Fig. 6. Expression of Ci-otx in different lineages in control and PD184352-treated embryos. (A) Control embryo; dorsal view, anterior upwards; 100% positive, n100. (B) PD184352-treated embryo; 100%, n=302. The size of the expression domain was variable. (C-J) cytochalasin cleavage-arrested embryos. (C,G) Schematic representation of 64-cell stage embryo showing fates of each blastomere with a spot of colour, key is in the top left-hand corner. Blastomeres are also indicated on the cleavage arrested embryos with an appropriate coloured dot. (D,H) Control-cleavage arrested, n=418. (E,F,I,J) Cleavage-arrested embryo+PD184352, n=379. (D) Control a-line (100% expression). Expression was also seen in the anterior epidermis precursor, a7.11 (99%). (E,F) +PD. Expression was generally lost in a-line 60% (E), persisting in 40% of cases (F). Individual percentages for four independent experiments are 85%, 21%, 18%, 0% positive (experiments 1-4 respectively). Thus, in three out of four experiments, expression of Ci-otx in a-line cells was largely inhibited. In the experiment where embryos expressed Ci-otx in a-line in 85% of cases, the same batch of embryos expressed neither Ci-ETR-1 nor Ci- tubulin in a-line cells. (H) Control A-line: 99% positive in A7.4; 0% positive in A7.8. (I,J) +PD A-line: 100% positive in A7.4; 75% positive for A7.8. Notochord expression: 40% (individual percentages for experiments 1-4; 85%, 20%, 19%, and 2%). Embryos with notochord expression also had low levels of expression in anterior endoderm. The variability of the Ci-otx expression domain in PD184352-treated embryos in a-line and notochord was batch dependent and higher concentrations of inhibitor had little effect on the type of phenotype found. Embryos treated with U0126 gave similar results. (K-P') DiI labelling showing difference in Ci-otx expression in control embryos and embryos treated with PD184352 from the eight-cell stage onwards. (K-P) DiI label; (K'-P') in situ hybridisation. Nuclear stain facilitates comparison between K-P and K'-P'. Embryos in K'-P' appear slightly smaller because of shrinkage during in situ hybridisation. The blastomere that was labelled with DiI is indicated on the left of the panels. In all cases both left and right blastomeres were labelled. Controls on left, PD184352-treated embryos on right panels. n=10-20 for each. It can be seen that embryos treated with PD184352 fail to gastrulate, with cells maintaining their relative positions from before the gastrula stage.

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