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Fig. 6. Distinct temporal requirements for signals during NC development in
vivo. (A,C,F) Embryos were manipulated just prior to
Snail2 expression at stage 11.5 then analysed at stage 14, to
determine the period of NC induction. (B,D,G) Embryos
manipulated at stage 15, after Snail2 expression has started, and
analysed at stage 18 prior to NC migration, to determine the NC maintenance
period. (C,D) Embryos were injected at the 32-cell stage into blastomeres
fated to become epidermis (A4) with the inducible construct Smad7GR to target
injections to epidermis. Activation of the construct by the addition of
dexamethasone during the gastrula stage results in ectopic expression of
Snail2 (arrow in C; 25%, n=40). However, when activated
during the maintenance phase, no ectopic expression is observed (D; 0%,
n=20). No effect is seen in the absence of dexamethasone (not shown;
0%, n=31). (F,G) Small groups of ectodermal cell expressing BMP4 were
grafted (arrows) next to the NC. When grafted during gastrulation, a strong
inhibition of the NC was observed (F; 100%, n=25). However, when BMP4
cell were grafted during the maintenance phase, no inhibition was observed (G;
0%, n=55). Grafts of cell injected with FDX alone had no effect (not
shown; 0%, n=42). The graft was recognised by immunostaining against
fluorescein, as FDX was used as a lineage tracer (blue). (E) Summary of
Smad7GR injections. (H) Summary of graft of BMP4-expressing cells.
(I-K) Positive control to ensure sufficient BMP4 is being released to
inhibit NC induction. (I) A small cluster of animal cap cells taken from an
embryo injected with FDX or BMP4 mRNA (blue) was conjugated with an explant of
stage 10.5 animal cap (AC) and DLMZ, in which NC markers are normally induced.
(J) Explants of AC/DLMZ conjugated with cells expressing FDX show strong
expression of Snail2 (85%, n=26). (K) Explants of AC/DLMZ conjugated
with cells expressing BMP4/FDX show a dramatic inhibition in Snail2
expression (10% expressing Snail2, n=35). (L)
Percentage of Sox2 or Snail2 expression after injecting
different amounts of Smad7GR into A4 blastomere of a 32-cell stage embryo, as
described in C,D. Each experiment was repeated at least three times.
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). (M) Double in situ hybridisation against Snail2
and Wnt8. (N) BMP4 expression. (O-Q) Sections of equivalent
embryos to those shown in L-N. n, notochord; S, somites; IM, intermediate
mesoderm; NC, neural crest. (O) Snail2/12-101 staining showing that
it is the IM and not the somite the tissue that underlay the NC. (P)
Wnt8 is expressed in the IM. (Q) BMP4 is expressed in the ectoderm
next to or within the NC.
