Transient Neuronal Populations Are Required to Guide Callosal Axons: A Role for Semaphorin 3C (original) (raw)
Figure 8
Sema3C acts through Npn-1 to attract and promote growth of cortical axons.
(A–C) Typical patterns of axonal outgrowth (Ai) from explants of E15.5 CCi cocultured with AP-control and AP-Sema3C–expressing HEK293T cells in the presence or absence of 20 µg/ml polyclonal anti–Npn-1 antibodies. (Aii) Quantification of the axonal guidance responses to AP-control and AP-Sema3C–expressing HEK293T cells. Data are expressed as a P/D ratio, where P and D are the mean lengths of axons in the quadrants proximal and distal to the cell aggregate [77]. In control conditions, a P/D ratio close to 1 (P/D ratio = 1.04±0.09) indicates radial outgrowth. AP-Sema3C causes strong chemoattraction of cortical axons (P/D ratio = 1.73±0.15). This effect is blocked by anti–Npn-1 antibodies (P/D ratio = 0.99±0.09). (Bi) Typical images of dissociated neurons from E15.5 CCi cultured in the presence or absence of 10 µg/ml polyclonal anti–Npn-1, and of 10 nM AP-Sema3C. (Bii) Histograms of Sema3C effects on the growth of dissociated neurons from CCi. Data are presented as mean axonal length ±SEM and are normalized to 100% with respect to values obtained in control conditions. The anti–Npn-1 antibodies prevent stimulation of axon growth by Sema3C. (Ci) Dissociated neurons of CCi electroporated with GFP-expressing vector together with either control siRNA or Npn-1 siRNAs 1 and 2 (see Materials and Methods) were cultured in the presence or absence of 10 nM AP-Sema3C. (Cii) Quantification of the effects of Npn-1 siRNAs on axon length. Knock-down of Npn-1 completely abolishes the growth-promoting effect of Sema3C. Data are mean axonal lengths ±SEM calculated as a percentage of mean values obtained in control conditions for each experiment. Double asterisks (**) indicate significantly different with p<0.01; triple asterisks (***) indicate significantly different with p<0.001.