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Fig. 2. Axonal outgrowth and survival in vitro. Dissociated E12 ventral midbrain of
control (mixture of
En1+/–;En2–/– or
En2–/–) and En double mutant in 3D collagen
matrix (A), on coverslips coated with laminin (B) and a membrane carpet
derived from wild-type E12 midbrain (C) after 1 day in culture stained against
TH. For the first 24 hours in vitro, dissociated En double mutant mDA neurons
are viable and extend axonal processes. (D) The mean length of the TH-positive
processes was the same for mutant and littermate controls. The difference was
not statistically significant (Student’s t-test,
P=0.35). Error bars are not shown, as axonal outgrowth varied between
0.3 and 78 µm. n=number of cells measured out of four independent
experiments for each genotype. (E) Average number of TH-positive En double
mutant cells counted at several time points. After dissociation, disappearance
of mDA neurons was arrested for 24 hours. Thereafter, the mutant cells follow
their in vivo counterparts such that almost no TH-positive cells are left 72
hours post dissociation. n=5 independent cell culture experiments for
each time point. Error bars indicate s.d. (F-I) Cell culture at about 48 hours
post dissociation stained against TH (F,F', green) and activated caspase
3 (G,G', red). Each sample was counterstained with DAPI (H-I') to
identify cell nucleus. Mutant mDA neurons (arrows) retract their processes,
round up and are positive for activated caspase 3. An additional sign for
apoptosis is the pyknotic nuclei. (F'-I') By contrast, TH-positive
cells derived from littermate controls possess elongated processes and show no
signs of apoptosis (arrows). (I,I') Magnification of H,H'. Scale
bars: 20 µm for A-C,F-H; 10 µm in I.
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