Fig. 7: RSPO2 requires ZNRF3 to antagonize BMP4-BMPR1A signaling.
From: R-spondins are BMP receptor antagonists in Xenopus early embryonic development
a BRE reporter assay in HEPG2 cells. Cells were transfected with siControl or siZNRF3/siRNF43, and BMP4 with or without RSPO1-4 were added overnight as indicated. Normalized BRE activity upon BMP4 without RSPO2 stimulation was set to 100%. n = 3 biologically independent samples. b BRE reporter assay in HEPG2 cells upon ZNRF3ΔR transfection, with or without overnight BMP4 and RSPO2 treatment as indicated. n = 3 biologically independent samples. c BMP-reporter (vent2) assay in Xenopus laevis St.15 neurulae. Embryos were injected animally with reporter plasmids and the indicated Mo with or without rspo2 mRNA at 4-cell stage. Normalized vent2 activity of control Mo injected embryos with reporter plasmids was set to 1. n = 3 biologically independent samples. d Immunofluorescence microscopy (IF) in Xenopus laevis animal cap explants for Bmpr1a (green) and the plasma membrane (red) from embryos injected with mRNA as indicated, with a representative cell (top) and magnification (inset). Scale bar, 20 μm. For scheme, see Fig. 6c. e Quantification of d. n = areas analyzed and data are displayed as mean ± SD. ns, not significant, ****P < 0.0001 from one-way ANOVA analysis. f–g BRE reporter assay in HEPG2 cells treated with BMP4 and RSPO2/RSPO2ΔFU1/RSPO2ΔFU2 overnight as indicated. For domain structure of RSPO2ΔFU1/RSPO2ΔFU2, see Supplementary Fig. 8a. n = 3 biologically independent samples. h IF for Bmpr1a (green) and plasma membrane (red) in animal cap explants injected as indicated, with a representative cell (top) and magnification (inset) showing the plasma membrane. Scale bar, 20 μm. For domain structure of Xenopus Rspo2 mutants, see Supplementary Fig. 8d. i Quantification of h. n = areas analyzed and data are displayed as mean ± SD. ns, not significant, ****P < 0.0001 from one-way ANOVA analysis. All data are displayed as mean ± SD. The P values for reporter assays (a–c, f, g) were determined from two-tailed unpaired t-test. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
