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The understanding of vertebrate development has greatly benefited from the study of gastrulation in the Xenopus embryo. Over the years, the molecular dissection of the Spemann organizer has proven to be a very fruitful source for gene discovery. Here, we report a comprehensive screen of gene expression in the Xenopus gastrula using cDNA macroarrays. Nylon filters containing more than 72000 cDNAs from a gastrula stage library were hybridized with differential probes from embryos in which organizer induction had been inhibited by reducing Nodal-related or maternal beta-Catenin signaling. Combining the changes in gene expression levels caused by these two major signaling pathways in a single graph identified both known and novel dorsoventral regulated genes. The most highly enriched organizer-specific genes were the secreted molecules chordin and Xnr-3, followed by the transmembrane protein paraxial protocadherin (PAPC). Ventral-specific abundant cDNAs included S10-40-H5, members of the Hyaluronan synthase family, Xvent-2 and XFD2/FoxI1. A differential probe of dorsal and ventral lips identified many more organizer-specific cDNAs than the screens inhibiting Nodal-related and beta-Catenin signaling, suggesting that additional, as yet uncharacterized signaling pathways, contribute to organizer formation. Finally, extension of this approach to the blastula preorganizer signaling center identified the transcription factor pintallavis/FoxA2 as a new preorganizer component.
Fig. 2. Macroarray screen of the maternal β-Catenin pathway. (A) xy-scatter plot of the intensities of the individual spots after hybridization with “LiCl” and “δN-XTcf-3” probes. Clones are categorized into five regions (I–V) depending on the extent of regulation by the maternal β-Catenin pathway. Note that chordin and Xnr-3 comprise most of the clones strongly regulated by β-Catenin (region V). (B–G) Whole-mount in situ hybridization of gastrula stage embryos left untreated (B, D, F) or treated with LiCl (C, E, G) and hybridized with xHAS-rs (B, C), XFD2/FoxI1a (D, E), and S10-45-H5 (F, G) probes. All embryos are shown from the side with dorsal to the right. (H) RT-PCR analysis of whole gastrula stage embryos injected radially with δN-XTcf-3 (800 pg, lane 2), siamois-ENR (120 pg, lane 3), and a combination of δN-XTcf-3 and siamois (200 pg, lane 4). EF1-α serves as RNA loading control.
Fig. 5. Macroarray analysis of dissected dorsal and ventral tissue. (A) Dorsal marginal zones (DMZ) and ventral marginal zones (VMZ) of stage 10.5 embryos were excised and dorsal- and ventral-specific probes prepared. (B) Graph of screen using DMZ and VMZ probes. Chordin and goosecoid were the two genes with greatest differential dorsal expression. The ventral genes XVent2, xHAS-1, and xHAS-rs are also indicated. The graph shows the analysis of a different filter from the one shown in the previous figures. (C–F) Whole-mount in situ hybridization of gastrula stage embryos with FRL-1 (C, D), B3C21 (E), and xPRMT1b (F) in lateral view (C, E, F) or vegetal view (D). (G) The 600 dorsal-most enriched spots were projected onto the combined organizer graph of Fig. 4. The results of three different differential screens of 18,000 gastrula cDNAs plated in duplicate onto a single nylon filter are integrated in this one graph. The wide range of expression of the 600 spots suggests that there are additional pathways besides β-Catenin and Nodal-related that affect dorsal-specific gene expression.
Fig. 3. Macroarray analysis of the Nodal-related signaling pathway. (A) xy-scatter plot of the spot intensities after hybridization of the macroarray filters with probes comparing wild-type embryos to those in which Nodal-related signaling was inhibited by injection of 150 pg cer-S mRNA into each blastomere at the four-cell stage. (B, C) Whole-mount in situ hybridization of gastrula stage embryos left uninjected (B) or injected with cer-S mRNA (C) with the novel gene P17F11 in vegetal view.
Fig. 6. The preorganizer graph. (A) The data for the maternal h-Catenin and the Nodal-related signaling pathways at blastula stage (8.5 h post fertilization) were plotted as in Fig. 4. Color coding indicates the spots encoding chordin, Xnr-3, pintallavis/FoxA4a, and several novel genes. Since the spot intensities using blastula stage RNAs were weaker than those at gastrula stage, background correction distorted the clone distribution and therefore this step was omitted. (B E) Whole-mount in situ hybridizations with pintallavis/FoxA4a on untreated (B, D) and LiCl-treated (C, E) blastula stage embryos, (B, C) animal (D, E) and lateral views. Like other preorganizer genes, pintallavis/FoxA4a expression is expanded over the entire animal cap upon activation of the maternal h-Catenin pathway.