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Fig. 5. Sulf1-/-;Sulf2-/- esophagi have defective
GDNF-dependent neurite outgrowth. Esophagi (
400 µm) were dissected
from E11.5 embryos and plated on collagen gel containing BSA, GDNF or
neurotrophins at various concentrations. After 4 days, the whole explant was
immunostained with the TuJ1 antibody. Explants that failed to attach to
collagen gel were not included in the assay. (A,B) Neurite
outgrowth of E11.5 esophageal explants was selectively dependent on GDNF, but
not on neurotrophin. Sulf1-/-;Sulf2-/- esophagi
failed to extend neurites at 10 ng/ml GDNF and showed reduced neurite
outgrowth at 20 ng/ml and 50 ng/ml GDNF. (C) Quantification of the
neurite outgrowth shown in A and B. The length of the extended neurite was
measured along six axes, 30° apart and the average was calculated to
represent the neurite outgrowth of one explant. Data represent the mean and
the standard deviation of a minimum of four individual cultures.
(D,E) Quantification of the total number of neurons in the
explants. The neurons in the explants (dark cell-body staining by the TuJ1
antibody, indicated by arrows) were quantified using the bright field at low
magnitude. Neurons were scattered, or even migrated out of the control
explants in the presence of 10 ng/ml GDNF. In control explants cultured in the
presence of BSA or NGF and in GDNF-treated
Sulf1-/-;Sulf2-/- explants, neurons tended to
form clusters. The large clusters of neurons in
Sulf1-/-;Sulf2-/- explants were quantified by
summing the neuronal numbers at different focal planes. **,
P<0.01 (two-tailed Student's t-test). Scale bars: 250
µm in A,B; 100 µm in D.
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