Figure 6. Conditional deletion of Pikfyve in OLs results in profound CNS hypomyelination.
(A-D’) Sagittal sections of P13 mouse brains. (A) Pikfyve control (Pikfyveflox/+ or Pikfyveflox/flox; n = 3) mice and (A’) Pikfyve conditional null (Pikfyveflox/flox,Olig2Cre; n = 3) mice stained with FluoroMyelin Green. In Pikfyveflox/flox,Olig2Cre, no myelin staining was observed, Scale bar, 1 mm. (B-D’) in situ hybridization for Plp1 shows virtual absence of mature OLs in P13 Pikfyveflox/flox,Olig2Cre brain tissue, including (B and B’) internal capsule and corpus callosum, (C’ and C’) hippocampus and corpus callosum and (D and D’) cerebellar white matter. Scale bar (B-D’) = 500 µm. (E-H) Cultures of primary OPCs/OLs isolated from Pikfyve control and Pikfyveflox/flox,Olig2cre mouse pups. (E, E’) At DIV2, cells were fixed and stained with anti-PDGFRα, anti-Ki67and Hoechst 33342 dye. (F, F’) After 3 days in differentiation medium, supplemented with T3, cells were fixed and stained with anti-MBP and Hoechst 33342. (G) Quantification of proliferating OPCs revealed a Pikfyve-dependent reduction in Ki67+/PDGFRα+ double-labeled cells (n = 3 experiments per genotype). (H) Quantification of MBP+ OLs normalized to Hoechst+ cells shows a highly significant decrease in the number of MBP+ OLs in Pikfyveflox/flox,Olig2cre cultures (n = 3 experiments per genotype). Unpaired Student’s t-test; mean value ± SEM. **p=0.011 and ****p<0.0001. Toluidine blue labeling of epoxy resin embedded optics nerves of Pikfyve control and Pikfyveflox/flox,Olig2cre conditional mutant mice is shown in Figure 6—figure supplement 1.
Figure 6—figure supplement 1. Optic nerve axons are not myelinated in Pikfyveflox/flox,Olig2Cre mice.
