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Fig. 7. Analysis of neural crest migration in vitro. (A) Neural crest was dissected
from an early neurula and cultured in vitro on fibronectin for several hours.
We analyzed migration, cell movement, cytoskeletal staining and cellular
morphology. (B) Control neural crest explant, showing normal migration
(n=60 explants). (C) Explant taken from an embryo injected with 1 ng
of Dsh-DEP+ mRNA. Note the absence of cell migration (n=30
explants). (D) Explant taken from an embryo injected with 2 ng of
dnWnt11 mRNA. Note the inhibition of cell migration (n=12
explants). (E) Summary of three independent experiments where the percentage
migration was analyzed. One hundred pecent of the control explants showed a
normal neural crest migration, whereas this process was inhibited in the
Dsh-DEP+ and dnWnt11-injected embryos. (F-I) Single
time-lapse frames showing representative cell behaviour; right, diagrams of
cells with time points. Cell bodies, black; lamellipodia, red. n=8
explants. (F,G) Control cells; (H,I) Dsh-DEP+ injected cells. (J)
Protrusive activity. Control cells extend more lamellipodia and
Dsh-DEP+ injected cells withdraw more protrusions. (K-T')
Analysis of lamellipodia was performed between 2 and 20 hours after culture,
by staining actin with Phalloidin-rhodamine (K,L,N-P) or with an antibody
against tubulin (M), or by SEM (R-T). Representative cells are shown for
control (K-M) and Dsh-DEP+ injected cells (N-P). (K,N) Representative
large lamellipodia. (L,M,O) Representative normal lamellipodia. (M) Tubulin
staining for the cell shown in L. (P) Representative cell extending filopodia
as its main protrusion. (Q) Types of protrusion. Control cells show large
lamellipodia and mainly normal lamellipodia, whereas Dsh-DEP+
injected cells show mainly filopodia. (R,R') Control neural crest;
yellow arrow indicates a large lamellipodia at the front of migration.
(S,S') Neural crest injected with Dsh-DEP+; red arrow indicates
filopodia. (T,T') Neural crest injected with dominant-negative
Wnt11; red arrow indicates filopodia. Scale bars in R-T': 10
µm.
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