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Fig. 1. Preparations for macroarray screening. (A) Pax2 is downregulated by XHR1-VP16-GR only in the presence of DEX (indicated by arrow). mRNAs for XHR1-VP16-GR (12.5 pg) and nβ-gal were coinjected into the dorsoanimal region of the left blastomere at the four-cell stage. Half the injected embryos were treated with DEX at the early gastrula stage (stage 10.5–11) (right) and the other half were untreated (left). Following nβ-gal staining (red), WISH was performed with a Pax2 probe at the early neurula stage (stage 13–14; purple). The images are dorsal views with the anterior to the top (the orientation of the following figures is the same as shown here unless otherwise stated). (B) Pax2 expression levels and gastrulation-arrest rates upon DEX treatment at different stages. Embryos were coinjected as described in panel (A) with mRNAs for XHR1-VP16-GR or β-globi n (100 pg) and nβ-gal, treated with DEX from the indicated stages to stage 13–14, and subjected to WISH for Pax2 expression. Colored bars indicate Pax2 expression levels on the injected side. Percentages of embryos with gastrulation arrest are shown on the right side of the graph. N, number of embryos. Each datum is from two or more individual experiments. (C) A schematic representation of the dissection procedure used to isolate the anterior neuroectoderm containing the pre-MHB region. Embryos were injected with mRNA at the four-cell stage, cut into halves in the middle of the body length at the late gastrula to early neurula stage (stage 12.5–13). The anterior neuroectoderm (ANE) together with the underlying anterior endomesoderm (AEM) was dissected and separated by collagenase digestion. (D, E) Dissected fragments containing the pre-MHB region as ascertained by XHR1 expression visualized by WISH (D), or by RT-PCR of extracted total RNA for XHR1 expression (E). Histone H4 was used as the loading control. |
