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Fig. 2. Changes in Barhl2 levels alter neural plate formation. (A) Sequence comparison between the Eh1 motifs of Gsc, Eng, Nkx, Anf-1, Hesx1 and Msx, and the FILs domains of Barhl proteins. Black shading indicates identical residues, gray shading indicates similarity. Most of the sequences, except for Barhl members, come from Smith and Jaynes (Smith and Jaynes, 1996). (B) Map of the RNA constructs. The two blue boxes indicate the FIL domains; the green box indicates the Myc epitope; the yellow box indicates the Engrailed repression domain. (C) Sequences of the Xbarhl2 antisense morpholinos. The five mispairs in Xbarhl2ASIII are in red and the ATG is in bold. For comparison, the N-terminal sequences of mouse Barhl2, Xbarhl1 and Xbarhl2 are shown. (D) In situ hybridization of embryos injected with mouse Barhl2, Barhl2{Delta}FIL, Barhl2{Delta}FIL-enR, Xbarhl2ASI, Xbarhl2ASII, Xbarhl2ASIII and analyzed at stage 15. Dorsal view, anterior end upwards. The broken line indicates the midline and `inj' indicates the injected side. Embryos labeled with Xsox3 as probe: (a) control; injected with mouse Barhl2 at (b) 10 pg and (c) 50 pg; (e) injected with mouse Barhl2{Delta}FIL (100 pg); (f) injected with mouse Barhl2{Delta}FIL-enR (50 pg); (g) injected with Xbarhl2ASI; (h) injected with Xbarhl2ASII; injected with Xbarhl2ASII at (i) 20 ng, (j) 50 ng and (k) 100 ng. Embryos labeled with XK81 as a probe: injected with mouse Barhl2 at 10 pg (d); injected with Xbarhl2ASII (l). (E) Two-cell embryos were injected in one blastomere and analyzed at stage 9. Representative embryos are shown under fluorescent light. (a) Embryos injected with Xbarhl2-myc (100 pg); (b) injected with Xbarhl2-myc (100 pg) together with Xbarhl2ASII (100 ng); (c) injected with Xbarhl2-myc (100 pg) together with Xbarhl2ASIII (100 ng). (F) Western analysis on protein extracts from stage 9 embryos injected with Xbarhl2-myc (100 pg) together with: Xbarhl2ASIII (100 ng) (a); Xbarhl2ASII (50 ng) (b) or Xbarhl2ASII (100 ng) (b') using anti-Myc antibody. (G) Embryos labeled with Xsox3 as probe. The percentage of embryos exhibiting the staining patterns: (a) injected with mouse Barhl2 (10 pg), 78%; (b) injected with Xbarhl2ASII (50 ng), 88%; (c) injected with mouse Barhl2 (10 pg) together with Xbarhl2ASII (50 ng), 60%.