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Development
1998 Dec 01;12523:4595-605. doi: 10.1242/dev.125.23.4595.
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Suppression of GATA factor activity causes axis duplication in Xenopus.
Sykes TG
,
Rodaway AR
,
Walmsley ME
,
Patient RK
.
Abstract
In Xenopus, the dorsoventral axis is patterned by the interplay between active signalling in ventral territories, and secreted antagonists from Spemann's organiser. Two signals are important in ventral cells, bone morphogenetic protein-4 (BMP-4) and Wnt-8. BMP-4 plays a conserved role in patterning the vertebrate dorsoventral axis, whilst the precise role of Wnt-8 and its relationship with BMP-4, are still unclear. Here we have investigated the role played by the GATA family of transcription factors, which are expressed in ventralmesendoderm during gastrulation and are required for the differentiation of blood and endodermal tissues. Injection ventrally of a dominant-interfering GATA factor (called G2en) induced the formation of secondary axes that phenocopy those induced by the dominant-negative BMP receptor. However, unlike inhibiting BMP signalling, inhibiting GATA activity in the ectoderm does not lead to neuralisation. In addition, analysis of gene expression in G2en injected embryos reveals that at least one known target gene for BMP-4, the homeobox gene Vent-2, is unaffected. In contrast, the expression of Wnt-8 and the homeobox gene Vent-1 is suppressed by G2en, whilst the organiser-secreted BMP antagonist chordin becomes ectopically expressed. These data therefore suggest that GATA activity is essential for ventral cell fate and that subsets of ventralising and dorsalising genes require GATA activity for their expression and suppression, respectively. Finally, using G2en, we show that suppression of Wnt-8 expression, in conjunction with blocked BMP signalling, does not lead to head formation, suggesting that the head-suppressing Wnt signal may not be Wnt-8.
Fig. 2. Phenotype of G2en injected embryos. (a) Stage 34 embryo injected into both ventral blastomeres at the 4-cell stage with 50 pg G2en (bottom) develops a secondary axis (black arrows), whereas G2f has no axis inducing activity (top), but occasionally produces vesicles in ventral tissues that are typical of RNA injection. (b) St 26 G2en-injected embryos have secondary neural tubes as shown by NCAM in situ hybridisation. (c) Transverse section through a G2en-injected embryo, showing lack of notochord formation in the secondary (right-hand) axis. Secondary axes contain ectopic gut lumen (gl), muscle (mu) and neural tube (nt). (d) G2en can partially rescue axis formation in embryos ventralised with UV treatment. Top is a stage 38 control embryo, left is an uninjected UV-treated embryo lacking all axial structures, right is a UV-treated G2en injected embryo showing partial axis rescue.
Fig. 3. G2en dorsalises mesoderm but not ectoderm. (a) G2en (50- 100 pg) and Enr (200-400 pg) did not induce neural tissue in ectoderm explants (animal caps) cultured to Stage 23, unlike injection of deltaBMPR (500 pg), as judged by the upregulation of the pan-neural marker NCAM, assayed by RNase protection. The low signal in the whole embryo lane reflects dilution by non-neural tissues compared to the ectoderm-only explants. (b) VMZ explants from embryos injected with the same dose of G2en as above (50 pg) became elongated (lower left, 20/22 explants) in a similar manner to dorsal marginal zone explants (DMZ, lower right, 21/22 elongated). VMZ explants from uninjected embryos remained spherical (top left, 0/22 elongated) as did VMZ explants from embryos injected with Enr (top right, 0/22 elongated). Explants are stage 23 equivalent. (c) The explants in b were probed for cardiac actin expression by RNase protection, using cytoskeletal actin as a loading control. G2en but not Enr induced cardiac actin expression in VMZ explants, to levels equivalent to those seen in DMZ explants, confirming dorsalisation.
Fig. 4. Expression of ventralising genes in G2en-injected embryos at stage 10.5. Panels are in pairs, with uninjected embryos on the left; all embryos are viewed from the vegetal pole, with dorsal at the top. (a) Vent-1 expression is seen in ventral and lateralmesoderm and in endoderm. (b) G2en injection represses expression of Vent-1 (16/20 embryos affected), in both mesoderm and endoderm. (c) Wnt-8 expression is seen in ventral and lateralmesoderm and in endoderm. (d) Injection of G2en represses expression of Wnt-8 in both mesoderm and endoderm (14/23 embryos affected). (e) Vent-2 expression is seen in ventral and lateralmesoderm but not endoderm. The probe used was from the Xom cDNA (Ladher et al., 1996), which is 100% identical to Vent-2. (f) Vent-2 expression is unaffected by G2en (0/13 embryos affected). (g) Quantitation of levels of gene expression in G2en-injected VMZ explants at stage 10.5 by RT-PCR. G2en injection results in the suppression of Wnt-8 in VMZ explants, whereas the expression of BMP-4 was unaffected (lanes 1 and 2). In addition, G2en induced the expression of chordin (chd). Note that in G2en-injected VMZ explants (lane 2) that the amplification products are slightly underloaded, as judged by levels of FGFR1 expression. All products were amplified over 27 cycles, except chordin (chd) which was amplified for 26. No non-specific products were seen in any reactions, and no products from genomic DNA were generated, as judged by lack of amplification in the −RT lane (lane 5). All amplifications were performed in the linear range of the reaction, as shown by two-fold increases in product from stage 10.5 whole embryo cDNA (lanes 6-8).
Fig. 5. Expression of dorsalising genes in G2en-injected embryos at stage 10.5. Panels are in pairs, with uninjected embryos on the left; all embryos are viewed from the vegetal pole, with dorsal at the top. (a) Goosecoid expression in the organiser. (b) Goosecoid is unaffected by G2en injection (0/25 embryos affected). (c) Noggin expression. (d) Noggin is also unaffected by G2en (0/22 embryos affected) (e) Chordin expression. (f) Ectopic chordin expression is seen on the ventral side in embryos injected with G2en (arrowhead) (12/19 embryos affected).
