Endothelial RNF20 regulates the self-renewal and differentiation of neural precursor cells during embryonic development

Purpose: To further explore how endothelial RNF20 regulates embryonic neurogenesis, RNA isolated from the endothelial cells of RNF20fl/fl and RNF20cKO-Tie2 brain cortex at E13 was analyzed by RNA-seq to determine the genome-wide changes. Methods: mRNA from E13 isolated endothelial cells of RNF20fl/fl and RNF20cKO-Tie2 mice was extracted. Specifically, Agilent 2100 Bioanalyze was used to quality controlled and quantified. then, mRNA was converted to cDNA and bound the library. RNA-sequencing analysis was used by the Illumina HiSeq 2500 platform in Annoroad Genomics Results: Approximately one thousand transcripts showed differential expression between the RNF20fl/fl and RNF20cKO-Tie2 mice brain cortex, with a fold change >= 2 and p value <0.05. Gene ontology analysis of downregulated genes showed the genes were enriched in terms related to neuron differentiation, cell communication and secretion by cells.Concomitantly, upregulated genes were enriched in terms related to negative regulation of cytokine production and cell development.The results showed that endothelial RNF20 is critical for neurogenesis. Conclusions: Endothelial RNF20 RNA-seq would provide a overall understanding how endothelial RNF20 regulates the self-renewal and differentiation of neural precursor cells during embryonic development mRNA profiles of E13 RNF20fl/fl and RNF20cKO-Tie2 mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.研究目的：为深入探究内皮细胞RNF20调控胚胎神经发生的分子机制，本研究对胚胎第13天（E13）RNF20fl/fl与RNF20cKO-Tie2小鼠脑皮层内皮细胞提取的总RNA开展RNA测序（RNA-seq），以分析全基因组水平的基因表达变化。 实验方法：提取RNF20fl/fl与RNF20cKO-Tie2小鼠E13脑皮层内皮细胞的信使RNA（mRNA）。采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）对提取的mRNA进行质量控制与定量。随后将mRNA反转录为互补DNA（cDNA）并构建测序文库。依托安诺优达基因科技（Annoroad Genomics）的Illumina HiSeq 2500平台完成RNA测序分析。 实验结果：RNF20fl/fl与RNF20cKO-Tie2小鼠脑皮层内皮细胞间共有约1000个转录本出现差异表达，差异倍数≥2且P值<0.05。对下调差异基因进行基因本体（Gene Ontology, GO）富集分析显示，这些基因显著富集于神经元分化、细胞通讯及细胞分泌相关的功能条目。与此同时，上调差异基因则显著富集于细胞因子产生负调控与细胞发育相关的功能条目。上述结果表明，内皮细胞RNF20对神经发生具有关键调控作用。 研究结论：本研究生成的内皮细胞RNF20相关RNA测序数据，可全面解析胚胎发育过程中内皮细胞RNF20如何调控神经前体细胞的自我更新与分化。本实验采用Illumina HiSeq 2500平台进行三次生物学重复测序，获得了RNF20fl/fl与RNF20cKO-Tie2小鼠E13脑皮层内皮细胞的mRNA表达谱。