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

Additional File 1:

SFRP_CRD peptides cannot rescue the Wnt8- or Wnt5-induced over-expression phenotype. All the panels are dorsal views of embryos at stage 19–20 (optic vesicle stage) injected with GFP mRNA alone or combined with Wnt8, Wnt5, Sfrp1_CRD-2, or Sfrp3_CRD mRNA as indicated. Note that Sfrp1_CRD-2 (b) behaves as Sfrp1_CRD (Figure 1c) in over-expression assays, while Sfrp3_CRD has no evident effect even at high concentrations (c; 300 ng/μl). Consistently, neither Sfrp1_CRD nor Sfrp3_CRD can rescue the phenotype induced upon Wnt8 (_D-F) or Wnt5 (_G-I) over-expression. See Tables 1 and 2 for details. Scale bar: 0.1 mm.

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Additional File 2:

Wnt8 binds to Sfrp1 and Sfrp1_NTR while Sfrp1_CRD binds to Frizzled 2. (a) Mixed conditioned media used in Figure 8a (see legend) were precipitated with a polyclonal anti-myc and blotted with a monoclonal anti-HA (upper panel). Controls for inputs (middle and lower panels) are the same as those described in Figure 8a(ii and iii). Wnt8-HA co-immunoprecipitated with both Sfrp1-myc and Sfrp1_NTR-myc while Sfrp1_CRD-myc did not. (b) HEK 293T cells were transiently co-transfected with Fz2-HA constructs together with Sfrp1-_myc, Sfrp1_CRD-myc or Sfrp1_NTR-myc. Proteins from cell lysates were precipitated with anti-HA and then blotted with anti-myc antibody. Note that Sfrp1 and Sfrp1_CRD (red asterisks) interact with Fz2 while the Sfrp1_NTR does not. (c) Conditioned media from mock transfected cells were mixed with Sfrp1-myc, Sfrp1_NTR-myc or Sfrp1_CRD-myc conditioned media (as above). Addition of anti-HA polyclonal antibodies did not cause unspecific immunoprecipitations as revealed by western blotting with anti-Myc monoclonal antibody. (d) Addition of anti-HA polyclonal antibodies did not cause unspecific immunoprecipitations in cell lysates from mock and Sfrp1-myc, Sfrp1_NTR-myc or Sfrp1_CRD-myc co-transfected cells as revealed by western blots with anti-Myc antibody.

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Additional File 3:

Wnt8/Fz5 mediated activation of β-catenin transcriptional activity in dissociated embryonic retinal cells is inhibited by soluble Sfrp1 and Sfrp1_NTR as well as by the Sfrp1_CRD. (a) E5 embryonic chick retinal cells were dissociated and co-transfected with a reporter plasmid containing 4xLef-1 responsive element, the control plasmid pRLTK and the effector plasmids for each condition. In retinal cells, endogenous β-catenin transcriptional activity is low and barely modified by transfection of Fz5 alone or by the co-transfection of Fz5 with Sfrp1, Sfrp1_CRD or Sfrp1_NTR. In contrast, strong reporter activation is observed upon Fz5 and Wnt8 co-transfection. (b) HEK 293T cells grown in 2% fetal calf serum were transfected with Sfrp1-myc, Sfrp1_CRD-myc, or Sfrp1_NTR-myc. Two days later the conditioned media were collected and similar amounts of proteins were added to dissociated retinal cell cultures co-transfected with a reporter plasmid (as above), pRLTK, Wnt8 and Fz5. TCF-luciferase activity was measured after 24 hours of incubation. Note how the conditioned media strongly inhibit reporter activities. Data represent means ± standard error from three separate experiments performed in triplicates. (c) Cells dissociated from E5 embryonic retinas were co-transfected with a reporter plasmid containing 4xLef-1 responsive element together with Wnt8, Fz5 (100 ng) in combination with the PCDNA plasmid alone (200 ng) or containing Sfrp1, zSizzled, Sfrp1_CRD or zSizzled_CRD as indicated in the graph. Wnt8/Fz5 co-transfection activated the reporter expression. This activation was significantly inhibited by the addition of Sfrp1 and Sfrp1_CRD while with less efficiency by the addition of zSizzled or zSizzled_CRD. Similar results were obtained with the Xenopus sizzled constructs. *p < 0.05; **p < 0.01; ***p < 0.001).

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