Brinkmann EM , Mattes B , Kumar R , Hagemann AI , Gradl D , Scholpp S , Steinbeisser H , Kaufmann LT , Özbek S .

Xla Wt + fzd7 MO (fig.4.a)
Xla Wt + fzd7 MO (fig.4.c)
Xla Wt + Hsa.sfrp2 (fig.1.a)
Xla Wt + Hsa.sfrp2 (fig.2.d)
Xla Wt + ror2 MO (fig.2.a, b, c)
Xla Wt + sfrp1 MO (fig.4.a)
Xla Wt + sfrp2 (fig.1.a)
Xla Wt + sfrp2 MO (fig.1.a)
Xla Wt + sfrp2 MO (fig.2.a, b, c, d)
Xla Wt + sfrp2 MO (fig.4.a,b)

	FIGURE 1: Gain and loss of sFRP2 impairs CE movements in Xenopus. A: Representative phenotypes of embryos injected into the dorsal equatorial zone at 4-cell stage with indicated morpholino oligonucleotides (Mo, 15ng) and synthetic mRNA (500pg sfrp2/hsfrp2 or 200pg for the Mo-rescue). B: Quantification of phenotypic analysis shown in A. Numbers of analyzed embryos are indicated on top of the bars. C: For the AC elongation assay, embryos were injected animally at 2-cell stage with indicated synthetic mRNAs (300pg) and excised at stage 9. ACs were cultured with or without Activin overnight to analyze elongation. D: Quantification of AC elongation shown in C. Categorized in not elongated (blue), partially elongated (white) and fully elongated (grey) ACs. Numbers of analyzed ACs are indicated on top of the bars. E: Analysis of xbra expression of 10 ACs as shown in C, harvested after two hours incubation in Activin. Chart E shows a representative with technical triplicates. This was confirmed by at least n=2 independent experiments with similar results using sibling animal caps of those shown in C+D. F: AC elongation assay of embryos injected with 100pg of each indicated synthetic mRNA. To balance injected mRNA quantities the respective amount of gfp mRNA was co-injected. Scoring and quantification as in C+D.
	FIGURE 2: sFRP2 is required for papc expression and enhances Ror2 mediated signaling A: Representative papc expression pattern analyzed by whole mount in situ hybridization of gastrula (stage 10.5) embryos injected at 4-cell stage in the dorsal equatorial region with indicated Mos (15ng). Arrowheads indicate site of injection. B: Quantification of in situ hybridization shown in A. Numbers of analyzed embryos are indicated on top of the bars. C+D: Relative expression of papc analyzed by qPCR in whole embryos injected with indicated Mos (15ng) and synthetic mRNA (200pg). E+F: Relative expression of papc analyzed by qPCR in AC explants injected with indicated synthetic mRNAs (500pg ror2/sfrp2 and 150pg wnt5a). G: Relative expression of ror2 in ACs from uninjected embryos cut at stage 9 and harvested after 14h with or without Activin (50ng/ml) when sibling embryos reached stage 25. H: ATF2 luciferase reporter assay of stage 12 gastrula embryos injected with the indicated synthetic mRNAs (500pg ror2/sfrp2 and 100pg wnt5a) and the ATF2 Firefly and TK Renilla luciferase reporter constructs. Charts C and G show the mean ± SD of three independent experiments. (*) indicates significance (p < 0.05) compared to controls. Charts D,E and F show a representative with technical triplicates. This was confirmed by at least n=2 (D and F) or n=3 (E) independent experiments in different batches of Xenopus laevis with similar results. Graph H shows the mean ± SD of biological triplicates of pools of 7 embryos each (*p < 0.05 to controls). This was confirmed in at least n=3 independent experiments in different batches of Xenopus laevis.
	FIGURE 3: sFRP2 interacts with Ror2 in HEK293T cells and stabilizes Wnt5a/Ror2 complexes. A: Co-IP in Hek293T cells transfected with Ror2-myc and HA-tagged sFRP2 (1μg each). Protein lysates were precipitated with a mouse antibody against myc or HA, respectively, and IgG as negative control. Western blot (WB) analysis with anti-HA antibody shows that sFRP2 is pulled down together with Ror2. B: IP from conditioned media generated in Hek293T cells transfected with either Ror2 ECD-flag or sFRP2-HA. Purified proteins were mixed and further precipitated with a mouse anti-HA antibody. WB analysis with rabbit anti-flag shows that purified sFRP2 binds to purified Ror2-ECD. As a negative control (Ctrl) Ror2 ECD-flag conditioned medium was precipitated with anti-HA-coupled magnetic beads and sFRP2-HA conditioned medium with anti-flag-coupled magnetic beads. C: The Wnt5a-V5 binding assay was performed in Hek293T cells transfected with Ror2 ECD-flag alone or co-transfected with sFRP2-HA, CRD-HA, DKK3-HA or Fz7-myc (1μg each). Each cell sample was treated with equal amounts of Wnt5a-V5 conditioned medium for 15 min before cells were lysed for rabbit anti-flag pull-down to precipitate Ror2. The samples were then analyzed on two different WBs: WB on top was analyzed with anti-V5 antibody for the Wnt5a-V5 fraction bound to precipitated Ror2- flag. The bottom WB was analyzed with mouse anti-flag and shows that equal amounts of Ror2 were precipitated in the different samples. 3D: The WB analyzed with mouse anti-HA/anti-myc antibody shows that all co-transfected proteins were expressed (input, first 4 lanes) and were co-precipitated with the Ror2-flag pull-down (IP: anti-flag, last 4 lanes). Only Dkk3 was not precipitated with Ror2. A scheme of the different proteins used in the experiment is shown below. sFRP family proteins are related to Fz receptors in the CRD. CRD: Cysteine-rich domain; NTR: netrin-like domain; TM: Transmembrane domain; CD: cytoplasmic domain; Cys: Cysteine-rich domain. E: Confocal microscopy analysis of live zebrafish embryos expressing 1ng mRNA of the indicated constructs at 30-50% epiboly stages shown in the indicated colours. Confocal images represent single z sections. Ror2-mCherry shows membrane localization regardless of the presence of Wnt5a or sFRP2. Wnt5a-GFP shows co-localization with Ror2-mCherry in discrete clusters at the membrane. Co-expression of sFRP2 with Ror2-mCherry/Wnt5a-GFP leads to an enhanced membrane localization of Wnt5a-GFP.
	FIGURE 4: sFRPs and Fz7 act redundantly to activate Ror2. A+B: Relative expression of papc analyzed by qPCR in gastrula stage 10.5 whole embryos injected dorsal equatorially with indicated Mos (15ng) and synthetic mRNA (200pg). C: papc expression of Fz7 morphants analyzed by whole mount in situ hybridization of gastrula stage embryos. Arrowheads indicate site of injection. D: Relative expression of papc analyzed by qPCR in AC explants injected with indicated synthetic mRNAs (500pg ror2/fz7 and 150pg wnt5a). Charts A, B and D show a representative with technical triplicates. This was confirmed by at least n=2 independent experiments in different batches of Xenopus laevis with similar results. E: Confocal microscopy analysis of DMZ explants of Xenopus embryos injected dorsal equatorially at 4- cell stage with 500pg Ror2-myc or 500pg sFRP2-HA. Endogenous Fz7 is shown in green and over-expressed Ror2 and sFRP2 are shown in the indicated colours.
	FIGURE 5: sFRP2 and Ror2 inhibit Fz7 mediated non-canonical Wnt signaling. A: Co-IP in Hek293T cells transfected with Fz7-myc and sFRP2-HA (1μg each). Protein lysates were precipitated with a mouse antibody against myc or HA respectively and IgG as negative control. WB analyzed with anti-HA antibody shows that sFRP2 is pulled down together with Fz7. B+C+D: qPCR analysis of AC explants of embryos injected with the indicated mRNAs (500pg n7c5/sfrp2/ror2/ror2 kd and 100pg wnt5a . E+F: ATF luciferase reporter assay of stage12 gastrula embryos injected with the indicated synthetic mRNAs (300pg fz7/ sfrp2/ror2/ror2 kd and 100pg wnt5a per embryo) and the ATF Firefly and TK Renilla luciferase reporter constructs. Charts B+C show the mean ± SD of n=3 independent experiments. (*) indicates significant difference (Student´s t test* p < 0.05, **p < 0.001) compared to controls. Chart D shows a representative with technical triplicates. This was confirmed by at least n=2 independent experiments in different batches of Xenopus laevis with similar results. Charts E+F show the mean of 3 ± SEM of biological triplicates of pools of 5 embryos each (*p < 0.05 to controls). This was confirmed in at least n=2 independent experiments in different batches of Xenopus laevis.
	FIGURE 6: sFRP2 and Ror2 inhibit Fz7 receptor endocytosis. A+D: Confocal microscopy analysis of live zebrafish embryos expressing 1ng mRNA of indicated constructs at 30%-50% epiboly stage together with 1ng mRNA of the membrane marker mem-mCherry (red) or GFP-GPI (blue). Fz7- CFP (green) is present at the membrane and in endocytic vesicles. Co-expression of Wnt5a leads to enhanced internalization. sFRP2 as well as Ror2-mCherry are able to decrease Wnt5a mediated endocytosis of Fz7-CFP. B+E: Relative quantification of Fz7-CFP/ membrane co-localization. Ctrl represents Fz7 alone. C: The Wnt5a-V5 binding assay was performed in Hek293T cells transfected with Fz7-myc alone or co-transfected with sFRP2-HA or Ror2-HA (1μg each). Each cell sample was treated with equal amounts of Wnt5a-V5 conditioned medium for 15 min before cells were lysed for rabbit anti-myc pull-down to precipitate Fz7. The samples were then analyzed on two different WBs: WB on top was analyzed with anti-V5 antibody for the Wnt5a-V5 fraction bound to precipitated Fz7- myc. The bottom WB was analyzed with a mouse anti-myc and shows that equal amounts of Fz7-myc were precipitated in the different samples. D: Co-expression of Wnt5a leads to enhanced internalization, which is repressed by full-length sFRP2 (also compare with Fig. 6A). The CRD of sFRP2 alone is unable to prevent Wnt5a induced stabilization of Fz7 at the membrane but appears to increase the size of Fz7 vesicles.
	Fig. 7: Model for selective Fz7- or Ror2-mediated activation of non-canonical Wnt signaling. A: When expressed alone, Fz7 or Ror2 receptors activate distinct non-canonical Wnt pathways stimulated by Wnt5a. B: When sFRP2 is present, Fz7 receptor endocytosis is prevented and Fz7 signaling is inhibited while Ror2 signaling is enhanced indicated by stabilized Wnt5a/Ror2 membrane complexes. C: When both receptors are present in the same cell the Ror2-CRD acts in a similar way as sFRP2 leading to increased Ror2 activation at the expense of Fz7 signaling.

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