Overexpression of a dominant-negative ADAM10 mutated in the metalloprotease domain (dn-ADAM10-me) promotes neuronal differentiation in the developing spinal cord.

After electroporation at E4, transverse sections at E6 were used for immunostaining. The dn-ADAM10-me (AM10-me) transfected cells are marked by green fluorescence and pCAGGS-GFP (pCA-GFP) transfection and untransfected side (left side) serve as controls. The immune reactive cells are stained by red color. Cell nuclei are labeled with DAPI (blue). (A–C) Representative Western blots (A) and semi-quantitative Western blot analyses (B, C) of ADAM10 protein, including a pre-mature (pADAM10) and a mature (mADAM10) form, and the cleaved Notch1 (cle Notch1) in the transfected (tran) and untransfected side (untran) of pCAGGS-ADAM10 (pCA-AM10) or dn-ADAM10-me (AM10-me) transfected embryos. GAPDH is used as a loading control. The amount of ADAM10 and cle Notch1 protein is normalized by the number of the control side, which is set to be 1. All data are presented as mean ± SEM from at least 3 independent samples (*p<0.05, **p<0.01 compared to control). (D–G) Apoptotic cells (red, arrowheads) measured by TUNEL assay. (H–A’) Immunostaining using antibodies against NeuN (H–K), MNR2 (L–S), and NKx6.1 (T–A’), respectively. Arrows in (H–K) and (P–S) indicate ectopic immune reaction in the prospective ventricular zone of the transfected region (green); in (L–O) and (T–W) no change in the transfected region (green); in (X–A’) decrease of endogenous NKx6.1 expression. Scale bar, 200 µm in (D) for (D–A’).