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Fig. 3. Time-lapse analysis of Eph/ephrin Fc-fusion protein injected embryos
reveals dynamic neural crest cell behavior. (A-D) Static images
taken from time-lapse movie. Premigratory neural crest cells injected with
EGFP. ephrinB1-Fc fusion protein injected in ovo at E3 when neural crest cells
were dispersed adjacent to the dorsal aorta. Asterisk indicates location of
injection. Sagittal explants cultured at E3.5 and imaged with time-lapse
confocal microscopy. At A, t=0; at D, t=5 hours. Cells extend and retract
numerous filopodia but fail to significantly translocate. Sympathetic ganglia
at axial level of fusion protein injection do not sort into ganglia but stay
continuous along the dorsal aorta. The dashed circle indicates a region of
presumptive sympathetic ganglia. The arrowhead indicates a presumptive
inter-ganglionic region. The black arrow indicates time increasing from left
to right. (E) An EphB2-Fc injected embryo after 5 hours of incubation.
Sympathetic ganglia at level of fusion protein injection (asterisk) do not
segregate, but sympathetic ganglia caudal to the injection site segregate
normally (brackets), indicating that the inhibitor is only acting at the axial
level of the injection. (F) A graph representing the average number of
filopodia on neural crest cells in control IgG-Fc injected embryos, and
ephrinB1-Fc injected embryos. Control embryos extended 3.6±0.79 (s.d.)
filopodia. ephrinB1-Fc injected embryos extended 6.0±0.6
(*P<0.001) filopodia with 5.5±1.0
(*P<0.001) further extensions off primary filopodia
(secondary) extended from the cell body. (G) A graph representing the
total distance migrated (blue bars) compared to net displacement (purple bars)
for individual control and ephrinB1-Fc-inhibited neural crest cells. Scale
bars: 40 µm in A-D; 20 µm in E. A, anterior; c, caudal somite; P,
posterior; r, rostral somite.
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