Tada M and Smith JC (2000), Xwnt11 is a target of Xenopus Brachyury: regula...

XB-ART-11183

Development 2000 May 01;12710:2227-38. doi: 10.1242/dev.127.10.2227.

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Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway.

Tada M , Smith JC .

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Gastrulation in the amphibian embryo is driven by cells of the mesoderm. One of the genes that confers mesodermal identity in Xenopus is Brachyury (Xbra), which is required for normal gastrulation movements and ultimately for posterior mesoderm and notochord differentiation in the development of all vertebrates. Xbra is a transcription activator, and interference with transcription activation leads to an inhibition of morphogenetic movements during gastrulation. To understand this process, we have screened for downstream target genes of Brachyury (Tada, M., Casey, E., Fairclough, L. and Smith, J. C. (1998) Development 125, 3997-4006). This approach has now allowed us to isolate Xwnt11, whose expression pattern is almost identical to that of Xbra at gastrula and early neurula stages. Activation of Xwnt11 is induced in an immediate-early fashion by Xbra and its expression in vivo is abolished by a dominant-interfering form of Xbra, Xbra-En(R). Overexpression of a dominant-negative form of Xwnt11, like overexpression of Xbra-En(R), inhibits convergent extension movements. This inhibition can be rescued by Dsh, a component of the Wnt signalling pathway and also by a truncated form of Dsh which cannot signal through the canonical Wnt pathway involving GSK-3 and (beta)-catenin. Together, our results suggest that the regulation of morphogenetic movements by Xwnt11 occurs through a pathway similar to that involved in planar polarity signalling in Drosophila.

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Species referenced: Xenopus
Genes referenced: dvl1 dvl2 gal.2 gsc myf5 odc1 tbxt wnt11 wnt11b
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	Fig. 1. Identification of Xwnt11 as a target of Xbra. (A) Induction of Xwnt11 by Xbra-GR. Animal caps derived from embryos injected with 50 pg Xbra-GR RNA or left uninjected were dissected at blastula stages and then treated with 10-6 M dexamethasone (DEX) for 3 hours or left untreated. RNA was extracted and analysed by northern blotting. ODC and Xbra served as loading and positive controls, respectively. Note that DEX alone does not induce expression of Xwnt11. (B-G) Comparison of expression patterns of Xwnt11 (B-D) and Xbra (E-G) at stage 11 (B,E), stage 12.5 (C,F) or stage 14 (D,G).
	Fig. 2. Inhibition of Xwnt11 expression by a dominant-interfering Xbra construct. (A) Expression of Xwnt11 is induced in animal pole regions by activin (8 units/ml) or Xbra-GR in the presence of DEX, and induction is inhibited by Xbra-EnR (500 pg RNA). (B,C) Downregulation of Xwnt11 by Xbra-EnR in the whole embryo. Embryos were injected with 500 pg b-gal RNA (B) or 500 pg Xbra- EnR RNA (C) into both blastomeres at the 2-cell stage. Expression of Xwnt11 was examined by in situ hybridisation at the early gastrula stage. Note that injection of Xbra-EnR RNA causes a dramatic reduction in Xwnt11 expression, although expression in dorsal superficial cells remains.
	Fig. 6. Effects of dn-wnt11 on whole embryos. (A) Control embryos injected with 2 ng b-gal RNA (upper) or embryos injected with 2 ng dn-wnt11 RNA (lower) cultured to stage 34. Note that dnwnt11 inhibits formation of posterior structures (76%, n=78). (D) Control b-gal-expressing embryo (left) or dn-wnt11-expressing embryo (right) cultured to stage 14. Note that dn-wnt11 inhibits gastrulation movements. (B,C) Control embryos injected with 2 ng b-gal RNA were stained at stage 34 with the notochord-specific monoclonal antibody MZ15 (B) or the muscle-specific monoclonal antibody 12/101 (C). (E,F) Embryos injected with dn-wnt11 RNA stained with MZ15 (E), or 12/101 (F). Dn-wnt11 does not affect notochord differentiation and although it reduces the size of the somites it does not affect muscle differentiation. (G,H,I) Embryos injected with 2 ng b-gal RNA into both blastomeres at the 2-cell stage stained for Xbra (G), goosecoid (H) or Myf-5 (I) at stage 10.5. (J,K,L) Embryos at stage 10.5 previously injected with 2 ng dn-wnt11 RNA and stained for Xbra (J), goosecoid (K) or Myf-5 (L). Note that dn-wnt11 affects none of these mesodermal markers.