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doi: 10.1242/10.1242/dev.00435

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Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Bisei Ohkawara1, Takamasa S. Yamamoto1, Masazumi Tada2 and Naoto Ueno1,*

1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan
2 Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK



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  		Fig. 1. Xenopus glypican 4 (Xgly4) is a novel member of the glypican family. (A) Phylogenetic tree of the glypican family. (B) The deduced 556 amino acid protein has a typical glypican structure with an N-terminal signal peptide (underline), an extracellular cysteine (red)-rich domain and three putative O-glycosylation sites (purple).

 


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  		Fig. 2. Xgly4 is expressed in the dorsal mesoderm and ectoderm. Expression pattern of Xgly4 during Xenopus early development was analyzed by northern blot analysis (A) and whole-mount in situ hybridization (B). Numbers indicate developmental stage. In addition to expression in the deep layer of dorsal ectoderm, expression of Xgly4 was detected in the dorsal mesoderm and also weakly detected in the ventral mesoderm (cross-section along DV axis at stage 11; upper right in B). V, ventral; D, dorsal; A, anterior; P, posterior.

 


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  		Fig. 3. Expression of Xgly4 is induced by dorsalizing signals during gastrulation. Injections of ß-gal (100 pg; red) mRNA alone into ventral (A) or dorsal (E) regions did not affect Xgly4 expression pattern. However, expression of Xgly4 (purple) was detected by whole-mount in situ hybridization in the ventral side of embryos dorsalized with mRNA injection of Xnr1 (100 pg; B), ß-catenin (100 pg; C) or the dominant-negative BMP receptor ALK3 (100 pg; D) with ß-gal mRNA into the ventral region. Expression of Xgly4 was suppressed in embryo ventralized with Bmp4 mRNA (100 pg; F) and ß-gal mRNA injected into the dorsal region. Arrows indicate the injection sites.

 


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  		Fig. 4. Inhibition of Xgly4 translation blocks gastrulation movements. (A) Western blot analysis of Xgly4 protein tagged with a Flag epitope at the C terminus using an anti-Flag antibody. Translation of the injected globin-Flag (50 pg) and Xgly4-Flag (500 pg) mRNA lacking the 5' UTR sequence was not inhibited by the morpholino oligonucleotide Xgly4Mo (41 ng), directed against the 5' untranslated region and the first methionine region of Xgly4 mRNA. This finding indicates that Xgly4Mo does not inhibit translation nonspecifically. (B) Embryos injected with Xgly4Mo (41 ng) and Xgly4 mRNA (1 ng) at stage 35/36. Xgly4 morpholino oligonucleotide, which was injected into dorsal region, blocked gastrulation movements. (C) In situ hybridization for Xvent1 at stage 11, XmyoD at stage 11, Xwnt11 at stage 11, Xbra at stage 11, Xen2 at stage 13 and XmyoD at stage 25 in uninjected embryos (left) and embryos injected with XglyMo (41ng; right). (D) Injection of Xgly4 mRNA rescues the phenotypes of the knypek (kny) mutant. Embryos from crossing of knym119 heterozygotes were injected with 20 pg Xgly4 RNA at one-cell stage and scored at pharyngula stage. Twenty-five percent of uninjected embryos showed the kny mutant phenotype (n=64) (Topczewski et al., 2001Go), whereas none of injected embryos showed the phenotype. Rather, injected embryos were indistinguishable from wild-type embryos except that they occasionally exhibited a slightly curly tip of the tail (n=112). This result suggests that Xgly4 is a functional homolog of the zebrafish kny gene.

 


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  		Fig. 5. Both Xgly4Mo and wild-type overexpression inhibit convergent extension movements. (A) Activin (0.25 pg)-induced elongation of AC explants (top right) mimics the convergent extension movements seen during gastrulation. This elongation was inhibited by Xgly4Mo (41 ng; middle left). The inhibition was rescued by a low dose (25 pg) injection of Xgly4 (middle right). A higher dose (500 pg) of Xgly4 inhibited activin-induced elongation (bottom left), but a low dose (25 pg) did not (bottom right). (B) Expression levels of Xbra, Xwnt11 and Histone (control) were analyzed by RT-PCR assay. The expression levels of Xbra and Xwnt11 were not affected in any of the explants shown in A.

 


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  		Fig. 6. (A) Xgly4 enhances the Xwnt11-induced convergent extension movements. (B) Severity of affected embryos. The phenotypes were graded according to the index of severity from 0 (normal) to 3 (spina bifida). Number of each grade was indicated as percentage. Although co-injection of Xgly4 mRNA at a low dose (0.5 pg) enhanced the defective gastrulation movements caused by injecting Xwnt11 mRNA (1 ng), co-injection of Xgly4 mRNA at a high dose (25 pg) rescued these defects. This indicates that the Xgly4 protein acts as positive modulator, but inhibits Xwnt11 signaling at a high dose, just as Knypek does in silberblick (zebrafish wnt11) signaling (Topczewski et al., 2001Go).

 


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  		Fig. 7. Inhibition of Xgly4 translation disturbs the membrane accumulation of the Xdsh protein in the dorsal mesoderm. (A-D) The Xdsh protein accumulated at the cell membrane of dorsal mesoderm cells during gastrulation (data not shown). Injection of the dominant-negative Xwnt11 mRNA (dn-Xwnt11; 500 pg; B), Xgly4Mo (41 ng; C), or Xgly4 mRNA (500 pg; D) disturbed the Xdsh protein localization. The accumulation of Xdsh protein was not affected by the control morpholino oligonucleotide (A). The smeared staining in cytoplasm was observed only in co-injected cells but not in cells injected with Xdsh alone, which had previously been injected with Xgly4Mo only in one side of dorsal blastmeres at the four-cell-stage (data not shown). (E) The intensity of Xdsh staining in the cytoplasm and membrane in each cell was quantitated by using NIH image.

 


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  		Fig. 8. Xgly4 inhibition of elongation is rescued by the activation of the non-canonical, but not the canonical, Wnt pathway. It has previously been shown that Xdsh{Delta}DIX cannot activate the canonical Wnt pathway, whereas {Delta}DEP cannot activate the non-canonical pathway. Inhibition of activin-induced elongation (top left) by Xgly4Mo (41 ng; top right) was rescued by {Delta}DIX (50 pg; middle right) but not by {Delta}DEP (50 pg; middle left), which suggests that Xgly4 regulates the convergent extension movements by acting as a positive modulator of the non-canonical Wnt pathway upstream of Dsh. When injected alone, {Delta}DEP (bottom left) and {Delta}DIX (bottom right) had no effect.

 


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  		Fig. 9. Physical and functional interaction of Xgly4 with Wnt11. (A) Xgly4 binds and co-precipitates Wnt11, Wnt5a or Wnt8. HA-tagged Wnts expressed in HEK293T cells were efficiently co-precipitated with Flag-tagged full-length Xgly4 as well as a Flag-tagged extracellular domain of Fz7. (B) Xgly4 interacts efficiently with the signaling receptor Fz7, but barely with ActRIB (ALK4) or with ActRIIA. (Top) Pull-down of each GST-fused receptor expressed in 293T cells with glutathione-Sepharose, followed by western blotting with anti-Flag antibody. (Middle) Expression levels of Xgly4 revealed with anti-Flag antibody. (Bottom) Expression levels of receptors revealed with anti-GST antibody. (C) The cysteine-rich domain (CRD) of Xgly4 protein functionally interacts with Wnt11 signaling. (Top) The construction of wild-type and mutant Xgly4 proteins. Line represents internal deletion, black box designates epitope flag-tag sequence, asterisks indicate the putative glycosylation sites and C represents cysteine residues. (Bottom) Activin-induced elongation (bottom left) was inhibited by co-injection with wild-type Xgly4 (500 pg; top middle), Xgly4{Delta}GAG (500 pg; bottom middle), or XGly4{Delta}C (500 pg; top right), but not with XGly4{Delta}CRD (500 pg; bottom right) mRNA.

 

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