Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.. Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.

Purpose: The cellular composition of the trigeminal ganglion is altered in the mouse model of 22q11.2 deletion syndrome (the LgDel mouse). The goal of this study is to use RNA-seq to identify transcriptional differences in the embryonic day 10.5 trigeminal ganglion that prefigure changes in mature cell identity in this cranial nerve ganglion. Methods: Trigeminal ganglion mRNA profiles of E10.5 trigeminal ganglia from WT and LgDel mice were generated by deep sequencing. Ganglia samples were prepared as pools of at least 6 ganglia each, from at least 3 embryos. 5 biological replicates of LgDel and WT pools were sequenced. Paired-end libraries were constructed according to the Illumina protocol for the HiSeq2000 platform. Each pooled CNgV RNA sample, defined as a single biological replicate, was fragmented prior to cDNA conversion to ensure transcript coverage. WT (n = 5 replicates) and LgDel (n = 5 replicates) were subjected to sequencing (~100 million paired-end reads/replicate, 100 bp read length) on the Illumina HiSeq2000 platform. Quality-filtered reads were aligned against the mouse reference genome (GRCm38/mm10) using HISAT2 (v.2.1.0). These alignments were indexed using SAMtools (v.1.4) and aligned reads assembled into transcripts using Cufflinks (v.2.2.1) (58). Differential expression analysis was performed using Cufflinks. Validation of key transcriptional differences was performed by SYBR Green qPCR. Results: For WT RNA-Seq, 111–123 million paired-end reads were generated after QC filtering, and 97.3–97.9% of the reads aligned to the mouse genome reference sequence. For LgDel RNA-Seq, 77–137 million paired-end reads were generated after QC filtering, and 94.3–97.8% of the reads aligned to the mouse genome reference sequence. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior–posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17 000 genes expressed in common in LgDel versus WT CNgV. Conclusions: Quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice. Overall design: RNA-seq analysis of trigeminal nucleus in LgDel mouse

研究目的：22q11.2缺失综合征小鼠模型（LgDel小鼠）的三叉神经节细胞组成发生改变。本研究旨在通过RNA测序（RNA-seq），鉴定胚胎第10.5天三叉神经节中的转录组差异，以揭示该颅神经节成熟细胞身份改变的早期预示特征。
研究方法：采用深度测序技术，获取野生型（Wild Type, WT）与LgDel小鼠E10.5期三叉神经节的mRNA表达谱。神经节样本以至少6个神经节为一组混合，取自至少3个胚胎，LgDel组与WT组各设置5个生物学重复样本进行测序。依照Illumina HiSeq2000平台的建库方案构建双端测序文库。每个混合颅神经V节（CNgV）RNA样本（即单个生物学重复）在进行cDNA合成前先进行片段化处理，以确保转录本覆盖度。WT组（n=5个重复）与LgDel组（n=5个重复）均在Illumina HiSeq2000平台上完成测序，每个重复约产生1亿条双端读段，读长为100 bp。使用HISAT2（v.2.1.0）将经过质量过滤的读段比对至小鼠参考基因组（GRCm38/mm10），随后通过SAMtools（v.1.4）对比对结果构建索引，再利用Cufflinks（v.2.2.1）将比对读段组装为转录本（参考文献58）。采用Cufflinks进行差异表达分析，并通过SYBR Green定量聚合酶链反应（qPCR）验证关键转录组差异。
研究结果：经质量控制过滤后，WT组RNA-seq共产生1.11~1.23亿条双端读段，其中97.3%~97.9%的读段可比对至小鼠参考基因组序列；LgDel组则产生7700万~1.37亿条双端读段，94.3%~97.8%的读段可比对至小鼠参考基因组序列。在神经节融合后的E10.5期，LgDel组与野生型颅神经V节（CNgV）转录组存在显著差异。部分差异与颅基板与颅神经嵴来源的CNgV细胞比例改变相关，其余差异则与LgDel小鼠颅神经分化异常相关的前后轴模式转变一致。但最显著的定量差异为：与WT组相比，LgDel组CNgV中共表达的17000余个基因的表达水平均存在统计学可验证的显著变异度升高。
研究结论：在颅神经V节分化起始阶段，功能相关基因的定量表达变化以及全转录组水平的随机变异度升高，预示了LgDel小鼠后续颅神经分化障碍与口咽功能异常。
整体实验设计：对LgDel小鼠的三叉神经核进行RNA-seq分析。