Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells

Abstract

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.

Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology

“…Indeed, kinetic experiments performed by adding recombinant Cripto protein directly to the culture medium of Cripto À/À ES cells indicate that Cripto is required in a precise moment during differentiation, after which it fails to specify the cardiac lineage. Actually, addition of Cripto protein during the 0-2 day interval effectively restored the differentiation ability of Cripto À/À ES cells; however, addition at later time points results in dramatically reduced cardiomyocyte differentiation (Figure 1) (Parisi et al, 2003). These functional data are supported by expression data showing that endogenous Cripto protein is present at the earliest stages of ES cell differentiation and that it is absent at stages where contracting EBs start to appear (Parisi et al, 2003).…”

“…Loss-of-function experiments performed using Nodal antagonists, either Cerberus or Cerberus-S, indicate that Nodal signaling is indeed required to support Cripto-regulated cardiac induction and differentiation in ES cells. Addition of Nodal-antagonist Cerberus Short, which specifically blocks Nodal by direct binding to the ligand (Piccolo et al, 1999), results in a strong inhibition of Cripto activity in promoting cardiogenesis (Parisi et al, 2003) and provides direct evidence for a functional role of Alk4/Nodal pathway in Cripto-mediated specification of the cardiac lineage (Figure 3).…”

“…As a first attempt to unravel the signaling pathways activated by Cripto in cardiomiogenesis, the controlled differentiation of Cripto À/À ES cells has provided a powerful system. Indeed, stimulation of Cripto À/À ES with recombinant Cripto has been shown to activate Smad2 (Parisi et al, 2003). Interestingly, acute stimulation by Cripto, while competent in activating Smad2, is insufficient to achieve proper terminal cardiac differentiation, thus indicating that not only the particular timing but also the duration of Cripto signaling contributes to cardiomyocyte specification (Figure 2).…”

“…Unfortunately, the early lethality observed in cripto À/À mice occurs before a stage in which the role of cripto in cardiogenesis can be evaluated. In vitro analysis of Cripto-deficient ES cells, by overcoming the complexity of the in vivo situation, led to novel insights into the functional role of cripto in cardiomiogenesis (Xu et al, 1998(Xu et al, , 1999Parisi et al, 2003).…”

Cardiac Repair by Embryonic Stem-Derived Cells

“…For example, a number of studies have implicated signaling of BMP and Wnt family members in cardiomyogenic differentiation of ES cells (Winnier et al 1995;Czyz and Wobus 2001;Behfar et al 2002;Kawai et al 2004;Terami et al 2004). In addition, signaling through the FGF (Dell'Era et al 2003;Kawai et al 2004), IGF-II (Morali et al 2000), Crypto (Parisi et al 2003), PDGFBB (Sachinidis et al 2003), CT-1 (Sauer et al 2004), TGF-beta (Behfar et al 2002), and dynorphin B (Ventura et al 2003) pathways enhanced cardiomyogenesis during EB differentiation. Perhaps not unexpectedly, cytokine/growth factor treatments were occasionally observed to promote cardiomyogenic differentiation via rather indirect mechanisms.…”