Figure 2
Loss of Engrailed induces neurotrophin requirement in mesDA neurons. (A-F) Double immunohistochemistry on dissociated cells derived from En2-/- (A-C) and En1-/-;En2-/- (EnDM) (D-F) E12 ventral midbrain using antibodies against tyrosine kinase (Trk)B (A, D), TrkC (B, E), P75NTR(C, F) and TH (green) counterstained with DAPI. TrkB, TrkC and P75NTR are expressed by TH+ cells from both genotypes; however, the immunohistochemistry is not sensitive enough to detect differences in P75NTR expression between genotypes. (G, H) Western blot of ventral midbrain tissue derived from different Engrailed genotypes. The two Trk receptors do not depend on Engrailed expression (G). Brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3 are not expressed in E12 ventral midbrain tissue, but they are in the adult (H). (I) Treatments (>10 ng/ml) for 72 hours with TrkB/C-specific neurotrophins – BDNF, NT4 and NT3 – greatly increases the survival rate of EnDMmesDA neurons (n ≥ 6; p < 0.001), whereas nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), transforming growth factor (TGF)-β and growth differentiation factor (GDF)-15 do not significantly alter survival rate. (J) Dose response curve: BDNF concentration plotted against survival rate showing saturation at approximately the 10 ng/ml. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.