Fig. 4: sFRP2 specifically increases Wnt3a attachment on cell surfaces in vivo.

a Schematic figures of mRNA microinjection into fertilized Xenopus eggs. GFP-Wnt3a mRNA and various mRNAs, including sFRP mRNAs without (a) or with (a’) heparinase III (HEPIII) mRNA were injected into different blastomeres at the four-cell stage of Xenopus embryos. In most experiments, except that shown in (j), injected embryos were fixed with MEMFA at stage 11.5. GFP-Wnt3a-expressing cells are marked by membrane-bound Ruby (mRuby) expression (red), while neighboring cells expressing sFRP or controls are identified by Lyn-mBFP expression (blue). b–e Effect of sFRP proteins on the distribution of GFP-Wnt3a on surfaces of neighboring cells. Images of mRuby (b–e) and GFP-Wnt3a (b’–e’), as well as their merged images (b”-e”) are indicated. A magnified image of the area surrounded by a white line in (c”) is also shown (c”’). Injection with sFRP2 (c), but not with sFRP3 (d) or sFRP4 (e), increases GFP-Wnt3a accumulation on the cell surface and internalization into the cell (white arrowheads), compared to controls (b). f, g Correlation in the spatial pattern of GFP-Wnt3a and HSPG at the surface of sFRP2-expressing cells. Immunostaining of fixed embryos injected with GFP-Wnt3a and sFRP2, with antibodies specific for N-acetyl-rich (NAH46; f) or N-sulfo-rich (HepSS-1; g) HS chains. Images of GFP-Wnt3a (f, g) and HS chains (f’, g’), as well as their merged images (f”, g”), are indicated. A magnified image of the areas surrounded by white lines in (f” and g”) is also shown in (f”’ and g”’). White arrowheads in (f”’) indicate co-localization of GFP-Wnt3a puncta and N-acetyl-rich HS puncta. h, i Effect of heparinase III (Hep III) on GFP-Wnt3a accumulation at the surface of sFRP2-expressing cells. Accumulation of GFP-Wnt3a on sFRP2-expressing cells was examined with (i) or without (h) expression of a membrane-tethered form of HepIII. Images of mRuby (h-1, i-1), GFP-Wnt3a (h-2, h-2), Lyn-mBFP (h-3, i-3), as well as merged images of mRuby and GFP-Wnt3a (h-5, i-5), are indicated. A magnified image of areas surrounded by white lines in (h-5 and i-5) is also shown in (h-6 and i-6). To confirm activity of HEPIII to remove the HS rich region, embryos were fixed and stained with NAH46 at stage 11.5 (h-4, i-4). Distribution of GFP-Wnt3a in sFRP2- expressing cells (h-2) was disrupted by expression of HEPIII (i-2). Scale bar; 100 μm. j Effect of sFRP2 in the exosome-mediated Wnt3a secretion in Xenopus embryos. Recovery of GFP-Wnt3a in the P100 fraction is shown. Equal numbers of embryos injected with GFP-Wnt3a with or without sFRP2 were dissociated at stage 11.5 and incubated another 5 h at 17 °C, followed by fractionation by ultracentrifugation at 100,000 × g. The P100 pellet was dissolved and subjected to Western blotting.