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Fig. 8. Roles of XMAN1 during Xenopus development. (A) XMAN1 antisense morpholino oligo (XMAN1-MO) inhibits the translation from synthetic XMAN1 mRNA. Extracts prepared from stage 10.5 and stage 25 embryos injected with Myc-tagged XMAN1 mRNA possessing the XMAN1-MO annealing sequence (5′UTR-XMAN1-MT) together with XMAN1-MO or XMAN1-4mmMO (doses in ng per embryo are in parentheses) were subjected to immunoblotting with anti-Myc antibody (upper panel) and with anti-β-tubulin antibody (lower panel). (B) XMAN1-MO suppresses neural induction by XMAN1. Real-time RT-PCR analysis for nrp1 with animal caps at the equivalent of stage 25. Doses of injected mRNA, 500 pg per embryos. Doses of XMAN1-MO are indicated in parentheses (ng per embryo). (C) Morphological appearance of injected embryos. XMAN1-4mmMO (b,f), XMAN1-MO (c,g) and XMAN1-MO plus MT-XMAN1 mRNA (d,h) were injected together with β-gal mRNA as a tracer (b-d,f-h) into two right animal blastomeres at the four-cell stage. XMAN1-MO injection caused a reduced eye on the injected side (c,g), which is rescued by co-injection of MT-XMAN1 mRNA (d,h). Injected doses (per blastomere): MOs, 25 ng; MT-XMAN1 mRNA, 67.5 pg; β-gal mRNA, 15 pg. a-d, dorsal view (anterior is upwards); e-h, lateral view of a-d, respectively, showing the right side of the embryos. (D) Whole-mount in situ analysis for Dlx3 (epidermis, stage 13), Rx2A (eye, stage 20), Xemx1 (telencephalon, stage 20) and nrp1 (pan-neural, stage 20) expression in uninjected, XMAN1-4mmMO- and XMAN1-MO-injected embryos as indicated. a-c, dorsal view (anterior is upwards); d-l, anterior view (dorsal is upwards). β-Gal mRNA was co-injected in b,c,e,f. |
