Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation ofcriptoF78A/F78Amutant mice by homologous recombination in ES cells. (A) Strategy for inserting the F78A substitution at the cripto locus. Numbered boxes represent wild-type exons (black) and the mutant exon carrying the F78A substitution (gray). Location of PCR primers (black arrowheads) and probes (black bars) used for genotyping are indicated. B, BamHI; E, EcoRV; H HindIII; P, PstI. (B) Genotyping of cell lines by Southern blot analysis. Genomic DNA from wild-type (+/+) and floxed (+/F78A) ES cells was digested with EcoRV and hybridized with the 5′ (RH5) and the 3′ (BE6) probes shown in A. The sizes of hybridized fragments are indicated in kilobases. (C) Genotypes of offspring from cripto+/F78A heterozygote intercrosses. Primers c and d amplified a 280-bp fragment of the wild-type allele and a 400-bp fragment of the Cre-deleted allele. (D) Spatial distribution of both wild-type (left) and F78A mutant (right) Cripto protein. Wild-type and criptoF78A/F78A embryos were immunostained with anti-Cripto antibodies at 6.75 dpc. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234230&req=5

fig1: Generation ofcriptoF78A/F78Amutant mice by homologous recombination in ES cells. (A) Strategy for inserting the F78A substitution at the cripto locus. Numbered boxes represent wild-type exons (black) and the mutant exon carrying the F78A substitution (gray). Location of PCR primers (black arrowheads) and probes (black bars) used for genotyping are indicated. B, BamHI; E, EcoRV; H HindIII; P, PstI. (B) Genotyping of cell lines by Southern blot analysis. Genomic DNA from wild-type (+/+) and floxed (+/F78A) ES cells was digested with EcoRV and hybridized with the 5′ (RH5) and the 3′ (BE6) probes shown in A. The sizes of hybridized fragments are indicated in kilobases. (C) Genotypes of offspring from cripto+/F78A heterozygote intercrosses. Primers c and d amplified a 280-bp fragment of the wild-type allele and a 400-bp fragment of the Cre-deleted allele. (D) Spatial distribution of both wild-type (left) and F78A mutant (right) Cripto protein. Wild-type and criptoF78A/F78A embryos were immunostained with anti-Cripto antibodies at 6.75 dpc. Bars, 50 μm.

Mentions: To unravel the complex network of molecular interactions of Cripto with its target proteins in vivo, the amino acid residue F78, which is located in the EGF-like domain, was substituted by alanine (F78A) using Cre/loxP-mediated recombination (Fig. 1, A–C). The resulting heterozygous cripto+/F78A mice appeared phenotypically normal and were fertile; however, homozygosity for the criptoF78A-targeted allele resulted in embryonic lethality. We first verified the expression of the mutated allele in vivo by whole-mount immunohistochemistry analysis. Cripto protein was consistently detected in homozygous criptoF78A/F78A embryos, although its expression remained confined to the proximal epiblast (Fig. 1 D). Although this result indicates that the alanine substitution does not abolish the synthesis or stability of Cripto protein, expansion of the expression domain to the distal epiblast is clearly compromised. Upon dissection, criptoF78A/F78A mutants were recovered at the expected mendelian ratio until 10.5 d past confluence (dpc) and later were resorbed (Table I). However, at 7.5 dpc they already displayed ectopic folds in the embryonic region (Fig. 2, A and A′). At 8.5 dpc, mutant embryos failed to turn and the neural folds were enlarged (Fig. 2, B and B′), apparently at the expense of mesodermal structures, because somites and a beating heart were absent. These results show that residue F78 of Cripto is essential for postimplantation development.


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)

Generation ofcriptoF78A/F78Amutant mice by homologous recombination in ES cells. (A) Strategy for inserting the F78A substitution at the cripto locus. Numbered boxes represent wild-type exons (black) and the mutant exon carrying the F78A substitution (gray). Location of PCR primers (black arrowheads) and probes (black bars) used for genotyping are indicated. B, BamHI; E, EcoRV; H HindIII; P, PstI. (B) Genotyping of cell lines by Southern blot analysis. Genomic DNA from wild-type (+/+) and floxed (+/F78A) ES cells was digested with EcoRV and hybridized with the 5′ (RH5) and the 3′ (BE6) probes shown in A. The sizes of hybridized fragments are indicated in kilobases. (C) Genotypes of offspring from cripto+/F78A heterozygote intercrosses. Primers c and d amplified a 280-bp fragment of the wild-type allele and a 400-bp fragment of the Cre-deleted allele. (D) Spatial distribution of both wild-type (left) and F78A mutant (right) Cripto protein. Wild-type and criptoF78A/F78A embryos were immunostained with anti-Cripto antibodies at 6.75 dpc. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234230&req=5

fig1: Generation ofcriptoF78A/F78Amutant mice by homologous recombination in ES cells. (A) Strategy for inserting the F78A substitution at the cripto locus. Numbered boxes represent wild-type exons (black) and the mutant exon carrying the F78A substitution (gray). Location of PCR primers (black arrowheads) and probes (black bars) used for genotyping are indicated. B, BamHI; E, EcoRV; H HindIII; P, PstI. (B) Genotyping of cell lines by Southern blot analysis. Genomic DNA from wild-type (+/+) and floxed (+/F78A) ES cells was digested with EcoRV and hybridized with the 5′ (RH5) and the 3′ (BE6) probes shown in A. The sizes of hybridized fragments are indicated in kilobases. (C) Genotypes of offspring from cripto+/F78A heterozygote intercrosses. Primers c and d amplified a 280-bp fragment of the wild-type allele and a 400-bp fragment of the Cre-deleted allele. (D) Spatial distribution of both wild-type (left) and F78A mutant (right) Cripto protein. Wild-type and criptoF78A/F78A embryos were immunostained with anti-Cripto antibodies at 6.75 dpc. Bars, 50 μm.
Mentions: To unravel the complex network of molecular interactions of Cripto with its target proteins in vivo, the amino acid residue F78, which is located in the EGF-like domain, was substituted by alanine (F78A) using Cre/loxP-mediated recombination (Fig. 1, A–C). The resulting heterozygous cripto+/F78A mice appeared phenotypically normal and were fertile; however, homozygosity for the criptoF78A-targeted allele resulted in embryonic lethality. We first verified the expression of the mutated allele in vivo by whole-mount immunohistochemistry analysis. Cripto protein was consistently detected in homozygous criptoF78A/F78A embryos, although its expression remained confined to the proximal epiblast (Fig. 1 D). Although this result indicates that the alanine substitution does not abolish the synthesis or stability of Cripto protein, expansion of the expression domain to the distal epiblast is clearly compromised. Upon dissection, criptoF78A/F78A mutants were recovered at the expected mendelian ratio until 10.5 d past confluence (dpc) and later were resorbed (Table I). However, at 7.5 dpc they already displayed ectopic folds in the embryonic region (Fig. 2, A and A′). At 8.5 dpc, mutant embryos failed to turn and the neural folds were enlarged (Fig. 2, B and B′), apparently at the expense of mesodermal structures, because somites and a beating heart were absent. These results show that residue F78 of Cripto is essential for postimplantation development.

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