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Fig. 1. Effects of altering SHH signaling in vitro: synergism between SHH and EGF
signaling and induction of gene expression. (A) Proliferation response of
plated nsps to different concentrations of EGF with (darker bars) or without
(lighter bars) added SHH. The total number of BrdU+ cells per well,
1 week after the initiation of EGF/SHH treatment, is shown. P values
from comparing control and +SHH samples are: 0.05 ng/ml EGF,
P<0.001; 0.25 ng/ml EGF, P<0.01; 0.5 ng/ml EGF,
P=0.001; 2.5 ng/ml EGF, P<0.001; and 5ng/ml EGF,
P<0.5. (B) Quantification of BrdU+ cells in a 24-hour
cell culture assay in the presence of 1 ng/ml of EGF and varying
concentrations of SHH. P values from comparing control and +EGF
samples are: 0.1 nM SHH, P=0.61; 0.5nM SHH, P=0.58; 1 nM
SHH, P<0.5; 5 nM SHH, P=0.013; and 25 nM SHH,
P=0.596. Similar results were obtained with 48-hour cultures. (C)
Cloning dilution assay (at 1 cell/µl, 2000 cells per well in 1/3
conditioned media and 10 ng/ml of EGF in uncoated 6-well plates) after a 1
week treatment of nsps in 2.5 ng/ml EGF, in the presence of either 5nM SHH or
5 µM cyc. The total number of clones per well were counted in triplicate
(P<0.0001). (D) RT-PCR analyses of E15.5 nsps, treated for 48
hours or 6 days in the presence of 5 nM SHH or 10 µM cyc. (E,G) Comparison
of the percentage of BrdU+ cells at different concentrations of
cyc, in the presence of 10ng/ml EGF, at E15.5 (E) and P2 (G) after 24 hours.
(F,H) 48-hour cloning assays in presence of 10 ng/ml of EGF and cyc as
indicated. (F) 12.5 E15.5 cells/µl plated in 1 ml media in uncoated 12-well
plates (n=3) and (H) 25 P2 cells/µl plated in 2 ml media in
uncoated 6-well plates (n=6). The experiments in A-H were performed
with cells selected from the beginning in EGF only. Similar differences were
obtained counting clones after one week in culture. (I) RT-PCR analyses of
fresh dissected cortex of control and cyc-treated embryos. Regulation of
Egfr expression by SHH was also seen at E17.5 and P3 (not shown). All
RT-PCR assays were repeated 2-3 times. (J) Quantification of BrdU
incorporation in acutely dissociated cultures plated in the absence of growth
factors from HBC- (control) or cyc-treated neocorteces in vivo. Cells were
grown in the presence of one-third conditioned media without additional cyc.
Histograms in all figures show mean±s.e.m.
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