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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 lateral mesoderm 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 lateral mesoderm 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 lateral mesoderm 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). |
