Myelin sheath in early developing mice and zebrafish is reduced after Trappc9 deficiency, but the myelin sheath formation was not affected. (A-B), RNA in situ hybridization and RT-qPCR analysis of mbpb expression in 72 hpf zebrafish embryos. The expression of mbpb in mutant (zTrappc9m/m) and morphant (MO) zebrafish at 72 hpf was significantly reduced. Representative images from n=20-26 embryos/group in 7 experiments (A); n=3 experiments (B). (C-D), PLP immunostaining of 72 hpf zebrafish embryos. The area percentage of PLP fluorescence in zebrafish hindbrain (red arrowheads) was significantly decreased in MO and zTrappc9m/m zebrafish embryos. Representative images from n=10 embryos/group (C); n=8 sections (D). (E-F), MBP immunostaining of P11 mouse cerebral cortex, showing the area percentage of MBP fluorescence in the corpus callosum was significantly decreased in mTrappc9m/m mice. Representative images from n=3 mice/group I; n=6 sections (F). (G-I), Transmission electron microscope (TEM) micrographs of adult mouse corpus callosum showing no significant difference in the axon perimeter (H) and G-ratio (I) in mTrappc9m/m mice. Representative images from n=13 sections/group (G); n=200-220 myelinated axons (H, I). (J-L), MBP and OLIG2 immunostaining of primary cultured oligodendrocytes, which were differentiated from brain-derived oligodendrocyte precursor cells (OPCs) of P8 mice. The morphology (sholl analysis, K) and branch numbers (L) of oligodendrocytes were not affected by mTrappc9 deficiency. Representative images from n=3 coverslips/group (J); n=6 neurons (K); n=12 neurons (L). Data are means ± SEM; t-tests (L); One-way ANOVA (B, D, F, H, I); Two-way ANOVA (K); ****P≤0.0001; **P≤0.01; ns, not significant (P>0.05). Scale bars: 2 μm (G); 10 μm (J); 50 μm (A, C, E).
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