Ablation of Dicer from murine Schwann cells increases their proliferation while blocking myelination

Abstract

The myelin sheaths that surround the thick axons of the peripheral nervous system are produced by the highly specialized Schwann cells. Differentiation of Schwann cells and myelination occur in discrete steps. Each of these requires coordinated expression of specific proteins in a precise sequence, yet the regulatory mechanisms controlling protein expression during these events are incompletely understood. Here we report that Schwann cell-specific ablation of the enzyme Dicer1, which is required for the production of small non-coding regulatory microRNAs, fully arrests Schwann cell differentiation, resulting in early postnatal lethality. Dicer(-/-) Schwann cells had lost their ability to myelinate, yet were still capable of sorting axons. Both cell death and, paradoxically, proliferation of immature Schwann cells was markedly enhanced, suggesting that their terminal differentiation is triggered by growth-arresting regulatory microRNAs. Using microRNA microarrays, we identified 16 microRNAs that are upregulated upon myelination and whose expression is controlled by Dicer in Schwann cells. This set of microRNAs appears to drive Schwann cell differentiation and myelination of peripheral nerves, thereby fulfilling a crucial function for survival of the organism.

Figure 1. Defective myelination following Schwann cell specific Dicer depletion.

Electron microscopy of sciatic nerves derived from 18-day old Dicer^fl/fl Dhh-Cre⁺ (C and F) and control Dicer^wt/fl Dhh-Cre⁺ (B and E) and Dicer^fl/fl Dhh-Cre⁻ (A and D) littermates. Low magnification figures (A–C) show normally myelinated nerves fibers in control mice (A and B) and normal Remak bundles of unmyelinated small-caliber axons (black stars). In Dicer depleted mice (C), most of the large caliber axons remain unmyelinated; only a few fibers are ensheathed by a thin myelin sheath (black arrowheads). Most of these nerve fibers show proper axonal sorting by Schwann cells; only a few large-caliber axons remain unsorted (white arrowheads). Higher magnification figures show normal myelinated and unmyelinated nerve fibers (D and E) in controls. A Schwann cell in Dicer^fl/fl Dhh-Cre⁺ mice containing non-sorted and unmyelinated large caliber axons is shown. Basal lamina formation by this Schwann cell is evident (black arrow), indicating development into immature Schwann cell (F). Scale bar in A–C = 10 µm and in D–F = 1 µm.

Figure 2. Histological and biochemical analysis of mice lacking Dicer in Schwann cells.

(A–C) Longitudinal sections of sciatic nerves from 22-day old Dicer^fl/fl Dhh-Cre⁺ and control Dicer^wt/fl Dhh-Cre⁺ littermates stained with haematoxylin and eosin (HE) or decorated with S100, MIB1, CD3, or CD68 antibody. At least four nerves were analyzed per genotype. Scale bars = 50 µm. S100 positivity indicates Schwann cell development progresses at least up to the stage of immature Schwann cells (A). Increased proliferation in Dicer mutant nerves, as indicated by mitotic events (black arrows in A) and increased percentage of positive nuclei in MIB1 immunohistochemistry (B). Quantification of MIB1-positive nuclei shows a significantly higher percentage of proliferating cells in Dicer^fl/fl Dhh-Cre⁺ compared to Dicer^wt/fl Dhh-Cre⁺ littermate nerves. Error bars indicate standard deviation, p = 0.0003, p value was determined using unpaired two-tailed student's t-test (B). Few CD3-positive T cells and an increased percentage of CD68-positive macrophages infiltrated the nerves of Dicer^fl/fl Dhh-Cre⁺ mice (C). Biochemical analysis of signal transduction pathways and myelin components by Western blot (D). Compared to control Dicer^wt/fl Dhh-Cre⁺ littermates, phospho-Akt and phospho-Erk were significantly increased in sciatic nerves of 18-day old mice lacking Dicer in Schwann cells, while total Akt and Erk protein levels were unchanged compared to controls, and NFκB was significantly decreased. In agreement with the histological findings, components of non-compact (CNPase) and compact myelin (MBP, PMP22) were nearly absent from Dicer mutant nerves. In addition, Ras levels were significantly lower in Dicer mutant nerves. GAPDH and β-actin served as loading controls.

Figure 3. Time course analysis of defective myelination following Schwann cell specific Dicer depletion.

Sciatic nerves of embryos (E17) and newborn mice (p4) were analyzed by electron microscopy for morphological effects of Schwann cell-specific Dicer depletion (A), 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–D). 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 (A). 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: p = 0.0026 (Oct6), p = 0.0028 (Egr2), p = 0.0003 (Brn2), p = 0.0028 (Sox10; B), no significant difference in expression of suppressors of myelination Sox2 and c-jun (C) and altered expression of Notch signaling components and p75NTR. P values for comparisons between p4 controls and mutants were: p = 0.061 (Delta1), p = 0.043 (Jagged1), p = 0.0001 (Jagged2), p = 0.02 (Notch3), p = 0.0061 (p75NTR; D). Dicer mRNA expression was significantly upregulated upon myelination in Dicer^wt/fl Dhh-Cre⁺ sciatic nerves (p4 in comparison with E17, p = 0.0004). At both time points, E17 and p4, significant Dicer mRNA depletion in Dicer^fl/fl Dhh-Cre⁺ was shown (E17: p = 0.0007; p4: p<0.0001; E). 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 (F). The wild type allele is 1.3 kb in length, and the recombined allele is approx. 500bp (G). 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.

Figure 4. Heat map of developmentally and/or Dicer dependently regulated miRNAs in sciatic nerves.

Expression of miRNAs in sciatic nerves of E17 and p4 Dicer^fl/fl Dhh-Cre⁺ and control Dicer^wt/fl Dhh-Cre⁺ littermates was analyzed by miRNA microarray. Four biological replicates for each group were analyzed on separate arrays. Of the 216 miRNAs expressed, 109 miRNAs (listed on the right side of the heat map) were differentially expressed, either in an age-dependent manner or in a manner dependent on the expression of Dicer in Schwann cells (p≤0.05, log₂ ratio≥0.5). Differential expression in log₂ ratio is color coded as indicated in the legend below the heat map (red = upregulation, green = downregulation). Based on hierarchical clustering, five groups, each containing miRNAs of similar expression pattern are indicated by gray bars on the left side. Group 1 contains miRNAs which are upregulated in Dicer^fl/fl Dhh-Cre⁺ compared to Dicer^wt/fl Dhh-Cre⁺ nerves at E17 and at p4. Group 2 contains miRNAs that are downregulated in both genotypes at p4 compared to E17. Group 3 includes miRNAs that are downregulated in Dicer^wt/fl Dhh-Cre⁺ nerves at p4 when compared to E17 and downregualted in Dicer^fl/fl Dhh-Cre⁺ compared to Dicer^wt/fl Dhh-Cre⁺ nerves at E17. Group 4 contains miRNAs which are downregulated in Dicer^fl/fl Dhh-Cre⁺ compared to Dicer^wt/fl Dhh-Cre⁺ nerves at p4 and are abundantly expressed also in Dicer^wt/fl Dhh-Cre⁺ nerves at E17. Group 5 includes miRNAs that are upregulated in Dicer^wt/fl Dhh-Cre⁺ nerves at p4 compared to both, Dicer^fl/fl Dhh-Cre⁺ and Dicer^wt/fl Dhh-Cre⁺ nerves at E17.

Figure 5. Real time PCR confirmation of miRNAs upregulated upon myelination.

Expression of miRNAs in sciatic nerves of E17 and p4 Dicer^fl/fl Dhh-Cre⁺ (fl/fl) and control Dicer^wt/fl Dhh-Cre⁺ (wt/fl) mice was analyzed by real time PCR with Taqman probes. The levels of miRNA expression were quantified in comparison with sno234 RNA as the endogenous control. Expression is shown as relative values compared to p4 Dicer^wt/fl Dhh-Cre⁺. Error bars indicate standard deviation. All miRNAs analyzed were significantly downregulated in Dicer^fl/fl Dhh-Cre⁺ nerves at p4: p≤0.0001 (miR-34a, miR-146b, miR-338-3p, miR-204, miR-27b, miR-140, miR-138, miR-30a), p = 0.0002 (miR-195). Furthermore, miRNAs were confirmed to be upregulated upon myelination: p≤0.0001 (miR-34a, miR-146b), p = 0.04 (miR-338-3p), p = 0.003 (miR-204), p = 0.0007 (miR-27b), p = 0.005 (miR-140), p = 0.0002 (miR-138), p = 0.01 (miR-195), p = 0.0004 (miR-30a). P values were determined using unpaired two-tailed student's t-test.

Figure 6. The role of Dicer and miRNAs in Schwann cell development.

In the absence of Dicer, Schwann cells develop normally to the pro-myelin stage with properly sorted axons (light yellow). The presence of Dicer is required for acquisition of myelination competence by Schwann cells (turquoise). When Dicer is removed from Schwann cells (red), reduction of miRNAs leads to Schwann cell hyperproliferation and degeneration, suggesting that miRNAs are essential both for driving maturing Schwann cells into cell cycle arrest and terminal differentiation, as well as for their survival.