NeuroD2 functions cell-autonomously to regulate thorny excrescence development in vivo. (A) Immunohistochemistry (IHC) for lentiviral GFP expression, pups injected at P5 and perfused at P16. (B) GFP-positive neuron filled with Lucifer Yellow (LY) dye and processed for IHC against LY (red); GFP-positive neurons are green. (C) Higher magnification image of LY IHC showing TE spines. (D) Overlaid image of LY and GFP IHC, demonstrating co-localization. (E) Immunoblot for myc-tagged NeuroD2 and NeuroD1 co-transfected with either NeuroD2 shRNA expressing lentiviral vector or control vector into 293T cells for 48 hours. Control is blotting for anti-GAPDH for same blot. Loading is duplicate wells. (F) Quantification of TE spine head density on primary and secondary dendrites of vector and shNeuroD2 lentivirus-infected neurons filled with LY dye (shVector, n = 14; shNeuroD2, n = 15) (G) Quantification of classical spine density on tertiary dendrites of lentivirus-infected neurons filled with LY dye (shVector, n = 15; shNeuroD2, n = 7). *P < 0.05, t-test. Scale bar: 50 μm (A, B); 5 μm (C, D). Error bars represent ± standard error of the mean.