RNA sequencing analysis of PINCH mutant sciatic nerves

During peripheral nervous system (PNS) development, Schwann cells (SC) depend on interactions between extracellular matrix proteins, integrins and growth factor signal transduction pathways for survival, sorting and wrapping of axon segments to form myelin. Together with integrin-linked-kinase and parvins, PINCH (Particularly-interesting-new-cystidin-hystidin-rich protein) forms a heterotrimeric complex referred to as the IPP complex, which functions as a signaling hub connecting integrins with various signaling pathways. Using SC-specific conditional gene targeting in mice, gene expression profiling by RNAseq, and other methodologies, we demonstrate distinct functional roles for PINCH1 and PINCH2 in SC. Total RNA was prepared from nerves collected from control, PINCH1 mutant and PINCH2 mutant (three biological replicates each). Each biological replicate consisted in a pool of four SN, from 1 male and 1 female to avoid gender-related differences). All procedures involving mice were in strict accordance to the guidelines approved by the Portuguese Veterinary Office (DGAV) and EU directives.在外周神经系统（peripheral nervous system, PNS）发育过程中，雪旺细胞（Schwann cells, SC）依赖细胞外基质蛋白、整合素与生长因子信号转导通路之间的相互作用，以完成存活、轴突节段的分选与包裹，进而形成髓鞘。PINCH蛋白（Particularly-interesting-new-cystidin-hystidin-rich protein）与整合素连接激酶（integrin-linked-kinase）及副蛋白（parvins）共同组成异三聚体复合物，即IPP复合物，其作为信号枢纽连接整合素与各类信号通路。本研究通过小鼠雪旺细胞特异性条件性基因靶向技术、RNA测序（RNAseq）基因表达谱分析及其他实验方法，阐明了PINCH1与PINCH2在雪旺细胞中截然不同的功能角色。研究中，我们从对照组、PINCH1突变体组与PINCH2突变体组小鼠的神经组织中提取总RNA，每组各设置3次生物学重复。每一次生物学重复均由4条坐骨神经（sciatic nerve, SN）组成，取材自1只雄性与1只雌性小鼠，以规避性别相关的实验差异。所有涉及小鼠的实验操作均严格遵循葡萄牙兽医办公室（DGAV）批准的实验指南及欧盟相关指令。