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Fig. 3. Loss of Xfoxi1a/b function results in an expansion of the neural plate and reduction of non-neural ectodermal tissues. (A) The Xfoxi1a/b-MO binding sites are shown with the underlines. The red box indicates the start codon. Identical nucleotides are marked with asterisks. (B) Flag-tagged Xfoxi1a mRNA (50 pg/cell) was injected with Xfoxi1a-MO (2.5 ng/cell), Xfoxi1b-MO (2.5 ng/cell) or the control five-base mismatched Xfoxi1a MO into animal cells of eight-cell embryos. (C) Flag-tagged Xfoxi1b mRNA (50 pg/cell) was injected with Xfoxi1b-MO (2.5 ng/cell), Xfoxi1a-MO (2.5 ng/cell) or control five-base mismatched Xfoxi1b MO into animal cells of eight-cell embryos. (D) Flag-tagged δ5′UTR-Xfoxi1a mRNA (50 pg/cell) was injected with Xfoxi1a-MO (2.5 ng/cell) or Xfoxi1b-MO (2.5 ng/cell) into animal cells of eight-cell embryos. Animal caps were excised at stage 9 and cultured until stage 11. Xfoxi1a-flag (B,D) or Xfoxi1b-flag (C) proteins were detected by western blot analysis using an anti-flag antibody. Hsp70 was used as the loading control.δ 5′UTR means that the synthetic mRNA contains only the coding sequence and not the target sequence of Xfoxi1a/b-MO. (E-U) Xfoxi1a-MO (12.5 ng/cell; i1aMO; E-M), five-base mismatched control MO of Xfoxi1a (12.5 ng/cell; 5mis; N-P), Xfoxi1a-MO (12.5 ng/cell) ± Xfoxi1a mRNA (25 pg/cell; Q,R) or Xfoxi1b-MO (12.5 ng/cell; i1bMO; S-U) was injected into two unilateral blastomeres of eight-cell embryos. Embryos were harvested at stage 14-15 (E-J,L-U) or stage 24 (K) and analyzed by whole-mount in situ hybridization with the probes indicated in each panel. (E-H,J,L-O,Q-T) Dorsal view (anterior towards the top); (I,K,P,U) anterior view (dorsal towards the top). Injected sides are marked with arrowheads. Dashes indicate the midline. NP, nasal placode. Double-headed arrows in G,Q show the expansion of Dlx3– and Six2+ regions, respectively. |
