Transcriptional profiling of endothelial cells lacking Flvcr2

Flvcr2 is a putative heme transporter expressed in endothelial cells of the brain. Mutations in this gene in humans is the cause of Fowler syndrome, in which newborns show malformed brain blood vessels. We generated a mouse model of this disease by knocking out the mouse ortholog of the gene (Flvcr2). These mouse mutants recapitulate the human phenotype. To understand the molecular changes associated with Flvcr2 deletion, we defined the transcriptional profile of heterozygous and mutant endothelial cells. We observed changes in gene expression consistent with altered angiogenesis and metabolic pathways necessary for endothelium homeostasis. Endothelial cells were isolated from E14.5 embryonic brains using fluorescent-activated cell sorting. Since the mutation in the Flvcr2 gene leads to expression of GFP, we sorted GFP+ CD31+ cells. Total RNA was extracted from 2000-5000 cells to synthesize PolyA+ libraries for sequencing in a HiSeq4000 using SE50. More than 40 million reads were obtained for each sample.Flvcr2是一种推定的血红素转运蛋白，在大脑内皮细胞中表达。人类该基因发生突变可引发Fowler综合征，患者新生儿会出现脑血管畸形症状。我们通过敲除该基因的小鼠同源基因（Flvcr2）构建了该病的小鼠模型，该小鼠突变体可重现人类患者的表型。为探究Flvcr2缺失相关的分子变化，我们解析了杂合子及突变型内皮细胞的转录谱，观察到基因表达的变化与血管生成通路异常及维持内皮稳态所必需的代谢通路改变相符。我们通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）从胚胎发育第14.5天（E14.5）的小鼠脑组织中分离内皮细胞：由于Flvcr2基因突变会诱导绿色荧光蛋白（GFP）的表达，因此我们分选了GFP阳性且CD31阳性的细胞。我们从2000~5000个细胞中提取总RNA，合成PolyA+文库，随后采用SE50测序模式在HiSeq4000测序平台上进行测序，每个样本可获得超过4000万条测序读段。