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Supplemental Figure 4 (Supports Figure 5). (A) Injection of 20 ng of HEB MO in both blastomeres at the 2-cell stage does not result in gross morphological defects at gastrulation (stage 10.5) or in early tailbud embryos (stage 22). (B) Co- injection of 10 ng of E2A MO with nuclear β-galactosidase mRNA in one blastomere at the 4-cell stage results in cell-autonomous inhibition of blastopore formation and of xbra expression. Red color represents red-gal substrate staining and marks the injected portion of the embryo. (C) Embryos co-injected with e2a MO and mouse e2a mRNA were compared to uninjected controls for expression of xbra (mesoderm) and sox17β (endoderm). While e2a MO injection led to downregulation of xbra, this was rescued by co-injection with mouse E2A mRNA. |
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Figure 5. E2A is essential for gastrulation and gene expression in X. tropicalis. (A, right) X. tropicalis embryos were injected with a translation-blocking MO (e2a MO) directed against e2a, and assayed morphologically at stages 10.5 and 25. These were compared with uninjected controls (shown at left). (B) Blasto- pore lip formation in e2a MO-injected embryos can be restored by subsequent injection of mouse e2a mRNA. Using either in situ hybridization (C) or qRTCR (D), e2a MO-injected embryos were compared with controls for expression of molecular markers. qRTCR results represent at least four biological replicates. (E) e2a morphants and uninjected controls were in- jected in the animal pole at the four-cell stage with 10 pg of Activin mRNA or 40 pg of Xnr1 mRNA, and assayed for the presence of ectopic bottle cells. Representative embryos are shown at stage 11 in animal views. |