Time course analysis of defective myelination following Schwann cell specific Dicer depletion.

<p>Sciatic nerves of embryos (E17) and newborn mice (p4) were analyzed by electron microscopy for morphological effects of Schwann cell-specific Dicer depletion (<b>A</b>), and by quantitative RT-PCR for mRNA expression of factors known to regulate myelination. Number of biological replicates used: Dicer^fl/fl Dhh-Cre⁺ E17 n = 4, Dicer^wt/fl Dhh-Cre⁺ E17 n = 3, Dicer^fl/fl Dhh-Cre⁺ p4 n = 4, and Dicer^wt/fl Dhh-Cre⁺ p4 n = 4 (<b>B–D</b>). No morphological difference was observed before the onset of myelination at E17 between Dicer^wt/fl Dhh-Cre⁺ and Dicer^fl/fl Dhh-Cre⁺ nerves. By p4, myelination had begun in Dicer^wt/fl Dhh-Cre⁺ mice, but not in Dicer^fl/fl Dhh-Cre⁺ nerves. Some unsorted fibers were visible in Dicer^fl/fl Dhh-Cre⁺ nerves. Scale bars = 2 µm (<b>A</b>). Quantitative RT-PCR for mRNA expression in Dicer mutant nerves showed lack of developmental upregulation for activators of myelination. P values for comparisons between p4 controls and mutants were: <i>p</i> = 0.0026 (Oct6), <i>p</i> = 0.0028 (Egr2), <i>p</i> = 0.0003 (Brn2), <i>p</i> = 0.0028 (Sox10; <b>B</b>), no significant difference in expression of suppressors of myelination Sox2 and c-jun (<b>C</b>) and altered expression of Notch signaling components and p75NTR. P values for comparisons between p4 controls and mutants were: <i>p</i> = 0.061 (Delta1), <i>p</i> = 0.043 (Jagged1), <i>p</i> = 0.0001 (Jagged2), <i>p</i> = 0.02 (Notch3), <i>p</i> = 0.0061 (p75NTR; <b>D</b>). Dicer mRNA expression was significantly upregulated upon myelination in Dicer^wt/fl Dhh-Cre⁺ sciatic nerves (p4 in comparison with E17, <i>p</i> = 0.0004). At both time points, E17 and p4, significant Dicer mRNA depletion in Dicer^fl/fl Dhh-Cre⁺ was shown (E17: <i>p</i> = 0.0007; p4: <i>p</i><0.0001; <b>E</b>). Presence of the recombined Dicer allele in E17 and p4 mice was demonstrated by PCR using primers that differentiate between wild type Dicer and the recombined allele (<b>F</b>). The wild type allele is 1.3 kb in length, and the recombined allele is approx. 500bp (<b>G</b>). Akt phosphorylation and expression were analyzed by Western blot in Dicer^wt/fl Dhh-Cre⁺ compared to Dicer^fl/fl Dhh-Cre⁺ at p4. P-Akt in relation to total Akt was reduced to 62±14% of control level in Dicer^fl/fl Dhh-Cre⁺ at p4. All p values were determined using an unpaired two-tailed student's t-test. All error bars indicate standard deviation.