Next Generation Sequencing of Control and Pbx Mutant Spinal Motor Neuron Transcriptomes

Purpose: Conducted expression profiling by RNA-seq as unbiased screen to identify genes that are altered in motor neurons of PbxMNdelta mice at e12.5 at brachial and thoracic levels of the spinal cord. Because loss of Pbx genes affects MN organization at all rostrocaudal levels, we focused on genes whose profiles were altered at both brachial and thoracic levels. Methods: We compared gene expression profiles in MNs isolated from control Hb9::GFP and PbxMNdelta; Hb9::GFP embryos at e12.5. MNs were purified by FACS, and RNA was extracted from 9 PbxMNdelta; Hb9::GFP and 9 control Hb9::GFP embryos at brachial and thoracic levels using the Arcturus Picopure RNA isolation kit. 10ng of RNA was pooled from 3 RNA samples of each genotype, and used to amplify 100ng of cDNA using Nugene's Ovation RNA-Seq System V2 kit, 100ng of cDNA for each sample was used as in input to prepare 12 bar coded libraries using the Ovation Ultralow Library system. We then performed expression profiling by RNA-seq. The samples were mixed into two pools and run on two 50-nucleotide paired end read rapid run flow cell lanes with the Illumina HiSeq 2500 sequencer. Generating on average 74 and 101 million reads passing filter for brachial and thoracic samples respectively. Results: This analysis yielded 64 brachial and 124 thoracic genes that were differentially expressed with a stringent cutoff of padj.<0.05. Of these genes, we found 31 genes in common between the two, brachial and thoracic, levels of the spinal cord that may play a role in motor neuron columnar organization. Furthermore our expression profiling of control brachial and control thoracic MNs identified 61 genes with (padj.<0.05), that represent distinct molecular profiles of MNs generated at brachial and thoracic levels which may be used to further characterize MNs involved in forelimb and thoracic innervation. Conclusions: Our study represents a detailed transcriptional analysis of embryonic spinal motor neurons and revealed a number of novel motor neuron-specific genes that are under transcriptional regulation of Pbx genes. Examination of embryonic spinal MN expression profiles at 2 different spinal cord levels, brachial and thoracic. From RNA collected from 9 pooled Control and 9 PbxMNdelta e12.5 Hb9::GFP FACS MNs.研究目的：通过RNA测序（RNA-seq）开展无偏筛选，以鉴定胚胎发育第12.5天（e12.5）的PbxMNdelta小鼠脊髓臂段与胸段运动神经元（motor neurons, MN）中表达发生改变的基因。鉴于Pbx基因缺失会影响所有前后轴水平的运动神经元排布，本研究聚焦于在臂段和胸段均出现表达异常的基因。 实验方法：对比对照Hb9::GFP胚胎与PbxMNdelta; Hb9::GFP胚胎中分离得到的运动神经元基因表达谱。通过荧光激活细胞分选（FACS）纯化运动神经元，使用Arcturus Picopure RNA分离试剂盒，从9个PbxMNdelta; Hb9::GFP胚胎和9个对照Hb9::GFP胚胎的臂段与胸段脊髓中提取RNA。将每种基因型的3份RNA样本混合为10ng总RNA，随后采用Nugene公司的Ovation RNA-Seq System V2试剂盒扩增得到100ng cDNA。以每份样本的100ng cDNA为起始材料，通过Ovation Ultralow Library系统构建12个带条形码的测序文库。随后通过RNA-seq进行表达谱分析。将样本混合为两个文库池，在Illumina HiSeq 2500测序仪的两个50核苷酸双端读长快速运行流通池通道中完成测序，臂段样本与胸段样本的平均过滤后有效读段数分别为7400万和1.01亿。 实验结果：以校正后P值（padj）<0.05作为严格筛选阈值，本分析共鉴定出64个臂段差异表达基因与124个胸段差异表达基因。其中31个基因在脊髓臂段和胸段的运动神经元中均存在表达差异，这类基因可能参与运动神经元柱的排布调控。此外，对对照臂段与对照胸段运动神经元的表达谱分析鉴定出61个padj<0.05的差异基因，这些基因代表了臂段与胸段运动神经元的独特分子特征，可用于进一步表征参与前肢和胸壁神经支配的运动神经元。 研究结论：本研究对胚胎脊髓运动神经元开展了详细的转录组分析，揭示了一批受Pbx基因转录调控的新型运动神经元特异性基因。本研究检测了胚胎脊髓运动神经元在两个不同脊髓节段（臂段与胸段）的表达谱，所用RNA样本来自经荧光激活细胞分选（FACS）得到的e12.5 Hb9::GFP阳性运动神经元，分别混合了9份对照胚胎与9份PbxMNdelta胚胎的样本。