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Fig. 7. Overexpression of ß-catenin overcomes density-dependent inhibition of
neural differentiation in an EB-independent protocol. ES cells transfected
with the constructs indicated were induced to differentiate into the neural
lineage in an EB-independent protocol (see Materials and methods) at a density
of 106 cells/cm2. Cells were fixed and stained 8-10 days
post-plating, with Hoechst dye (blue), and anti-nestin (green) and
anti-ß-tubulin 3 (red) antibodies. (A) Empty vector transfected cells;
(B) ß-catenin 
N transfected cells. Control cultures were devoid of
nestin- and ß-tubulin 3-positive cells, whereas overexpression of
ß-catenin N resulted in many nestin- and ß-tubulin 3-positive
cells, suggesting that ß-catenin exerts both proneural and proneuronal
effects. (C) Quantification of cells immunoreactive to antibodies specific to
the antigens indicated in the key. (D) BrdU incorporation of ES cells. Left
panel (plus Lif): undifferentiated ES cells stably transfected with either
empty vector (black) or ß-catenin N (gray) were pulsed for 3 hours
with BrdU and stained with anti-BrdU antibodies. No statistical difference was
found. Right panel: ES cells stably transfected with either empty vector
(black) or ß-catenin N (gray) were differentiated in vitro and
pulsed with BrdU on day 6 post-Lif withdrawal. ß-catenin N
expression resulted in decreased BrdU incorporation
(*P<0.05; **P<0.01, by
t-test; ND, none detected). Cells transfected with empty vector (E)
or ß-catenin N (F) seeded at high density were analyzed for
ß-catenin localization by optical sectioning. High-density cultures had a
highly membrane localized stain for ß-catenin, whereas in the
ß-catenin N overexpressor, the staining was more diffuse. To
substantiate the visual observation, fluorescent intensities over a random
cross section of the cells were plotted. In the control cells, there are clean
peaks and valleys (E'), whereas in the ß-catenin
N-transfected cells, the fluorescence is noisier (F').
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