Fig 1.
Differentiating Oligodendrocytes present PC.
PC markers coincide with γ-tubulin staining on single rod-shaped structures on the surface of rat differentiating OPCs. Specific PC markers; (A) Glutamylated tubulin; (B) Acetylated Tubulin; (in green) were detected by immunofluorescence on the surface of rat oligodendrocytes 2 hrs after plating (0 hrs in differentiation medium) and for the following 24, 48 and 72 hrs. γ-tubulin staining is shown in red in all pictures. Bars: 2 μm in A and 5 μm in B.
Fig 2.
γ-Tubulin positive axonemes are undetectable in OPCs expressing the differentiation marker MBP.
Isolated rat Oligodendrocytes were cultured in differentiation medium over 72 hrs. At set time-points (A: 0 hrs; B: 24 hrs; C: 48 hrs; D: 72 hrs) cultures were fixed and stained for γ-tubulin (A, B) and MBP (C, D). DAPI was used for nuclei stain. Bars: 2 μm (E). Random fields were photographed in the 488 nm (γ-tubulin) and 543 nm (MBP) channels and cells were scored double blind. The graph represents the mean percentage of marker (+) cells ± s.d. of three independent experiments. *p< 0,01 (Kruskal Wallis test).
Fig 3.
Cyclopamine, a Hedgehog pathway antagonist, inhibits in vitro differentiation of Oligodendrocytes.
(A) Shh antagonist (Cyclopamine, 5μM) significantly inhibits OPC differentiation at 24, 48 and 72 hrs as assayed by process extension and branching evaluated through Scholl analysis. Each graph represents the distribution of cell processes crossing superimposed concentric circles marking distance from soma ± s.d. of three independent experiments. *p< 0,01 (Kruskal Wallis test). (B) Scholl analysis on color-inverted image of α-tubulin stained differentiating OPC with superimposed concentric circles marking distance from soma. (C) Cyclopamine (5μM for 72 hrs) significantly reduced MBP-rich membrane expansions. The graph represents the mean percentage of MBP+ cells ± s.d. of three independent experiments. Representative images of differentiated control and Cyclopamine-treated oligodendrocyte stained for MBP and α-tubulin (D, E). Bars: 10 μm. Cyc: Cyclopamine.
Fig 4.
Cyclopamine inhibits PDGF-AA mediated PC proliferation, but PDGF-Rα does not localize to the PC.
Isolated OPCs proliferate in response to PDGF-AA (10 ng/ ml), but in the presence of cyclopamine (5 μM) cell proliferation is inhibited as evidenced by a reduction of phosphorylated Histone-3 (pH3) (A) and BrdU incorporation (B) Bars: 10 μm. Graphs represents the mean percentage of marker (+) cells ± s.d. of three independent experiments. *p< 0,01 (Kruskal Wallis test). (C) PDGFR (green) and γ-tubulin (red and insets) localization was assessed by immunofluorescence in differentiating OPCs in the presence or absence of 5 μM cyclopamine (Cyc 5 μM). Bars: 20 μm.
Fig 5.
Shh rescues PC and Oligodendrocyte morphology from ciliobrevin.
A. Proliferating OPCs (medium supplemented with PDGF-AA) were pretreated for 3 hrs with ciliobrevin (30 μM) or with proliferation medium, after which they received 3.3 μg/ml of recombinant Shh and were cultured for 24 hrs before assessing PDGF-Rα (green) and Arl-13B (red) localization by immunofluorescence. Shh supplementation restored PC presence and structure. Bars: 20 μm. B. Quantification of 30 random fields scored for the presence of Arl-13B (+) and counted as a percent from total Dapi (+) cells. Graph shows percent of Arl-13B positive cells ± s.e.m. *P< 0,01 (Kruskal Wallis test).