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

Analysis of gastrulation defects in criptoF78A/F78A mutants reveals a hypomorphic phenotype. Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos at 7.5 dpc stained by whole-mount mRNA in situ hybridization. All embryos are shown with anterior side toward the left. (A, A′, and A″) Double staining of Brachyury (blue) and Otx2 (red) mRNAs in the primitive streak and anterior neurectoderm, respectively, reveals normal A–P patterning in wild-type (A) and criptoF78A/F78A embryos (A′). In contrast, in cripto- mutants (A″), cells expressing Brachyury and Otx2 remain in the proximal and distal epiblast, respectively. (B, B′, and B″) Double staining for Nodal (blue) and Otx2 (red) in wild-type embryos marks the node and the anterior neuroectoderm, respectively (B). In criptoF78A/F78A embryos (B′), Nodal mRNA is expressed in a group of posterior cells, whereas it is confined to the embryonic–extraembryonic boundary in cripto- mutants (B″). (C, C′, and C″) Double staining of Foxa2 (blue) and Brachyury (red). (C) Foxa2 is expressed in the node and axial mesendoderm of wild-type embryos. (C′) In criptoF78A/F78A embryos, Foxa2 marks both the distal primitive streak and the anterior side of the embryo. (C″) Foxa2 is not expressed in cripto- embryos. (D, D′, and D″) Double staining for Cer-1 (blue) and Brachyury (red) reveals that Cer-1 marks the anterior definitive endoderm in both wild-type (D) and criptoF78A/F78A (D′) embryos. In contrast, Cer-1 expression is absent in cripto- mutants (D″). (E, E′, and E″) Double staining for Chordin (blue) and Brachyury (red). Chordin marks the axial mesendoderm in wild-type (E) and criptoF78A/F78A (E′, arrowhead) embryos; however, the axial mesoderm is not expressed in cripto- mutants (E″). (s1 and s2) Transverse sections taken from the embryo in C′. Foxa2 signal is localized throughout the anterior proximal region of the embryo, revealing the presence of mesoderm and definitive endoderm (s1). Brachyury expression is confined to posterior embryonic and extraembryonic mesoderm. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234230&req=5

fig3: Analysis of gastrulation defects in criptoF78A/F78A mutants reveals a hypomorphic phenotype. Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos at 7.5 dpc stained by whole-mount mRNA in situ hybridization. All embryos are shown with anterior side toward the left. (A, A′, and A″) Double staining of Brachyury (blue) and Otx2 (red) mRNAs in the primitive streak and anterior neurectoderm, respectively, reveals normal A–P patterning in wild-type (A) and criptoF78A/F78A embryos (A′). In contrast, in cripto- mutants (A″), cells expressing Brachyury and Otx2 remain in the proximal and distal epiblast, respectively. (B, B′, and B″) Double staining for Nodal (blue) and Otx2 (red) in wild-type embryos marks the node and the anterior neuroectoderm, respectively (B). In criptoF78A/F78A embryos (B′), Nodal mRNA is expressed in a group of posterior cells, whereas it is confined to the embryonic–extraembryonic boundary in cripto- mutants (B″). (C, C′, and C″) Double staining of Foxa2 (blue) and Brachyury (red). (C) Foxa2 is expressed in the node and axial mesendoderm of wild-type embryos. (C′) In criptoF78A/F78A embryos, Foxa2 marks both the distal primitive streak and the anterior side of the embryo. (C″) Foxa2 is not expressed in cripto- embryos. (D, D′, and D″) Double staining for Cer-1 (blue) and Brachyury (red) reveals that Cer-1 marks the anterior definitive endoderm in both wild-type (D) and criptoF78A/F78A (D′) embryos. In contrast, Cer-1 expression is absent in cripto- mutants (D″). (E, E′, and E″) Double staining for Chordin (blue) and Brachyury (red). Chordin marks the axial mesendoderm in wild-type (E) and criptoF78A/F78A (E′, arrowhead) embryos; however, the axial mesoderm is not expressed in cripto- mutants (E″). (s1 and s2) Transverse sections taken from the embryo in C′. Foxa2 signal is localized throughout the anterior proximal region of the embryo, revealing the presence of mesoderm and definitive endoderm (s1). Brachyury expression is confined to posterior embryonic and extraembryonic mesoderm. Bars, 50 μm.

Mentions: Loss-of-function analysis has shown that cripto converts proximal-distal patterning into an A–P axis and promotes primitive streak formation (Ding et al., 1998; Liguori et al., 2003). To assess whether criptoF78A/F78A embryos have defects in axis formation, we examined the expression of asymmetrically expressed marker genes such as Brachyury and Otx2 at 7.5 dpc. In normal embryos, Brachyury marks the primitive streak, whereas expression of the anterior neural marker Otx2 by this stage is restricted to the opposite pole (Fig. 3 A; Wilkinson et al., 1990; Simeone et al., 1993). By comparison, cripto- mutants largely consist of anterior neuroectoderm (Ding et al., 1998; Liguori et al., 2003) and, therefore, ectopically express Otx2 throughout the distal embryonic region (Fig. 3 A″; Ding et al., 1998; Liguori et al., 2003), whereas the mesodermal marker Brachyury is only activated in a few cells along the embryonic–extraembryonic boundary (Fig. 3 A″; Ding et al., 1998). In contrast, in criptoF78A/F78A mutant embryos, Brachyury expression was normally posteriorized and persisted until 8.5 dpc, indicating the presence of posterior mesoderm populations that are missing in cripto- mutants (Fig. 3 A′; and Fig. S1, A, A′, and A″, available at http://www.jcb.org/cgi/content/full/jcb.200709090/DC1). In addition, Otx2 mRNA was consistently localized in the anterior region (Fig. 3 A′; and Fig. S1, A and A′), suggesting that A–P patterning is relatively normal. To monitor posterior neuroectoderm, we also analyzed the expression of Krox20, a marker of rhombomeres three and five, which is absent in cripto- mutants (Ding et al., 1998). Krox20 mRNA was clearly detected in criptoF78A/F78A embryos at 8.5 dpc (Fig. S1, B and B′). In addition, Mox1, a marker of paraxial mesoderm that fails to be induced in cripto- mutants, was expressed in the posterior region of criptoF78A/F78A embryos (Fig. S1, C and C′). These results demonstrate that criptoF78A/F78A homozygotes establish an A–P axis and arrest development at a later stage compared with 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)

Analysis of gastrulation defects in criptoF78A/F78A mutants reveals a hypomorphic phenotype. Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos at 7.5 dpc stained by whole-mount mRNA in situ hybridization. All embryos are shown with anterior side toward the left. (A, A′, and A″) Double staining of Brachyury (blue) and Otx2 (red) mRNAs in the primitive streak and anterior neurectoderm, respectively, reveals normal A–P patterning in wild-type (A) and criptoF78A/F78A embryos (A′). In contrast, in cripto- mutants (A″), cells expressing Brachyury and Otx2 remain in the proximal and distal epiblast, respectively. (B, B′, and B″) Double staining for Nodal (blue) and Otx2 (red) in wild-type embryos marks the node and the anterior neuroectoderm, respectively (B). In criptoF78A/F78A embryos (B′), Nodal mRNA is expressed in a group of posterior cells, whereas it is confined to the embryonic–extraembryonic boundary in cripto- mutants (B″). (C, C′, and C″) Double staining of Foxa2 (blue) and Brachyury (red). (C) Foxa2 is expressed in the node and axial mesendoderm of wild-type embryos. (C′) In criptoF78A/F78A embryos, Foxa2 marks both the distal primitive streak and the anterior side of the embryo. (C″) Foxa2 is not expressed in cripto- embryos. (D, D′, and D″) Double staining for Cer-1 (blue) and Brachyury (red) reveals that Cer-1 marks the anterior definitive endoderm in both wild-type (D) and criptoF78A/F78A (D′) embryos. In contrast, Cer-1 expression is absent in cripto- mutants (D″). (E, E′, and E″) Double staining for Chordin (blue) and Brachyury (red). Chordin marks the axial mesendoderm in wild-type (E) and criptoF78A/F78A (E′, arrowhead) embryos; however, the axial mesoderm is not expressed in cripto- mutants (E″). (s1 and s2) Transverse sections taken from the embryo in C′. Foxa2 signal is localized throughout the anterior proximal region of the embryo, revealing the presence of mesoderm and definitive endoderm (s1). Brachyury expression is confined to posterior embryonic and extraembryonic mesoderm. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Analysis of gastrulation defects in criptoF78A/F78A mutants reveals a hypomorphic phenotype. Wild-type (top), criptoF78A/F78A (middle), and cripto- (bottom) embryos at 7.5 dpc stained by whole-mount mRNA in situ hybridization. All embryos are shown with anterior side toward the left. (A, A′, and A″) Double staining of Brachyury (blue) and Otx2 (red) mRNAs in the primitive streak and anterior neurectoderm, respectively, reveals normal A–P patterning in wild-type (A) and criptoF78A/F78A embryos (A′). In contrast, in cripto- mutants (A″), cells expressing Brachyury and Otx2 remain in the proximal and distal epiblast, respectively. (B, B′, and B″) Double staining for Nodal (blue) and Otx2 (red) in wild-type embryos marks the node and the anterior neuroectoderm, respectively (B). In criptoF78A/F78A embryos (B′), Nodal mRNA is expressed in a group of posterior cells, whereas it is confined to the embryonic–extraembryonic boundary in cripto- mutants (B″). (C, C′, and C″) Double staining of Foxa2 (blue) and Brachyury (red). (C) Foxa2 is expressed in the node and axial mesendoderm of wild-type embryos. (C′) In criptoF78A/F78A embryos, Foxa2 marks both the distal primitive streak and the anterior side of the embryo. (C″) Foxa2 is not expressed in cripto- embryos. (D, D′, and D″) Double staining for Cer-1 (blue) and Brachyury (red) reveals that Cer-1 marks the anterior definitive endoderm in both wild-type (D) and criptoF78A/F78A (D′) embryos. In contrast, Cer-1 expression is absent in cripto- mutants (D″). (E, E′, and E″) Double staining for Chordin (blue) and Brachyury (red). Chordin marks the axial mesendoderm in wild-type (E) and criptoF78A/F78A (E′, arrowhead) embryos; however, the axial mesoderm is not expressed in cripto- mutants (E″). (s1 and s2) Transverse sections taken from the embryo in C′. Foxa2 signal is localized throughout the anterior proximal region of the embryo, revealing the presence of mesoderm and definitive endoderm (s1). Brachyury expression is confined to posterior embryonic and extraembryonic mesoderm. Bars, 50 μm.
Mentions: Loss-of-function analysis has shown that cripto converts proximal-distal patterning into an A–P axis and promotes primitive streak formation (Ding et al., 1998; Liguori et al., 2003). To assess whether criptoF78A/F78A embryos have defects in axis formation, we examined the expression of asymmetrically expressed marker genes such as Brachyury and Otx2 at 7.5 dpc. In normal embryos, Brachyury marks the primitive streak, whereas expression of the anterior neural marker Otx2 by this stage is restricted to the opposite pole (Fig. 3 A; Wilkinson et al., 1990; Simeone et al., 1993). By comparison, cripto- mutants largely consist of anterior neuroectoderm (Ding et al., 1998; Liguori et al., 2003) and, therefore, ectopically express Otx2 throughout the distal embryonic region (Fig. 3 A″; Ding et al., 1998; Liguori et al., 2003), whereas the mesodermal marker Brachyury is only activated in a few cells along the embryonic–extraembryonic boundary (Fig. 3 A″; Ding et al., 1998). In contrast, in criptoF78A/F78A mutant embryos, Brachyury expression was normally posteriorized and persisted until 8.5 dpc, indicating the presence of posterior mesoderm populations that are missing in cripto- mutants (Fig. 3 A′; and Fig. S1, A, A′, and A″, available at http://www.jcb.org/cgi/content/full/jcb.200709090/DC1). In addition, Otx2 mRNA was consistently localized in the anterior region (Fig. 3 A′; and Fig. S1, A and A′), suggesting that A–P patterning is relatively normal. To monitor posterior neuroectoderm, we also analyzed the expression of Krox20, a marker of rhombomeres three and five, which is absent in cripto- mutants (Ding et al., 1998). Krox20 mRNA was clearly detected in criptoF78A/F78A embryos at 8.5 dpc (Fig. S1, B and B′). In addition, Mox1, a marker of paraxial mesoderm that fails to be induced in cripto- mutants, was expressed in the posterior region of criptoF78A/F78A embryos (Fig. S1, C and C′). These results demonstrate that criptoF78A/F78A homozygotes establish an A–P axis and arrest development at a later stage compared with 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