Figure 2.

Hap1 and Tsc1 regulate hippocampal pyramidal neuron axon specification and positioning. A, Lysates of hippocampal neurons from 0 to 5 DIV were subjected to immunoblotting using the Tsc1, Hap1, or Tubulin (β-III type) antibody. B, E18 rat hippocampal neurons transfected 2 h after plating with the control U6 RNAi or Hap1 RNAi or Tsc1 RNAi plasmids were subjected to immunocytochemistry on DIV5 using the GFP (green) and the axonal antigen Tau1 (red) antibodies. Scale bar, 20 μm. The i.1, i.2, i.4 nomenclature refers to individual shRNAs. C, Quantification of axon specification as percentage of neurons. Hap1 or Tsc1 knockdown promoted specification of supernumerary axons (n = 3 experiments). ***p < 0.001 (two-way ANOVA and post hoc Bonferroni test). No axon spec., neurons that failed to specify a Tau1-immunoreactive axon. D, Lysates of HEK293T cells transfected with Myc-Hap1 and control U6 RNAi or Hap1 RNAi (top) or DIV5 hippocampal neurons infected 2 h after plating with U6 RNAi or Tsc1 RNAi (bottom) were subjected to immunoblotting. E, E15 mice were electroporated with Hap1 RNAi, Tsc1 RNAi, or scrambled control RNAi and VENUS-IRES-EGFP plasmids. Brains were harvested on P3 and subjected to immunohistochemistry using the GFP (green) antibody and Hoechst 33258 (purple). Scale bar, 50 μm. SP, Stratum pyramidale; SO, stratum oriens. F, Quantification of neuronal position as percentage of neurons. Knockdown of Tsc1 or Hap1 impaired neuronal positioning into the SP in the hippocampus in vivo (n ≥ 3). ***p < 0.001 (two-way ANOVA and post hoc Bonferroni test). Number of animals analyzed per condition is shown. G, Lysates of mouse neuro2a cells transfected for 3 d with control U6 RNAi, Hap1 RNAi, or Tsc1 RNAi were subjected to immunoblotting.