Abstract
Pericytes, the mural cells of blood microvessels, regulate microvascular development and function and have been implicated in many brain diseases. However, due to a paucity of defining markers, pericyte identification and functional characterization remain ambiguous and data interpretation problematic. In mice carrying two transgenic reporters, Pdgfrb-eGFP and NG2-DsRed, we found that double-positive cells were vascular mural cells, while the single reporters marked additional, but non-overlapping, neuroglial cells. Double-positive cells were isolated by fluorescence-activated cell sorting (FACS) and analyzed by RNA sequencing. To reveal defining patterns of mural cell transcripts, we compared the RNA sequencing data with data from four previously published studies. The meta-analysis provided a conservative catalogue of 260 brain mural cell-enriched gene transcripts. We validated pericyte-specific expression of two novel markers, vitronectin (Vtn) and interferon-induced transmembrane protein 1 (Ifitm1), using fluorescent in situ hybridization and immunohistochemistry. We further analyzed signaling pathways and interaction networks of the pericyte-enriched genes in silico. This work provides novel insight into the molecular composition of brain mural cells. The reported gene catalogue facilitates identification of brain pericytes by providing numerous new candidate marker genes and is a rich source for new hypotheses for future studies of brain mural cell physiology and pathophysiology.
Highlights
All vertebrate blood vessels contain two principal cell types, endothelial cells (EC) and mural cells
Pdgfrb and NG2 are two commonly used markers for mural cells, neither of which are completely PC-specific. Their non-mural cell expression in the brain appeared non-overlapping, (Fig. 1A,D) with abundant Pdgfrb-enhanced green fluorescent protein (eGFP) expression noticeable in the subventricular zone and NG2-DsRed expression scattered throughout the brain in cells that likely correspond to oligodendrocyte progenitors (OPC)[43]
Cells with concurrent expression of Pdgfrb-eGFP and NG2-DsRed were invariably associated with the blood vessel endothelium, and had the expected location, density, distribution and morphology of vascular mural cells (Fig. 1A)
Summary
All vertebrate blood vessels contain two principal cell types, endothelial cells (EC) and mural cells. We reasoned that by comparing published data with direct profiling of isolated brain mural cells, we would derive significantly deeper and more authentic information on the brain mural cell transcriptome. To this end, we generated a double transgenic www.nature.com/scientificreports/. The double reporter mice (Pdgfrb-eGFP/NG2-DsRed mice) expressed both reporters only in mural cells in the brain, whereas the single reporters were expressed in other neuroglial cells in non-overlapping patterns This allowed us to isolate brain mural cells by FACS and analyze the cells with high throughput next-generation RNA sequencing (RNA-seq). The resulting gene catalogue provides direct insights into the unique molecular composition, signaling pathways and protein interaction networks within vascular mural cells of the brain
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