Transcriptome analyses of miRNA pathway mutants and injury response in developing and adult sciatic nerves

We addressed the requirement of DGCR8, DROSHA and DICER functions in developing and adult Schwann cells (SCs) using mouse mutants. We found that the microprocessor components DGCR8 and DROSHA are crucial for axonal radial sorting and to establish correct SC numbers upon myelination. Transcriptome analysis revealed that the microprocessor is essential to prevent aberrant accumulation and de novo expression of injury-response genes. Those genes are predicted targets of stage-specifically enriched miRNAs. In agreement, DGCR8 and DICER are required for proper maintenance of the myelinated SC state. We conclude that the miRNA pathway is crucial for preventing inappropriate activity of injury response genes in developing and adult SCs. At post natal day 1, sciatic nerves of Schwann cell specific conditional mutants for DICER, DGCR8, DROSHA and respective controls were analyzed by RNA sequencing in triplicate. Sciatic nerves of induced Schwann cell specific mutants for DICER and DGCR8 were analyzed 3 months after administration of tamoxifen and compared to P0-CreERT2-negative controls in triplicate. In addition, wild-type adult sciatic nerves were analyzed 3 days post crush injury (in quadruplicate) in comparison to contralateral (in quadruplicate) and uncrushed sciatic nerve controls (in triplicate).本研究借助小鼠突变体模型，探究了DGCR8、DROSHA与DICER在发育及成熟施万细胞（Schwann cell, SC）中的功能需求。研究发现，miRNA微处理器复合物（microprocessor）的组分DGCR8与DROSHA对于轴突径向分选以及髓鞘形成过程中维持正常施万细胞数量至关重要。转录组分析显示，该微处理器复合物对于阻断损伤应答基因的异常积累与从头表达不可或缺，上述损伤应答基因被预测为阶段特异性富集miRNA的靶标。与此一致，DGCR8与DICER对于维持有髓鞘施万细胞的正常状态同样必需。综上，本研究证实miRNA通路对于阻断发育中及成熟施万细胞内损伤应答基因的异常激活至关重要。 实验设计如下：于出生后第1天，对DICER、DGCR8及DROSHA施万细胞特异性条件性敲除小鼠及其对应野生型对照的坐骨神经开展三次生物学重复RNA测序分析；在他莫昔芬给药3个月后，对诱导型DICER与DGCR8施万细胞特异性敲除小鼠的坐骨神经进行三次生物学重复RNA测序分析，并与P0-CreERT2阴性对照小鼠进行对比；此外，对成年野生型小鼠坐骨神经挤压损伤3天后的样本进行四次生物学重复RNA测序分析，并分别与损伤侧对侧坐骨神经样本（四次生物学重复）及未损伤坐骨神经对照样本（三次生物学重复）进行比对。