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Fig. 5. Expression of neural markers in embryos treated with MEK inhibitor from the
eight-cell stage onwards. Arrows indicate gene expression in palps.
(A,A') Ci-HB9/Mnx. Late gastrula stage embryo; dorsal view,
anterior upwards. Expression is lost in PD184352-treated embryos (+PD) in
neural plate cells (n=174). (B-K) Control embryo; lateral view,
dorsal upwards. (B'-K') PD184352-treated embryos. (B,B')
Cihox5 (+PD, 0% expression, n=116). (C,C')
Cilv38e16 (+PD, 0% expression, n=139). (D,D')
Citb8a22 (+PD, 0% expression, n=36). (E,E')
Ci-islet (+PD, 0% expression, n=20). Arrowhead indicates the
visceral ganglion expression. (F,F') Ci-otx (+PD, 100%
expression, n=80). (G,G') Ci-ETR-1 (+PD, 100%
expression, n=108). Expression in tail epidermal sensory neurones was
not affected (arrowheads). (H,H') Ci-gsx (control, 72%
expression; +PD,46% expression, n=278). (I,I') 08C09
(+PD, 91% expression, n=103). (J,J') 24H09 (+PD, 0%
expression, n=103). (K,K') Ci-TRP (+PD, 0% expression,
n=30). In all cases where gene expression was lost, this was not
simply a delay of expression, as the onset of gene expression was either much
earlier than the stage shown, or loss of expression was verified at a later
developmental stage. Similar results were obtained with U0126. (L-O) Dorsal
views of embryos showing expression of Ci-ETR-1, 08C09, 24H09 and
Ci-TRP to indicate relative expression domains along the
anteroposterior axis. A bar indicates the posterior limit of the sensory
vesicle. (P) DiI labelling of A7.4 at the 64-cell stage. At tailbud stages,
label is seen in the posterior sensory vesicle (posterior to the pigment
cells) and in the ventral tail nerve cord (not seen in this focal plane). (Q)
DiI labelling of A7.8 at the 64-cell stage. At tailbud stages, label is seen
in the visceral ganglion, lateral tail nerve cord and a few muscle cells (not
visible in picture). (R) A schematic summary of the data shown in this figure.
The central nervous system consists of a sensory vesicle, visceral ganglion
and tail nerve cord. In black are the pigment cells. A-line cells are coloured
grey. The relative expression domains along the anteroposterior axis of genes
used in this figure is indicated. + and - indicate the expression status of
the genes in PD184352-treated embryos.
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100. (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.
1 hour and 45 minutes after 110-cell stage. Detection of
neurones is expressed as a percentage. (C) An example of `neurones in both
head and tail'. (D) An example of a `good network'. Experiments with U0126
produced similar results. (I) Embryos were cleavage-arrested at the 64-cell
stage and placed in inhibitor at the time points (
