Cerebellar gene expression changes in Pdgfc and Pdgfra mutant

Platelet-derived growth factor-C (PDGF-C) is one of three known ligands for the tyrosine kinase receptor PDGFR-alpha. Analysis of Pdgfc null mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFR-alpha ligands might hide additional functions. In search of further developmental roles for PDGF-C, we generated mice that were double mutants for Pdgfc -/- and Pdgfra GFP/+. These mice display a range of severe phenotypes including cerebellar malformation, neuronal over-migration in the cerebral cortex, spina bifida and lung emphysema. We focused our analysis on the central nervous system (CNS), where PDGF-C was identified as a critical factor for the formation of meninges and assembly of the glia limitans basement membrane. Cerebellum from E13.5 and E14.5 embryos were freely dissected. Tissues were stored in RNA later (Ambion), RNA was isolated using RNeasy micro kit (Qiagen) and quality checked in a 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcription profiling was performed with Gene Chip Mouse Gene 1.0 ST array. We compared mice with a cerebellar phenotype (Pdgfc-/-; Pdgfra GFP/+) with a control group consisting of all littermates (Pdgfc+/+, Pdgfc+/-, Pdgfc-/-, Pdgfc+/+; Pdgfra GFP/+ and Pdgfc+/-; Pdgfra GFP/+). One litter from E13.5 (3 mutants and 5 controls) and one litter from E14.5 (2 mutants and 7 controls) were used.血小板衍生生长因子-C（Platelet-derived growth factor-C，PDGF-C）是酪氨酸激酶受体PDGFR-α的三种已知配体之一。对Pdgfc纯合敲除小鼠的研究表明，PDGF-C在腭闭合与脑室形成过程中发挥关键功能，但由于其与其他PDGFR-α配体存在功能冗余，可能掩盖了PDGF-C的额外生物学作用。为进一步探究PDGF-C在发育过程中的未知功能，我们构建了同时携带Pdgfc-/-与Pdgfra GFP/+双突变的小鼠模型。该双突变小鼠表现出一系列严重表型，包括小脑畸形、大脑皮层神经元过度迁移、脊柱裂与肺气肿。我们将分析重点聚焦于中枢神经系统（Central Nervous System，CNS），研究发现PDGF-C是脑膜形成与胶质界膜基底膜组装的关键调控因子。我们精准分离了胚胎发育第13.5天（E13.5）与第14.5天（E14.5）的小鼠小脑组织，随后将组织储存在RNAlater RNA保存液（Ambion公司）中，使用RNeasy微量RNA提取试剂盒（Qiagen公司）提取总RNA，并通过2100生物分析仪（安捷伦科技有限公司，美国加利福尼亚州圣克拉拉市）对RNA质量进行检测。我们采用小鼠基因1.0 ST芯片（Gene Chip Mouse Gene 1.0 ST array）开展转录组分析，以携带小脑表型的双突变小鼠（Pdgfc-/-; Pdgfra GFP/+）为实验组，以所有同窝仔鼠作为对照组，对照组涵盖以下基因型：Pdgfc+/+、Pdgfc+/-、Pdgfc-/-、Pdgfc+/+; Pdgfra GFP/+以及Pdgfc+/-; Pdgfra GFP/+。本研究共使用两窝小鼠样本：E13.5阶段的一窝（含3只突变体与5只对照小鼠）以及E14.5阶段的一窝（含2只突变体与7只对照小鼠）。