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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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Effector genes in the Nodal pathway are only partially inhibited in criptoF78A/F78A mutants. (A, A′, and A″) Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos stained for Nodal at 6.75 dpc. In the wild type, Nodal marks the primitive streak and the posterior mesoderm (A), whereas it is confined to the rim of the proximal epiblast in both criptoF78A/F78A (A′) and cripto- embryos (A″). (B, B′, and B″) Expression of Lefty1 (blue) and 2 (red) mRNA at 6.75 dpc. In the wild type, Lefty1 marks the anterior visceral endoderm, whereas Lefty2 is expressed in the nascent mesoderm (B). Lefty1 and 2 are weakly expressed in criptoF78A/F78A embryos (B′) but are completely absent in cripto- mutants (B″). (C, C′, and C″) At 7.5 dpc, wild-type embryos express both Lefty2 (blue) and Fgf8 (red) in the primitive streak (C). In criptoF78A/F78A embryos, Lefty2 expression is detectable at reduced levels at the posterior pole of the embryo. Conversely, Fgf8 expression is up-regulated and ectopically extends into the extraembryonic region (C′). In sharp contrast, cripto- mutants express neither Lefty2 nor Fgf8 (C″). Bars, 50 μm.
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fig4: Effector genes in the Nodal pathway are only partially inhibited in criptoF78A/F78A mutants. (A, A′, and A″) Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos stained for Nodal at 6.75 dpc. In the wild type, Nodal marks the primitive streak and the posterior mesoderm (A), whereas it is confined to the rim of the proximal epiblast in both criptoF78A/F78A (A′) and cripto- embryos (A″). (B, B′, and B″) Expression of Lefty1 (blue) and 2 (red) mRNA at 6.75 dpc. In the wild type, Lefty1 marks the anterior visceral endoderm, whereas Lefty2 is expressed in the nascent mesoderm (B). Lefty1 and 2 are weakly expressed in criptoF78A/F78A embryos (B′) but are completely absent in cripto- mutants (B″). (C, C′, and C″) At 7.5 dpc, wild-type embryos express both Lefty2 (blue) and Fgf8 (red) in the primitive streak (C). In criptoF78A/F78A embryos, Lefty2 expression is detectable at reduced levels at the posterior pole of the embryo. Conversely, Fgf8 expression is up-regulated and ectopically extends into the extraembryonic region (C′). In sharp contrast, cripto- mutants express neither Lefty2 nor Fgf8 (C″). Bars, 50 μm.

Mentions: Several studies in mice, X. laevis, and zebrafish link Cripto to the Nodal pathway (Shen and Schier, 2000). Therefore, to assess the role of residue F78 of Cripto, we analyzed the expression pattern of Nodal and its target genes, Lefty1 and 2, in criptoF78A/F78A and cripto- mutants at 6.75 dpc. At this stage, Nodal is expressed throughout the primitive streak and posterior mesoderm in wild-type embryos (Fig. 4 A; Conlon et al., 1994; Collignon et al., 1996). In contrast, in both criptoF78A/F78A and cripto- mutants, Nodal expression was reduced and remained at the rim of the proximal epiblast (Fig. 4, A′ and A″). Next, to assess whether Nodal signaling was induced, we analyzed the expression of Lefty1 and 2. In wild-type embryos at 6.75 dpc, Lefty1 is expressed in the anterior visceral endoderm, whereas Lefty2 marks the nascent mesoderm generated from the primitive streak (Fig. 4 B; Meno et al., 1997). Expression of both Lefty1 and 2 was absent in cripto- mutants (Fig. 4 B″). Interestingly, both genes were induced in criptoF78A/F78A embryos, although below normal levels (Fig. 4 B′). To determine whether Nodal signaling is also maintained at later stages in criptoF78A/F78A embryos, we analyzed the expression pattern of Lefty2, a direct Nodal target gene, and Fgf8 at 7.5 dpc. As expected, both genes were readily detectable in the primitive streak of wild-type embryos (Fig. 4 C) but not in cripto- mutants (Fig. 4 C″). In contrast, Lefty2 mRNA was detected in a subset of cells in the posterior side of criptoF78A/F78A embryos (Fig. 4 C′). Furthermore, Fgf8 was expressed in criptoF78A/F78A mutants and its expression domain was even enlarged and extended into the extraembryonic region (Fig. 4 C′). Collectively, these data strongly suggest that the strength or duration of Nodal signaling in criptoF78A/F78A embryos is perturbed compared with wild-type embryos, although it significantly exceeds that observed in cripto- mutants.


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)

Effector genes in the Nodal pathway are only partially inhibited in criptoF78A/F78A mutants. (A, A′, and A″) Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos stained for Nodal at 6.75 dpc. In the wild type, Nodal marks the primitive streak and the posterior mesoderm (A), whereas it is confined to the rim of the proximal epiblast in both criptoF78A/F78A (A′) and cripto- embryos (A″). (B, B′, and B″) Expression of Lefty1 (blue) and 2 (red) mRNA at 6.75 dpc. In the wild type, Lefty1 marks the anterior visceral endoderm, whereas Lefty2 is expressed in the nascent mesoderm (B). Lefty1 and 2 are weakly expressed in criptoF78A/F78A embryos (B′) but are completely absent in cripto- mutants (B″). (C, C′, and C″) At 7.5 dpc, wild-type embryos express both Lefty2 (blue) and Fgf8 (red) in the primitive streak (C). In criptoF78A/F78A embryos, Lefty2 expression is detectable at reduced levels at the posterior pole of the embryo. Conversely, Fgf8 expression is up-regulated and ectopically extends into the extraembryonic region (C′). In sharp contrast, cripto- mutants express neither Lefty2 nor Fgf8 (C″). Bars, 50 μm.
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Related In: Results  -  Collection

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fig4: Effector genes in the Nodal pathway are only partially inhibited in criptoF78A/F78A mutants. (A, A′, and A″) Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos stained for Nodal at 6.75 dpc. In the wild type, Nodal marks the primitive streak and the posterior mesoderm (A), whereas it is confined to the rim of the proximal epiblast in both criptoF78A/F78A (A′) and cripto- embryos (A″). (B, B′, and B″) Expression of Lefty1 (blue) and 2 (red) mRNA at 6.75 dpc. In the wild type, Lefty1 marks the anterior visceral endoderm, whereas Lefty2 is expressed in the nascent mesoderm (B). Lefty1 and 2 are weakly expressed in criptoF78A/F78A embryos (B′) but are completely absent in cripto- mutants (B″). (C, C′, and C″) At 7.5 dpc, wild-type embryos express both Lefty2 (blue) and Fgf8 (red) in the primitive streak (C). In criptoF78A/F78A embryos, Lefty2 expression is detectable at reduced levels at the posterior pole of the embryo. Conversely, Fgf8 expression is up-regulated and ectopically extends into the extraembryonic region (C′). In sharp contrast, cripto- mutants express neither Lefty2 nor Fgf8 (C″). Bars, 50 μm.
Mentions: Several studies in mice, X. laevis, and zebrafish link Cripto to the Nodal pathway (Shen and Schier, 2000). Therefore, to assess the role of residue F78 of Cripto, we analyzed the expression pattern of Nodal and its target genes, Lefty1 and 2, in criptoF78A/F78A and cripto- mutants at 6.75 dpc. At this stage, Nodal is expressed throughout the primitive streak and posterior mesoderm in wild-type embryos (Fig. 4 A; Conlon et al., 1994; Collignon et al., 1996). In contrast, in both criptoF78A/F78A and cripto- mutants, Nodal expression was reduced and remained at the rim of the proximal epiblast (Fig. 4, A′ and A″). Next, to assess whether Nodal signaling was induced, we analyzed the expression of Lefty1 and 2. In wild-type embryos at 6.75 dpc, Lefty1 is expressed in the anterior visceral endoderm, whereas Lefty2 marks the nascent mesoderm generated from the primitive streak (Fig. 4 B; Meno et al., 1997). Expression of both Lefty1 and 2 was absent in cripto- mutants (Fig. 4 B″). Interestingly, both genes were induced in criptoF78A/F78A embryos, although below normal levels (Fig. 4 B′). To determine whether Nodal signaling is also maintained at later stages in criptoF78A/F78A embryos, we analyzed the expression pattern of Lefty2, a direct Nodal target gene, and Fgf8 at 7.5 dpc. As expected, both genes were readily detectable in the primitive streak of wild-type embryos (Fig. 4 C) but not in cripto- mutants (Fig. 4 C″). In contrast, Lefty2 mRNA was detected in a subset of cells in the posterior side of criptoF78A/F78A embryos (Fig. 4 C′). Furthermore, Fgf8 was expressed in criptoF78A/F78A mutants and its expression domain was even enlarged and extended into the extraembryonic region (Fig. 4 C′). Collectively, these data strongly suggest that the strength or duration of Nodal signaling in criptoF78A/F78A embryos is perturbed compared with wild-type embryos, although it significantly exceeds that observed in cripto- mutants.

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