Darras S et al. (1997), Animal and vegetal pole cells of early Xenopus ...

XB-ART-15924

Development 1997 Nov 01;12421:4275-86. doi: 10.1242/dev.124.21.4275.

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Animal and vegetal pole cells of early Xenopus embryos respond differently to maternal dorsal determinants: implications for the patterning of the organiser.

Darras S , Marikawa Y , Elinson RP , Lemaire P .

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The maternal dorsal determinants required for the specification of the dorsal territories of Xenopus early gastrulae are located at the vegetal pole of unfertilised eggs and are moved towards the prospective dorsal region of the fertilised egg during cortical rotation. While the molecular identity of the determinants is unknown, there are dorsal factors in the vegetal cortical cytoplasm (VCC). Here, we show that the VCC factors, when injected into animal cells activate the zygotic genes Siamois and Xnr3, suggesting that they act along the Wnt/beta-catenin pathway. In addition, Siamois and Xnr3 are activated at the vegetal pole of UV-irradiated embryos, indicating that these two genes are targets of the VCC factors in all embryonic cells. However, the consequences of their activation in cells that occupy different positions along the animal-vegetal axis differ. Dorsal vegetal cells of normal embryos or VCC-treated injected animal cells are able to dorsalise ventral mesoderm in conjugate experiments but UV-treated vegetal caps do not have this property. This difference is unlikely to reflect different levels of activation of FGF or activin-like signal transduction pathways but may reflect the activation of different targets of Siamois. Chordin, a marker of the head and axial mesoderm, is activated by the VCC/Siamois pathway in animal cells but not in vegetal cells whereas cerberus, a marker of the anterior mesendoderm which lacks dorsalising activity, can only be activated by the VCC/Siamois pathway in vegetal cells. We propose that the regionalisation of the organiser during gastrulation proceeds from the differential interpretation along the animal-vegetal axis of the activation of the VCC/beta-catenin/Siamois pathway.

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Species referenced: Xenopus
Genes referenced: cer1 chrd fgfr1 nodal3.1 nodal3.2 sia1 tbxt

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	Fig. 1. Animal caps injected with vegetal cortical cytoplasm express Siamois and Xnr3 but not Xbra. Animal caps from embryos injected with vegetal cortical cytoplasm (VCC) or animal cortical cytoplasm (ACC) were analysed by RT-PCR at stage 10.5 for the expression of Siamois, Xnr3 and Xbra. Endogenous FGFR1 transcripts were used here as an internal standard. Analysis of the same samples by RNase protection gave similar results, as did independent northern analysis of Xnr3. + and – refers in this and the following figures to the presence or absence of reverse transcriptase, as a control for DNA contamination. WE, whole embryo; An, uninjected animal caps.
	Fig. 2. Expression of Xnr3 and Siamois in UV-irradiated gastrulae. (A) Expression in whole embryos. Fertilised eggs were irradiated and the expression of Xnr3, Siamois and the FGFR1 genes was assayed by RT-PCR at stage 10.25. In the experiment shown, the average dorsoanterior index (DAI) of the embryos was 0.27 at the tadpole stage. C, control embryo; U.V., irradiated embryo. (B) Distribution of the transcripts in control (C) or UV-irradiated (U.V.) early gastrulae (DAI=0.26). Vegetal pole explants were dissected at stage 10, and RNA from both these explants and the rest of the embryos was prepared at stage 10.25. Analysis of transcript abundance was carried out by RT-PCR. Siamois and Xnr3 transcripts are enriched at the vegetal pole of UV-irradiated embryos. (C) In situ hybridisation with an Xnr3 antisense RNA probe on sectioned control or UV-irradiated early gastrulae. Due to a better penetration of the probe on sectioned embryos, it can be observed that the distribution of Xnr3 transcripts in normal early gastrulae is not restricted to the epithelial outer cell layer, but extends into the deep marginal and vegetal cells. In UV treated embryos, Xnr3 transcripts are detected in the perinuclear space of cortical cells. A similar result was obtained with a Siamois probe (not shown). Note that in normal embryos, vegetal expression of Xnr3 extends nearly down to the vegetal pole.
	Fig. 3. Animal caps injected with vegetal cortical cytoplasm secrete a dorsalising signal. (A) Diagrammatic representation of the experiment. Ventral marginal zone explants (composed mostly of ventral mesodermal cells) were taken from embryos (stage 10.25) previously injected with the lineage tracer FLDx. The explants were immediately combined with blastula animal caps (stage 8.5) derived from embryos previously injected with animal or vegetal cortical cytoplasm (ACC, VCC). The conjugates were cultured until their mesodermal component reached the equivalent of stage 32 and they were then immunostained with the muscle antibody 12/101. (B) Photographs of sections through conjugates composed of a ventral marginal zone explant labelled with fluoresceine lysinated dextran (FLDx) combined with an animal cap derived either from uninjected embryos (none), from embryos injected with vegetal cortical cytoplasm (VCC), or from embryos injected with animal cortical cytoplasm (ACC). Left panels, bright-field images; middle panels, position of FLDx-labelled cells; right panels, 12/101 staining. Muscle-specific staining is seen only in the progeny of the FLDx-labelled marginal zone cells which were conjugated to the VCC-injected animal cap.
	Fig. 4. Dorsalising activity of dorsal marginal zone cells, dorsal vegetal cells or vegetal pole cells from UV-irradiated embryos. (A) Experimental strategy. Explants of ventral marginal zone (VMZ) were taken at stage 10 from embryos injected with the lineage tracer rhodamine lysinated dextran (RLDx). They were combined with either vegetal pole from UV-treated embryos (UV Veg Pole), dorsal vegetal cells from stage 8.5-9 embryos (D. Veg) or stage 10 dorsal marginal zone (DMZ). The conjugates were cultured until stage 30-35 and immunostained with the muscle antibody 12/101. (B) Photographs of sections through conjugates of ventral marginal zone explants (VMZ) with UV vegetal pole (top), dorsal vegetal cells (middle) or dorsal marginal zone explants (bottom). While DMZ and dorsal vegetal cells have a dorsalising activity, UV vegetal poles fail to dorsalise VMZ explants. Notochordal differentiation, monitored by the presence of highly vacuolated cells, is found in the DMZ explants but never observed in the dorsal vegetal explants. Left panels, 12/101 staining; right panels, position of RLDx labelled cells.
	Fig. 5. Dorsalising activity of animal or vegetal pole cells injected with Siamois or chordin mRNA. (A) Experimental strategy. Stage 10 ventral marginal cells (derived from embryos injected with RLDx) were combined either with animal cap (stage 8.5-9) derived from embryos previously injected in the animal pole with 900 pg of chordin mRNA or 100 pg Siamois mRNA or with vegetal cap derived from embryos injected similarly in the vegetal pole. The conjugates were cultured until stage 30-35 and immunostained with the muscle antibody 12/101. (B) Photographs of sections through conjugates of VMZ explants with chordin mRNA injected animal caps (chd an) or vegetal poles (chd veg), or with Siamois mRNAinjected animal caps (Sia An) or vegetal poles (Sia veg). While injection of chordin mRNA confers dorsalising activity to both animal and vegetal pole cells, injection of Siamois mRNA leads to the secretion of dorsalising signals in animal caps cells only. chd, chordin; Sia, Siamois. Left panels: 12/101 staining; right panels: RLDx labelled cells.
	Fig. 6. Activation of chordin and cerberus by Siamois is context dependent. (A) Cerberus is a target of Siamois. 4-cell embryos were injected with 100 pg of Siamois mRNA in a ventral vegetal location or with 100 pg of enR-Sia mRNA in a dorsal vegetal position. Control and injected embryos were reared until stage 10 before excising ventral vegetal cells when needed. Whole embryos (WE) and ventral vegetal explants from uninjected (VVZ) or Siamois-injected embryos (VVZ+Sia) were analysed at stage 11 for cerberus expression. (B) Chordin, but not cerberus, is activated by the VCC in animal caps. The same cDNA samples as in Fig. 1 were subjected to RT-PCR analysis for the expression of chordin and cerberus. Abbreviations are as in Fig. 1. (C) cerberus is expressed at the vegetal pole of UV-irradiated embryos. Control or UV-irradiated (U.V.) whole embryos (WE) as well as explants were analysed at stage 11 by RT-PCR. In the experiment shown on the left panel, chordin expression was undetectable in the UV-irradiated whole embryos (not shown). enR-Sia: embryos injected at the vegetal pole with 100 pg of enRSia mRNA.