Lagna G et al. (1999), Negative regulation of axis formation and Wnt s...

XB-ART-13299

Mech Dev 1999 Jan 01;801:101-6. doi: 10.1016/s0925-4773(98)00208-1.

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Negative regulation of axis formation and Wnt signaling in Xenopus embryos by the F-box/WD40 protein beta TrCP.

Lagna G , Carnevali F , Marchioni M , Hemmati-Brivanlou A .

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Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.

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Species referenced: Xenopus
Genes referenced: btrc ctnnb1 gsk3b nodal3.2 not sia1 tbxt wnt1 wnt8a

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	Fig. 1. The 58DP clone induces partial secondary axes. Dorsal view of stage 35 tadpoles. The red arrows point to the dorsal axes induced by injection of 1 ng of 58DP RNA in the ventral side of 4-cell stage Xenopus embryos.
	Fig. 3. x-bTrCP-2 inhibits dorsal axis formation. 5 ng of full-length x- bTrCP-2 RNA were injected either in the ventral or the dorsal side of four- cell stage Xenopus embryos. Tadpoles injected dorsally display a severely reduced axis. Ventral injection did not phenotypically affect development. Injection of less than 5 ng of RNA resulted in less severe phenotypes; the requirement of a high dose of full-length x-bTrCP-2 RNA is presumably due to the low amount of protein expressed from this construct (data not shown).
	Fig. 4. The dominant negative mutant DN209 induces secondary dorsal axes. One nanogram of DN209-x-bTrCP-2 RNA was injected either in the ventral (VMZ) or the dorsal (DMZ) marginal zone of 4-cell stage Xenopus embryos. VMZ injection resulted in a high frequency of secondary axes (81%; n = 48). Dorsally injected tadpoles have slightly thickened noto- chords, but are otherwise normal.
	Fig. 5. Dominant negative x-bTrCP-2 induces Wnt-responsive genes in ectodermal explants. RNAs encoding Xwnt8 (16 pg) and the DN209-xbTrCP- 2 mutant (1 ng) were injected in the animal pole of two-cell stage embryos. Ectodermal explants were prepared at blastula stage and processed for RT-PCR when sibling embryos reached the early gastrula stage. Both Xwnt8 and DN209 induce siamois and xnr3, but not the mesodermal marker brachyury. EF-1a is the loading control.