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Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal autoinduction.

D'Andrea D, Liguori GL, Le Good JA, Lonardo E, Andersson O, Constam DB, Persico MG, Minchiotti G - J. Cell Biol. (2008)

Bottom Line: However, how ALK4-independent Cripto pathways function in vivo has remained unclear.In sharp contrast to cripto- mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements.Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Fate Laboratory, Institute of Genetics and Biophysics A. Buzzati-Traverso, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy.

ABSTRACT
The EGF-CFC gene cripto governs anterior-posterior (A-P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII-activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal-ALK4-Smad2 signaling both in embryonic stem cells and cell-based assays. In cripto(F78A/F78A) mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto- mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway.

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CriptoF78A does not support the activation of Smad2,3 by Nodal or GDF3 in cell-based assays. (A) Upon transfection in 293T cells, wild-type Cripto potentiates Nodal and GDF3 activity in a Smad3-dependent luciferase assay, whereas the F78A mutant is inactive. 293T cells were transfected with the luciferase reporter CAGA-Luc. The plasmids encoding native ligands, either GDF3 or Nodal, were transfected with or without wild-type or F78A Cripto or secreted Cripto-His constructs. Experiments were performed in triplicate. The expression of input proteins, as detected by Western blotting, is shown at the bottom. Secreted forms of wild-type and F78A Cripto lacking the GPI anchor were inactive in this assay. Error bars represent SD of three experiments. RLU, relative luciferase unit. (B) Wild-type, but not F78A, Cripto activates Smad2 in ES cells. 2-d-old Cripto−/− EBs were serum starved for 3 h and then treated with either wild-type or F78A recombinant Cripto protein for 20 min or left untreated (NI) as indicated. Wild-type ES cell–derived EBs were used as a control. Smad2 activation was detected by Western blot analysis using anti–phospho-Smad2 antibody. Levels of total Smad2 were also compared. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). Smad2 phosphorylation was expressed as the ratio between arbitrary densitometric units (ADU) of P-Smad2 and total Smad2. (C) F78A recombinant Cripto retains its ability to activate phospho-ERK. Serum-starved Cripto−/− EBs were treated with 2 μg/ml of either wild-type or F78A recombinant Cripto protein for 10 min or left untreated (NI) as indicated. ERK activation was detected by Western blot analysis using anti–phospho-ERK antibody. Densitometry analyses were performed as in B.
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fig5: CriptoF78A does not support the activation of Smad2,3 by Nodal or GDF3 in cell-based assays. (A) Upon transfection in 293T cells, wild-type Cripto potentiates Nodal and GDF3 activity in a Smad3-dependent luciferase assay, whereas the F78A mutant is inactive. 293T cells were transfected with the luciferase reporter CAGA-Luc. The plasmids encoding native ligands, either GDF3 or Nodal, were transfected with or without wild-type or F78A Cripto or secreted Cripto-His constructs. Experiments were performed in triplicate. The expression of input proteins, as detected by Western blotting, is shown at the bottom. Secreted forms of wild-type and F78A Cripto lacking the GPI anchor were inactive in this assay. Error bars represent SD of three experiments. RLU, relative luciferase unit. (B) Wild-type, but not F78A, Cripto activates Smad2 in ES cells. 2-d-old Cripto−/− EBs were serum starved for 3 h and then treated with either wild-type or F78A recombinant Cripto protein for 20 min or left untreated (NI) as indicated. Wild-type ES cell–derived EBs were used as a control. Smad2 activation was detected by Western blot analysis using anti–phospho-Smad2 antibody. Levels of total Smad2 were also compared. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). Smad2 phosphorylation was expressed as the ratio between arbitrary densitometric units (ADU) of P-Smad2 and total Smad2. (C) F78A recombinant Cripto retains its ability to activate phospho-ERK. Serum-starved Cripto−/− EBs were treated with 2 μg/ml of either wild-type or F78A recombinant Cripto protein for 10 min or left untreated (NI) as indicated. ERK activation was detected by Western blot analysis using anti–phospho-ERK antibody. Densitometry analyses were performed as in B.

Mentions: Previous analysis of ES cell–derived EBs suggested that F78 is essential for Cripto to stimulate the in vitro differentiation of cardiomyocytes (Parisi et al., 2003). Similarly, substitution of F78 by alanine entirely blocks the ability of Cripto to rescue gastrulation of oep mutant zebrafish embryos (Minchiotti et al., 2001). Given these reports, it was surprising that substitution of F78 by alanine only partially inhibited Cripto activity in the mouse embryo. To determine whether CriptoF78A can stimulate Nodal signaling in cell culture, we monitored its effect on CAGA-luc, a well characterized and sensitive luciferase reporter of ALK4–Smad3 signaling. Although transfection of wild-type cripto potently stimulated the activity of Nodal, CriptoF78A was completely inactive in this assay (Fig. 5 A). Analogous results were obtained using the activin response element (ARE)–luc reporter construct in conjunction with wild-type Nodal or a more potent supercleaved and stabilized derivative (Nsc-g; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200709090/DC1; Yan et al., 2002; Le Good et al., 2005; Chen et al., 2006; Andersson et al., 2007). These results suggest that CriptoF78A is unable to activate a Nodal–ALK4–Smad signaling complex.


Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal autoinduction.

D'Andrea D, Liguori GL, Le Good JA, Lonardo E, Andersson O, Constam DB, Persico MG, Minchiotti G - J. Cell Biol. (2008)

CriptoF78A does not support the activation of Smad2,3 by Nodal or GDF3 in cell-based assays. (A) Upon transfection in 293T cells, wild-type Cripto potentiates Nodal and GDF3 activity in a Smad3-dependent luciferase assay, whereas the F78A mutant is inactive. 293T cells were transfected with the luciferase reporter CAGA-Luc. The plasmids encoding native ligands, either GDF3 or Nodal, were transfected with or without wild-type or F78A Cripto or secreted Cripto-His constructs. Experiments were performed in triplicate. The expression of input proteins, as detected by Western blotting, is shown at the bottom. Secreted forms of wild-type and F78A Cripto lacking the GPI anchor were inactive in this assay. Error bars represent SD of three experiments. RLU, relative luciferase unit. (B) Wild-type, but not F78A, Cripto activates Smad2 in ES cells. 2-d-old Cripto−/− EBs were serum starved for 3 h and then treated with either wild-type or F78A recombinant Cripto protein for 20 min or left untreated (NI) as indicated. Wild-type ES cell–derived EBs were used as a control. Smad2 activation was detected by Western blot analysis using anti–phospho-Smad2 antibody. Levels of total Smad2 were also compared. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). Smad2 phosphorylation was expressed as the ratio between arbitrary densitometric units (ADU) of P-Smad2 and total Smad2. (C) F78A recombinant Cripto retains its ability to activate phospho-ERK. Serum-starved Cripto−/− EBs were treated with 2 μg/ml of either wild-type or F78A recombinant Cripto protein for 10 min or left untreated (NI) as indicated. ERK activation was detected by Western blot analysis using anti–phospho-ERK antibody. Densitometry analyses were performed as in B.
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fig5: CriptoF78A does not support the activation of Smad2,3 by Nodal or GDF3 in cell-based assays. (A) Upon transfection in 293T cells, wild-type Cripto potentiates Nodal and GDF3 activity in a Smad3-dependent luciferase assay, whereas the F78A mutant is inactive. 293T cells were transfected with the luciferase reporter CAGA-Luc. The plasmids encoding native ligands, either GDF3 or Nodal, were transfected with or without wild-type or F78A Cripto or secreted Cripto-His constructs. Experiments were performed in triplicate. The expression of input proteins, as detected by Western blotting, is shown at the bottom. Secreted forms of wild-type and F78A Cripto lacking the GPI anchor were inactive in this assay. Error bars represent SD of three experiments. RLU, relative luciferase unit. (B) Wild-type, but not F78A, Cripto activates Smad2 in ES cells. 2-d-old Cripto−/− EBs were serum starved for 3 h and then treated with either wild-type or F78A recombinant Cripto protein for 20 min or left untreated (NI) as indicated. Wild-type ES cell–derived EBs were used as a control. Smad2 activation was detected by Western blot analysis using anti–phospho-Smad2 antibody. Levels of total Smad2 were also compared. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). Smad2 phosphorylation was expressed as the ratio between arbitrary densitometric units (ADU) of P-Smad2 and total Smad2. (C) F78A recombinant Cripto retains its ability to activate phospho-ERK. Serum-starved Cripto−/− EBs were treated with 2 μg/ml of either wild-type or F78A recombinant Cripto protein for 10 min or left untreated (NI) as indicated. ERK activation was detected by Western blot analysis using anti–phospho-ERK antibody. Densitometry analyses were performed as in B.
Mentions: Previous analysis of ES cell–derived EBs suggested that F78 is essential for Cripto to stimulate the in vitro differentiation of cardiomyocytes (Parisi et al., 2003). Similarly, substitution of F78 by alanine entirely blocks the ability of Cripto to rescue gastrulation of oep mutant zebrafish embryos (Minchiotti et al., 2001). Given these reports, it was surprising that substitution of F78 by alanine only partially inhibited Cripto activity in the mouse embryo. To determine whether CriptoF78A can stimulate Nodal signaling in cell culture, we monitored its effect on CAGA-luc, a well characterized and sensitive luciferase reporter of ALK4–Smad3 signaling. Although transfection of wild-type cripto potently stimulated the activity of Nodal, CriptoF78A was completely inactive in this assay (Fig. 5 A). Analogous results were obtained using the activin response element (ARE)–luc reporter construct in conjunction with wild-type Nodal or a more potent supercleaved and stabilized derivative (Nsc-g; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200709090/DC1; Yan et al., 2002; Le Good et al., 2005; Chen et al., 2006; Andersson et al., 2007). These results suggest that CriptoF78A is unable to activate a Nodal–ALK4–Smad signaling complex.

Bottom Line: However, how ALK4-independent Cripto pathways function in vivo has remained unclear.In sharp contrast to cripto- mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements.Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Fate Laboratory, Institute of Genetics and Biophysics A. Buzzati-Traverso, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy.

ABSTRACT
The EGF-CFC gene cripto governs anterior-posterior (A-P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII-activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal-ALK4-Smad2 signaling both in embryonic stem cells and cell-based assays. In cripto(F78A/F78A) mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto- mutants, cripto(F78A/F78A) embryos establish an A-P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal-Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A-P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal-ALK4-Smad2 signaling pathway.

Show MeSH
Related in: MedlinePlus