Figure 12.

Sustained Sox9 expression inhibits OPC differentiation. A, Experimental design for B–G. Cortical primary OPCs in growth medium were transfected with Sox9-expressing plasmid (Sox9, n = 3) or EV (n = 3), then differentiated for 24 h in serum-free differentiating medium. B, qRT-PCR quantification of Sox9 mRNA expression. C, Representative immunocytochemical images of Sox9/Sox10. Arrowheads point to Sox9⁺/Sox10⁺ oligodendrocytes and arrows point to Sox9⁺/Sox10^– cells, which are presumably astrocytes. Scale bar, 20 μm. D, Percentage of Sox10⁺ oligodendroglial lineage cells that express Sox9 (top) and Sox9-immunoreactive intensity normalized to Sox10 (bottom). E, qRT-PCR assay of exon3b-containing Plp (Plp-E3b), which specifically expresses in differentiated OLs, and myelin regulatory factor (Myrf), a potent prodifferentiation gene. F, Representative immunocytochemical images of MBP/Sox10 and density (#/mm²) of Sox10⁺ oligodendroglial cells of complex ramified morphology indicated by MBP staining (F, arrowheads), Scale bar, 10 μm. G, Western blot images and quantification of MBP and internal loading control β-actin. H, Experimental designs for I–M, primary OPCs were treated with HIFα stabilizer DMOG and transfected with Sox9 siRNA twice with 24 h apart. I, J, Expression of Sox9 (I) and HIFα target gene Bnip3 (J) quantified by qRT-PCR. One-way ANOVA followed by Tukey's test: Sox9, F_(3,8) = 10.33, p = 0.004; Bnip3: F_(3,8) = 20.83, p = 0.0004. K, Representative immunostaining images of differentiated OLs labeled by MBP and Sox10. Scale bar, 10 µm. L, M, Density (#/mm²) of MBP⁺/Sox10⁺ differentiated OLs (L) and qRT-PCR assay of Mbp mRNA expression (M). One-way ANOVA followed by Tukey's test: MBP⁺/Sox10⁺, F_(3,8) = 33.29, p < 0.0001; Mbp, F_(3,8) = 97.96, p < 0.0001. N, Previously proposed and our alternative working model explaining the role of HIFα in OPC differentiation. Endogenous HIFα regulates CNS myelination by controlling upstream OPC differentiation but not downstream OL maturation in perinatal and early postnatal CNS, whereas aberrant HIFα activation (via VHL mutation of hypoxia-ischemia insult) inhibits OPC differentiation through the Wnt-independent and Sox9-dependent pathway.