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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Mol Cell Neurosci. 2010 Mar 16;44(2):135–153. doi: 10.1016/j.mcn.2010.03.003

Figure 6. Sema3F is a strong repellent of DA axons.

Figure 6

E14 VM rat explants were co-cultured with 293T-Sema3A producing cell aggregates for 2-3 days. Cultures were fixed and double-stained for TH (red) and tubulin (green) to distinguish DA axons. Untransfected 293T cell aggregates were used in control co-cultures. (A) The typical response elicited by Sema3F on VM explants. Note the strong repulsive effect of Sema3F on TH+ axons in contrast to the weaker effect on TH- axons. Inset: The effect of Sema3F was measured as the ratio between the proximal (P) and distal (D) hemiareas covered by axons with respect to the 293T cell aggregate. Calibration bar indicates 100 μm. (B) The graph represents the average P/D ratios ± SEM of ventro-mesencephalic (VM), brain stem (BrSt) or hippocampal (Hc) explants co-cultured with 293T or Sema3F aggregates. • p<0.05; •• p< 0.005 and ••• p<0.0001 when compared with controls (293T). (C) The P/D ratio of each co-culture was scored as repulsive when it was below -1SD from the 293T mean, as attractive when above +1SD, or un-responsive when it was within ± 1 SD. The bars represent the average percentage of cultures responding in one or another direction ± SEM. Note that more than 80% of the VM cultures analyzed were repelled by Sema3F. The number under the bars represents the number of independent cultures assayed. *: p < 0.0001 ANOVA followed by Student-Newman-Keul comparison test. (D) The repulsive effect of Sema3F on DA axons is lost in VM explants from Nrp2-/- mice. VM explants from wild type (Nrp2+/+), heterozygous (Nrp2+/-), and homozygous (Nrp2-/-) mice were co-cultured with Sema3F-producing cell aggregates for 3 days, fixed and stained for TH. Cultures P/D ratios were measured and scored as repulsion when the P/D ratio was below 75. Calibration bar: 200 μm.