Figure 5: MAP6 is required for the growth-promoting activity of Sema3E on subicular neurons.

(a) Typical images of subicular neurons cultured in the absence (CTL) or in the presence of 5 nM Sema3E for 24 h, electroporated with GFP-expressing vector and control siRNA or MAP6 siRNAs. To measure the axon length, neurons were stained with anti-α-tubulin (green) and phalloidin (red). Scale bar, 30 μm. The histogram shows quantifications of the relative axonal length in subicular neurons in the absence (CTL) or the presence of 5 nM recombinant Sema3E (Sema3E). Error bars, s.e.m., values corresponded to mean axonal length (measured in at least 60 neurons for each condition) and normalized to 100% for values obtained in control conditions. (b) Typical images of WT and MAP6-KO dissociated subicular neurons cultured in the absence (CTL) or the presence of 5 nM Sema3E. Scale bar, 30 μm. The histogram shows quantifications of relative axonal length of cultured subicular neurons in the absence (CTL) or the presence of 5 nM recombinant Sema3E. Error bars, s.e.m., values corresponded to mean axonal length (measured in at least 60 neurons for each condition) and normalized to 100% for values obtained in control conditions. (c) The histogram shows quantifications of relative axonal length after exogenous expression of rat MAP6-E-GFP. Error bars, s.e.m., values corresponded to mean axonal length (measured in at least 60 neurons for each condition) and normalized to 100% for values obtained in control conditions. (d) Sema3E axonal guidance response. Representative images of subiculum (Sub) explants from E17.5 WT or KO embryo co-cultured in a plasma clot with an aggregate of HEK cells transfected with Sema3E-AP and GFP. Scale bar, 200 μm. Quantification of the guidance response, graph shows mean guidance index (see methods) calculated for each condition (WT, n=11 and KO, n=8). Positive values indicate attraction. (e) Quantifications of axonal length in cultured cortical neurons in the absence or the presence of 5 nM recombinant Sema3E electroporated with GFP-expressing vector and control siRNA or MAP6 siRNAs. Error bars, s.e.m. Values corresponded to mean axonal length (measured in at least 60 neurons for each condtion) and normalized to 100% for values obtained in control conditions. (f) Quantifications of axonal length in cultured WT and MAP6-KO cortical neurons in the absence (CTL) or the presence of 5 nM recombinant Sema3E. Error bars, s.e.m., values corresponded to mean axonal length (measured in at least 60 neurons for each condition) and normalized to 100% for values obtained in control conditions. All quantifications have been repeated at least three times using independent neuronal cultures yielding consistent results. *P<0.05; **P<0.01; ***P<0.00; Student’s t-test (5a,b,c,e,f); non-parametric Mann–Whitney U-test (5 d).