Fig. 2. Expression of Ci-ETR-1 and Ci-Epi-1 at the neurula stage in embryos treated with or without PD184352 from the eight-cell stage onwards. (A,B) Schematic representation of 64-cell stage embryo showing fates of each blastomere with a spot of colour, key on left. Neural blastomere names are shown. Blastomere identities are also indicated on the cleavage-arrested embryos with an appropriate coloured dot. (C-L) In situ hybridisation was carried out for the genes indicated on the left of the panels. Cleaving embryos are shown in the left-hand panel. Embryos in which cleavage has been arrested from the 64-cell stage are shown in the middle and right hand panels. Application of PD184352 is indicated on the right: left-hand panels, dorsal views with anterior upwards (PD184352 treated embryos are effectively a vegetal view); middle panels, animal views; right-hand panels, vegetal views. Only animal views are shown for Ci-Epi-1 because this gene is not expressed in vegetal cells. (C-H) Ci-ETR-1. (C) Control, 100% positive, n>100. (D,E) Cleavage arrested control, n=156. (D) a-line, 100% positive. (E) A-line, 100% positive. (F) Cleaving embryo+PD184352, 100% positive, n=268. (G,H) Cleavage-arrested embryo+PD184352, n=271. (G) a-line, 0% expression. (H) A-line: 100% in A-line neural precursors; 96% in notochord. Expression of neural markers is detected in the notochord because of the conversion of notochord into neural cells in the absence of FGF or MEK signalling, as previously reported (see Introduction). Expression of Ci- tubulin was similar, except low levels of expression were also detected in the anterior endoderm (not shown). (I-L) Ci-Epi-1. (I) Control, n>100. (J) Control cleavage-arrested embryo, n=55. (K) Cleaving embryo+PD184352, n=71. (L) Cleavage arrested+PD184352, n=42. Embryos treated with U0126 produced similar results.

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