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Fig. 6-S2
Administration of Pgk1 could delay NMJ denervation caused by overexpression of Rtn4al in adult zebrafish muscle.
NMJ phenotype of transgenic zebrafish Tg(Zα:TetON-Rtn4al) harboring Rtn4al/NogoA cDNA driven by a Dox-inducible muscle-specific alpha-actin promoter was observed after intramuscular injection of (A–C) GFP protein (served as a control) and (D–F) Pgk1 protein. Histopathological examination of the muscle tissue around the injection site of adult fish was performed after fish were treated for one, two and three weeks as indicated. (A–F) Transverse section. Green fluorescent signal was used to detect synaptic vesicle glycoprotein 2A (SV2)-labeled peripheral motor neuron, while red fluorescence signal was used to detect α-Bungarotoxin (α-BTX)-labeled acetylcholine receptor on motor endplate. Arrowheads indicate the location of NMJ denervation. Scale bar, 20 μm. (G) Diagram depicts places where GFP and Pgk1 were injected and where the muscle samples were taken after soaking with Dox in adult transgenic zebrafish Tg (Zα:TetON-Rtn4al). (H) Quantitative analysis of the colocalization of SV2 and α-BTX signals. Data were averaged from three adult fish. Each fish datum was averaged from three images, whereas each image was counted by using Metamorph software to quantify SV2 and α-BTX signal colocalization area (400 × 400 pixels) from 600X magnification, followed by conversion to percentage. Student’s t-test was used for statistical analysis (***, significant difference at p<0.001; *, p<0.05).