| |
Fig. 2. Organizer formation is impaired by Zic3 overexpression. EGFP RNA alone (A; EGFP, 400 pg), EGFP RNA (400 pg) with Myc-tagged Zic3 RNA (200 pg) (B; EGFP + Zic3), or Myc-tagged Zic3 RNA alone (200 pg) (D; Zic3) were injected into the dorsal marginal zone of the dorsal two blastomeres of embryos at the 4-cell stage. The injected embryos and an uninjected sibling embryo (C) were observed at stage 10 (A, B) or stage 9.5 (C, D). (A, B) Green EGFP signals derive from the injected RNA. Dorsal lips were frequently defective in embryos with Zic3 RNA injection (73%, n = 16). (C, D) Expression of an organizer marker gene (Goosecoid) was analyzed by WMISH. Reduction of Goosecoid expression was frequently observed in the Zic3 RNA-injected embryos (76%, n = 27). (E) RT-PCR analysis. EGFP RNA alone (EGFP, 400 pg), EGFP RNA (400 pg) with Myc-tagged Zic3 RNA (200 pg) (Zic3), or EGFP RNA (400 pg) with Zic morpholino oligos (30 ng each) (Zic2MO + Zic3MO) was injected into the dorsal marginal zone of the dorsal two blastomeres of embryos at the 4-cell stage. At stage 10.5, the dorsal part was dissected as in bottom cartoon and used for total RNA extraction. Direct targets of β-catenin (Bra, Twn, Siamois, and Nr3) were downregulated by the Zic3 RNA injection, whereas genes in the Wnt/β-catenin signaling cascade (β-catenin, Axin, Dsh, GSK3β, CKα and CKε) and a housekeeping gene (ODC) were not changed. The cDNA synthesis reaction for the sample injected with EGFP RNA alone was also performed without reverse transcriptase (-RT) to confirm the absence of chromosomal DNA contamination. |
