Fig. 2

Zebrafish Bcl2-L1 overexpression prevented human 4R-tau-GFP and zebrafish 3R-tau-GFP induced Neuronal death. a GFP-labeled neuronal cells and axons were observed at 24 and 48 hpf in embryos co-injected with pHuC-zBcl2-L1-HA-2A-mCherry and pHuC-z3R-tau-GFP (b) or pHuC-h4R-tau-GFP (d). For comparison, embryos co-injected with pHuC-mCherry (panels a and c) were used as the control. The boxed regions are enlarged (a′-d′′) to show the GFP-labeled neuronal cells in 24 and 48 hpf embryos from the lateral view. Scale bars: 100 µm. The protection effect of zBcl2-L1 against neuronal death by human tau-GFP or zebrafish tau-GFP was presented in panel e to show higher percentage, 69 % and 66 % of zebrafish embryos expressing zBcl2-L1 with more neuronal cells, compared to 21 % and 18 % without zBcl2-L1. b GFP signals (panel a) and mCherry signals (panel a′) in neuronal cells and axons in embryos co-injected with pHuC-h4R-tau-GFP and pHuC-zBcl2-L1-HA-2A-mCherry were colocalized (panel a′′). Scale bar: 50 µm. c Double immunostaining of h4R-tau-GFP (GFP antibody, panel a) and Bcl2-L1-HA (HA antibody, panel a′) in spinal cord neurons of the aforementioned zebrafish embryos. The phosphorylation state of h4R-tau-GFP was detected using antibody pT212 (panel b) and antibody AT8 (panel b′). Embryos are shown from the lateral view with the anterior to the left and dorsal to the top. Scale bar: 50 µm. d Double immunostaining of zebrafish embryos expressing h4R-tau-GFP at different developmental stages was performed using polyclonal antibody against GFP and monoclonal antibody AT8. Scale bar: 50 µm. e Western blot analysis of total protein extract of zebrafish embryos expressing h4R-tau-GFP at 24 hpf was performed using polyclonal antibody against GFP and monoclonal antibody AT8