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The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate.

Lopez-Rios J, Esteve P, Ruiz JM, Bovolenta P - Neural Dev (2008)

Bottom Line: This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR.In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC, Dr. Arce 37, Madrid, 28002, Spain. Javier.Lopez-Rios@unibas.ch

ABSTRACT

Background: Secreted frizzled related proteins (SFRPs) are multifunctional modulators of Wnt and BMP (Bone Morphogenetic Protein) signalling necessary for the development of most organs and the homeostasis of different adult tissues. SFRPs fold in two independent domains: the cysteine rich domain (SfrpCRD) related to the extracellular portion of Frizzled (Fz, Wnt receptors) and the Netrin module (SfrpNTR) defined by homologies with molecules such as Netrin-1, inhibitors of metalloproteinases and complement proteins. Due to its structural relationship with Fz, it is believed that SfrpCRD interferes with Wnt signalling by binding and sequestering the ligand. In contrast, the functional relevance of the SfrpNTR has been barely addressed.

Results: Here, we combine biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to show that the Sfrp1NTR mimics the function of the entire molecule, binds to Wnt8 and antagonizes Wnt canonical signalling. This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.

Conclusion: On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

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Related in: MedlinePlus

Sfrp1NTR but notSfrp1CRD, rescues the phenotype induced by knocking-down Sfrp1. (a-e') All the panels are dorsal (a-e) and lateral (a'-e') views of embryos at stage 19–20 injected with GFP mRNA alone (a,a'), Mo-olSfrp1 alone (b,b') or co-injected with Sfrp1 (c,c'), Sfrp1CRD (d,d') or Sfrp1NTR (e,e') mRNAs as indicated in the panels. Embryos were hybridised for rx3 (eye field) and foxA2 (axial mesoderm) both visualised in blue. Optic vesicles fail to develop in embryos injected with Mo-Sfrp1, as judged by the reduction in rx3 expression (b,b'). This defect is reverted by the co-injection of Sfrp1 and Sfrp1NTR mRNAs in 50% of the embryos (c,c',e,e',f) but not by that of Sfrp1CRD (d,d',f) mRNA, where the reduction of the eye field is even more pronounced than that observed with the Mo-Sfrp1 alone. Note that Sfrp1 mRNA not only rescues the effect of Mo-Sfrp1 but also induces a partial over-expression phenotype (compare (c,c') with Figure 2a,j). (f) Quantification of the rescue efficiency in the different conditions. Scale bar: 0.2 mm.
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Figure 4: Sfrp1NTR but notSfrp1CRD, rescues the phenotype induced by knocking-down Sfrp1. (a-e') All the panels are dorsal (a-e) and lateral (a'-e') views of embryos at stage 19–20 injected with GFP mRNA alone (a,a'), Mo-olSfrp1 alone (b,b') or co-injected with Sfrp1 (c,c'), Sfrp1CRD (d,d') or Sfrp1NTR (e,e') mRNAs as indicated in the panels. Embryos were hybridised for rx3 (eye field) and foxA2 (axial mesoderm) both visualised in blue. Optic vesicles fail to develop in embryos injected with Mo-Sfrp1, as judged by the reduction in rx3 expression (b,b'). This defect is reverted by the co-injection of Sfrp1 and Sfrp1NTR mRNAs in 50% of the embryos (c,c',e,e',f) but not by that of Sfrp1CRD (d,d',f) mRNA, where the reduction of the eye field is even more pronounced than that observed with the Mo-Sfrp1 alone. Note that Sfrp1 mRNA not only rescues the effect of Mo-Sfrp1 but also induces a partial over-expression phenotype (compare (c,c') with Figure 2a,j). (f) Quantification of the rescue efficiency in the different conditions. Scale bar: 0.2 mm.

Mentions: Morpholino (Mo)-based knock-down of Sfrp1 expression results in embryos with a reduced eye field associated, in the most affected embryos, with a shortening and widening of the antero-posterior axis [13] (compare Figure 4b,b' with Figure 4a,a'). Low concentrations of Sfrp1 mRNA are sufficient to completely rescue this phenotype in a large part of the embryos [13] (Figure 4c,c',f). If Sfrp1NTR can mimic the effect of the entire molecule, it should also be able to rescue the effects of Mo interference. Supporting this hypothesis, co-injection of Mo-Sfrp1 and Sfrp1NTR mRNA rescued the size of the eye field of the treated embryos with efficiency similar to that of Sfrp1 (Figure 4e,e',f). In contrast, Sfrp1CRD mRNA did not counteract the Mo-Sfrp1 induced phenotype (Figure 4d,d',f) and even appeared to exacerbate it, in line with the over-expression studies.


The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate.

Lopez-Rios J, Esteve P, Ruiz JM, Bovolenta P - Neural Dev (2008)

Sfrp1NTR but notSfrp1CRD, rescues the phenotype induced by knocking-down Sfrp1. (a-e') All the panels are dorsal (a-e) and lateral (a'-e') views of embryos at stage 19–20 injected with GFP mRNA alone (a,a'), Mo-olSfrp1 alone (b,b') or co-injected with Sfrp1 (c,c'), Sfrp1CRD (d,d') or Sfrp1NTR (e,e') mRNAs as indicated in the panels. Embryos were hybridised for rx3 (eye field) and foxA2 (axial mesoderm) both visualised in blue. Optic vesicles fail to develop in embryos injected with Mo-Sfrp1, as judged by the reduction in rx3 expression (b,b'). This defect is reverted by the co-injection of Sfrp1 and Sfrp1NTR mRNAs in 50% of the embryos (c,c',e,e',f) but not by that of Sfrp1CRD (d,d',f) mRNA, where the reduction of the eye field is even more pronounced than that observed with the Mo-Sfrp1 alone. Note that Sfrp1 mRNA not only rescues the effect of Mo-Sfrp1 but also induces a partial over-expression phenotype (compare (c,c') with Figure 2a,j). (f) Quantification of the rescue efficiency in the different conditions. Scale bar: 0.2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542364&req=5

Figure 4: Sfrp1NTR but notSfrp1CRD, rescues the phenotype induced by knocking-down Sfrp1. (a-e') All the panels are dorsal (a-e) and lateral (a'-e') views of embryos at stage 19–20 injected with GFP mRNA alone (a,a'), Mo-olSfrp1 alone (b,b') or co-injected with Sfrp1 (c,c'), Sfrp1CRD (d,d') or Sfrp1NTR (e,e') mRNAs as indicated in the panels. Embryos were hybridised for rx3 (eye field) and foxA2 (axial mesoderm) both visualised in blue. Optic vesicles fail to develop in embryos injected with Mo-Sfrp1, as judged by the reduction in rx3 expression (b,b'). This defect is reverted by the co-injection of Sfrp1 and Sfrp1NTR mRNAs in 50% of the embryos (c,c',e,e',f) but not by that of Sfrp1CRD (d,d',f) mRNA, where the reduction of the eye field is even more pronounced than that observed with the Mo-Sfrp1 alone. Note that Sfrp1 mRNA not only rescues the effect of Mo-Sfrp1 but also induces a partial over-expression phenotype (compare (c,c') with Figure 2a,j). (f) Quantification of the rescue efficiency in the different conditions. Scale bar: 0.2 mm.
Mentions: Morpholino (Mo)-based knock-down of Sfrp1 expression results in embryos with a reduced eye field associated, in the most affected embryos, with a shortening and widening of the antero-posterior axis [13] (compare Figure 4b,b' with Figure 4a,a'). Low concentrations of Sfrp1 mRNA are sufficient to completely rescue this phenotype in a large part of the embryos [13] (Figure 4c,c',f). If Sfrp1NTR can mimic the effect of the entire molecule, it should also be able to rescue the effects of Mo interference. Supporting this hypothesis, co-injection of Mo-Sfrp1 and Sfrp1NTR mRNA rescued the size of the eye field of the treated embryos with efficiency similar to that of Sfrp1 (Figure 4e,e',f). In contrast, Sfrp1CRD mRNA did not counteract the Mo-Sfrp1 induced phenotype (Figure 4d,d',f) and even appeared to exacerbate it, in line with the over-expression studies.

Bottom Line: This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR.In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC, Dr. Arce 37, Madrid, 28002, Spain. Javier.Lopez-Rios@unibas.ch

ABSTRACT

Background: Secreted frizzled related proteins (SFRPs) are multifunctional modulators of Wnt and BMP (Bone Morphogenetic Protein) signalling necessary for the development of most organs and the homeostasis of different adult tissues. SFRPs fold in two independent domains: the cysteine rich domain (SfrpCRD) related to the extracellular portion of Frizzled (Fz, Wnt receptors) and the Netrin module (SfrpNTR) defined by homologies with molecules such as Netrin-1, inhibitors of metalloproteinases and complement proteins. Due to its structural relationship with Fz, it is believed that SfrpCRD interferes with Wnt signalling by binding and sequestering the ligand. In contrast, the functional relevance of the SfrpNTR has been barely addressed.

Results: Here, we combine biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to show that the Sfrp1NTR mimics the function of the entire molecule, binds to Wnt8 and antagonizes Wnt canonical signalling. This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.

Conclusion: On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

Show MeSH
Related in: MedlinePlus