Endothelial Pd1 orchestrates cerebrovasculature homeostasis and oligodendrogenesis during brain development

Purpose: To explore further into the mechanisms by which endothelial PD1 controls cerebrovascular development and oligodendrogenesis, we employed RNA-seq to examine genome-wide alterations in endothelial cells from P0 conditional PD1 knockout mice, comparing them with their wild-type littermates. Methods: mRNA was extracted from P0 isolated endothelial cells of Pd1fl/fl and Pd1cKO-Tie2 mice, and the quality and quantity were assessed using the Agilent 2100 Bioanalyzer. Subsequently, the mRNA was converted to cDNA and Libraries were prepared. RNA-sequencing was performed on the Illumina HiSeq 2500 platform at Annoroad Genomics. Results: Approximately one thousand transcripts showed differential expression between the Pd11fl/fl and Pd1cKO-Tie2 mice brain endothelial cell, with a fold change 2 and p value <0.05.Geneontology analysis of the downregulated genes were enriched in terms related to positive regulation of angiogenesis, positive regulation of vascular endothelial growth factor production and positive regulation of inflammatory response. Concomitantly, upregulated genes were enriched in terms related to endothelial cell migration, sequestering of BMP from receptor via BMP binding and regulation of establishment of blood-brain barrier. These results reflected endothelial PD1 plays vital roles in cortex development. Conclusions: Endothelial PD1 RNA-seq would provide a overall understanding how endothelium-derived PD1regulates vessel development and oligodendrogenesis during brain development mRNA profiles of P0 Pd1fl/fl and Pd1cKO-Tie2 mice were generated by deep sequencing, in duplicate, using Illumina HiSeq 2500.研究目的：为进一步探究内皮PD1（endothelial PD1）调控脑血管发育与少突胶质细胞生成的分子机制，本研究采用RNA测序（RNA-seq）技术，对出生后第0天（P0）条件性PD1敲除小鼠及其野生型同窝仔鼠的内皮细胞进行全基因组表达差异分析。 实验方法：从Pd1fl/fl与Pd1cKO-Tie2小鼠P0时期分离的内皮细胞中提取mRNA，使用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）评估mRNA的质量与浓度。随后将mRNA反转录为cDNA并构建测序文库，随后在安诺优达基因科技（Annoroad Genomics）的Illumina HiSeq 2500平台上完成RNA测序。 研究结果：Pd1fl/fl与Pd1cKO-Tie2小鼠脑内皮细胞间约有1000个转录本存在显著表达差异（倍数变化≥2，p值<0.05）。对下调基因进行基因本体（Gene Ontology, GO）富集分析发现，其显著富集于血管生成正调控、血管内皮生长因子产生正调控以及炎症反应正调控等生物学过程。与此同时，上调基因则显著富集于内皮细胞迁移、通过BMP结合从受体隔离骨形态发生蛋白（BMP）以及血脑屏障建立调控等生物学过程。上述结果表明，内皮PD1在大脑皮层发育过程中发挥关键调控作用。 研究结论：本研究产生的内皮PD1 RNA测序数据，可全面阐明脑发育过程中内皮源性PD1如何调控血管发育与少突胶质细胞生成的机制。本数据集包含采用Illumina HiSeq 2500平台完成的双重复测序得到的P0时期Pd1fl/fl与Pd1cKO-Tie2小鼠脑内皮细胞mRNA表达谱。