XB-ART-42875Dev Cell. March 15, 2011; 20 (3): 303-14.
Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.
The R-Spondin (Rspo) family of secreted Wnt modulators is involved in development and disease and holds therapeutic promise as stem cell growth factors. Despite growing biological importance, their mechanism of action is poorly understood. Here, we show that Rspo3 binds syndecan 4 (Sdc4) and that together they activate Wnt/PCP signaling. In Xenopus embryos, Sdc4 and Rspo3 are essential for two Wnt/PCP-driven processes-gastrulation movements and head cartilage morphogenesis. Rspo3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK. Rspo3 functions by inducing Sdc4-dependent, clathrin-mediated endocytosis. We show that this internalization is essential for PCP signal transduction, suggesting that endocytosis of Wnt-receptor complexes is a key mechanism by which R-spondins promote Wnt signaling.
PubMed ID: 21397842
Article link: Dev Cell.
Genes referenced: acss2.2 adam11 dvl2 fzd7 mapk8 rspo3 sdc4 tbxt wnt5a
Morpholinos referenced: ap2m1 MO1 dvl2 MO1 fzd7 MO1 lrp6 MO1 rspo3 MO1 sdc4 MO1 sdc4 MO2 wnt5a MO1
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|Figure 2. R-Spondin 3 Is Required for Xenopus Gastrulation and Noncanonical Wnt Signaling (A) Loss of Rspo3 function causes gastrulation defects in Xenopus embryos. Top, 4-cell stage embryos were micro- injected equatorially into two dorsal blastomeres with 40 ng per embryo of Rspo3 Mo. Note the spina bifida phenotype with two tail ends (arrowheads) in embryos in- jected with Rspo3 Mo (61%, n = 66) but not control (0%, n = 45) embryos. Middle, in situ hybridization for Xbra at gastrula stage (stage 11). Control and Rspo3 Mo embryos showed 100% (n = 20) and 95% (n = 20) normal Xbra stain- ing, respectively. However, note the enlarged blastopore in the Rspo3 Morphant. Bottom, Rspo3 Mo inhibits elon- gation of Activin injected animal caps. (B) Rspo3 signaling requires Sdc4 and Wnt5a to induce gastrulation defects. Embryos were injected into two dorsal blastomeres at 4-cell stage with Morpholinos and/ or mRNA as indicated (40 ng Rspo3 Mo; 100 pg wild- type or dominant negative hSdc4 mRNA; 10 or 20 ng of Sdc4 Mo; 2.5 or 10 ng of Wnt5a Mo [+, and ++]); 250 pg xRspo3 mRNA per embryo were used). (E) Confocal microscopy cell protrusion assay. Dorsal marginal zones at stage 10.5 from embryos injected with membrane-RFP mRNA and 40 ng Rspo3 Mo or 10 ng Wnt5a Mo and/or 50 pg hRspo3 mRNA per embryo were dissociated. White arrowheads indicate protrusions. Right, Quantification of protrusions from three indepen- dent experiments (between 300 and 600 cells counted for each bar). Standard deviation of the mean is indicated. VMZ, ventral marginal zone for control. (F) Top, nuclear phospho-JNK immunostaining in stage 10.5 dorsal mesoderm from embryos dorsally injected with indicated Morpholinos or mRNAs. Bottom, nuclear Hoechst stain.|
|Figure 3. R-Spondin 3 Is Required for Head Cartilage Morpho- genesis (A and B) Tadpole stage Xenopus embryos injected at 8-cell stage with 10 ng per embryo of Rspo3 Mo into the animal four blastomeres. Note reduced head in Rspo3 Morphants (68%, n = 98) compared to Co Mo (0.1%, n = 85). (C, D, G, and H) Ceratobranchial cartilage was stained with Alcian blue and dissected from stage 46 embryos injected at 8-cell stage animally with either 10 ng of Rspo3 Mo or 2.5 ng of Wnt5a Mo per embryo. Note compact cartilage elements. (E, F, I, and J) Ceratobranchial cartilage was dissected from Morpholino injected embryos and flattened. The length-to-width ratios (R) of chondrocytes were determined and are indicated. (E0 and F0) Schematic drawing of cell outlines of (E) and (F). Abbreviations: br, ceratobranchial cartilage; ba, basihyal cartilage.|
|Figure 5. Rspo3 Requires Clathrin-Mediated Endocytosis to Induce Phospho-JNK (A) Confocal microscopy of dissociated animal cap cells treated with hRspo3DC-SNAP549 protein for 1 hr (orange, arrowheads in CoMo). 4-cell stage embryos were injected animally with membrane-bound Venus mRNA (green) and indicated Morpholinos (Mo) (20 ng of Sdc4; 20 ng of Fz7; 5 ng of Wnt5a). The average number of vesicles per cell is indicated in the graph. Data with standard error of the mean are shown. (B) Confocal microscopy of Dvl-GFP (green) in stage 8 Xenopus animal caps. 4-cell stage embryos were injected animally with indicated Mo and/or mRNAs together with Dvl-GFP mRNA. (C and D) Confocal microscopy of animal cap (AC) cells from embryos injected at 4-cell stage with indicated Mo or mRNA (C) or treated with endocytosis inhibitors (D) as described in Experimental Procedures. Dissociated AC cells, embryos were injected animally with membrane-bound Venus mRNA (green); ACs were explanted and dissociated at stage 8, incubated for 1 hr with hRspo3DC-SNAP549 (red), fixed, and analyzed. AC tissue, whole stage 8 ACs were treated either with Protein A (top two panels) or recombinant hRspo3DC-streptag-PrA2 (Rspo3 treatment) and immunostained with anti-pJNK antibody, as described in Experimental Procedures. Cells and tissues were counter-stained with Hoechst (blue). Note that clathrin inhibitorreated cells (AP2m2 Mo, MDC) bind and cluster Rspo3 at the surface but fail to internalize the protein and to induce phospho-JNK.|