Cerebral 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 PDGFRalpha. Analysis of Pdgfc null mice has demonstrated roles for PDGF-C in palate closure and the formation of cerebral ventricles, but redundancy with other PDGFRalpha 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. Meninges freely dissected and released from P0 cerebrum. 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 Pdgfc-/-; Pdgfra GFP/+ mice, with a control group consisting of all littermates (Pdgfc+/+, Pdgfc+/-, Pdgfc-/-, Pdgfc+/+; Pdgfra GFP/+ and Pdgfc+/-; Pdgfra GFP/+). One litter was used, including 4 mutants and 9 controls血小板衍生生长因子C（Platelet-derived growth factor-C, PDGF-C）是目前已知的三种酪氨酸激酶受体PDGFRα（tyrosine kinase receptor PDGFRalpha）配体之一。对Pdgfc基因敲除小鼠（Pdgfc null mice）的分析已证实，PDGF-C在腭闭合与脑室形成过程中发挥关键作用，但其与其他PDGFRα配体间的功能冗余可能掩盖了PDGF-C的额外生物学功能。为探究PDGF-C更多的发育调控功能，我们构建了同时携带Pdgfc-/-与Pdgfra GFP/+基因型的双突变小鼠。该双突变小鼠呈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），在此区域中PDGF-C被证实为脑膜形成与胶质界膜基底膜（glia limitans basement membrane）组装的关键调控因子。可从出生后第0天（P0）的大脑中自由解离并分离得到脑膜。总RNA采用RNeasy微量试剂盒（RNeasy micro kit, Qiagen）提取，并通过2100生物分析仪（2100 BioAnalyzer, 安捷伦科技公司，美国加利福尼亚州圣克拉拉市）完成质量检测。转录谱分析（Transcription profiling）采用基因芯片小鼠基因1.0 ST阵列（Gene Chip Mouse Gene 1.0 ST array）开展。我们以Pdgfc-/-; Pdgfra GFP/+突变小鼠为实验组，对照组包含所有同窝仔鼠，其基因型涵盖Pdgfc+/+、Pdgfc+/-、Pdgfc-/-、Pdgfc+/+; Pdgfra GFP/+与Pdgfc+/-; Pdgfra GFP/+。本实验使用单窝仔鼠，其中包含4只突变个体与9只对照个体。