cases, two independent Cripto−/− ES clones (DE7 and DE14) were used. Data are representative of at least two independent experiments.
	Figure 5. Cripto promotes cardiomyocyte differentiation and inhibits neural differentiation of ES cells according to the timing of exposure. (A) Cardiomyocyte versus neuronal differentiation of Cripto−/− EBs as revealed by indirect immunofluorescence. 2-d-old Cripto−/− EBs, derived from DE7 cell line, were either left untreated (a and c) or treated for 24 h with 10 μg/ml of recombinant Cripto protein (b and d). On day 12 of in vitro differentiation, expression of either sarcomeric myosin or βIII-tubulin was revealed by immunofluorescence using anti–MF-20 (red, a and b) or βIII-tubulin (green, c and d) antibodies, respectively. Data are representative of at least two independent experiments. Comparable results were obtained with Cripto−/− DE14 ES cell line. (B) Cardiomyocyte versus neuronal differentiation of Cripto−/− EB–derived cells depends on the timing of exposure to Cripto. Percentage of Cripto−/− EBs stained for βIII-tubulin (red plot) or MF-20 (blue plot) after addition of recombinant Cripto protein at different time points. 10 μg/ml of recombinant Cripto protein was added to EBs at 24-h intervals starting from time 0 of the in vitro differentiation assay. On day 12 of in vitro differentiation, EBs were stained for either βIII-tubulin or MF-20 antibodies. Data are representative of two independent experiments.
	Figure 6. Activation of Smad2 in Cripto−/− cell aggregates treated with recombinant Cripto protein. 2-d-old Cripto−/− EBs were serum starved for 3 h and then treated with 10 μg/ml of recombinant Cripto protein for 30', 60', or 120' or left untreated, as indicated. Smad2 activation was detected by Western blot analysis using anti–phospho-Smad2 antibody. Levels of total Smad2 were also compared.
	Figure 7. Expression profile of Nodal, Alk4, and ActRIIB during cardiomyocyte differentiation and their effects on cardiac induction. (A) RNA expression levels of Nodal, Alk4, and ActRIIB genes during in vitro differentiation of ES cells. RT-PCR analysis was performed on RNA extracted from either undifferentiated ES or EBs (either wt or Cripto−/−) throughout a differentiation period of 10 d (days 2–10). HPRT gene was used as an internal control. (B) Western blot analysis of total lysates from 293EBNA cells transfected with either wt or ca form of HA-tagged human Alk4. Cells were cotransfected with Jun-HA expression vector as an internal control. A monoclonal anti-HA antibody was used to detect protein levels. (C) RNA expression profile of the αMHC gene during differentiation of Cripto−/− ES cells (days 5, 7, and 10) overexpressing wt and activated forms of either Alk4 or Taram-A. HPRT gene was used as an internal control.
	Figure 8. Identification of Cripto key residues required for cardiac induction and differentiation. (A) Schematic representation of wt and mutant Cripto derivatives. (B) Western blot analysis of total lysates from 293EBNA cells transfected with either wt or mutant cripto derivatives. Jun-HA expression vector was cotransfected as an internal control. Either polyclonal anti-Cripto or monoclonal anti-HA antibodies were used to detect protein levels. (C) RNA expression levels of the cardiac αMHC and MLC2v genes during in vitro differentiation of Cripto−/− ES cells (days 5, 7, and 10) overexpressing either wt or mutant cripto derivatives. Expression level of HPRT gene was analyzed as an internal control.
	Figure 9. Modulation of Cripto activity by O-fucosylation. Dose-dependent activity of T72A mutant Cripto compared with wt Cripto as assayed in cardiomyocyte differentiation assay. 2-d-old Cripto−/− EBs were treated with increasing amounts of either recombinant soluble T72A mutant or wt Cripto protein for 24 h and then cultured for the remaining days. Appearance of beating areas was scored from day 8 to 12 of the in vitro differentiation. Data are representative of two independent experiments.
	Figure 10. Exposure to Cerberus inhibits Cripto activity in cardiomyocyte differentiation assay. (A) Cerberus inhibits Cripto-dependent cardiomyocyte differentiation of Cripto−/− EBs. 2-d-old Cripto−/− EBs were cultured for 24 h in the presence of 100% (vol/vol) of media supernatant from 293T cells transiently expressing either Cerberus, Cerberus-S, or empty vector as control, in the presence of increasing amounts of recombinant Cripto protein. The number of Cripto−/− EBs containing beating areas was scored from day 8 to 12 of in vitro differentiation. Data are representative of two independent experiments. (B) Inhibition of Cripto by Cerberus-S is dose dependent. 2-d-old Cripto−/− EBs were cultured for 24 h with 10 μg/ml of soluble Cripto protein in the presence of increasing amounts (vol/vol) of media from 293T cells transiently expressing Cerberus-S. Cells were examined for cardiac differentiation as described above. Data are representative of two independent experiments.

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