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. 2016 Oct 11:6:35108.
doi: 10.1038/srep35108.

Analysis of the brain mural cell transcriptome

Liqun He  1 , Michael Vanlandewijck  1   2 , Elisabeth Raschperger  1   2 , Maarja Andaloussi Mäe  1 , Bongnam Jung  1 , Thibaud Lebouvier  1 , Koji Ando  1 , Jennifer Hofmann  1 , Annika Keller  3 , Christer Betsholtz  1   2
Affiliations

Analysis of the brain mural cell transcriptome

Liqun He et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. Specific labeling of central nervous mural cells in Pdgfrb-eGFP/NG2-DsRed mice.
(A) Expression pattern of Pdgfrb-eGFP and NG2-DsRed in cerebrum of Pdgfrb-eGFP/NG2-DsRed mice. In the lower panels, cells lining capillaries and with concurrent expression of both signals are pericytes (PC). The scale bars in the top and lower panels are 100 μm and 50 μm, respectively. (B) Pdgfrb-eGFP positive cells line blood vessels visualized by CD31 staining (in white) in the cerebrum of Pdgfrb-eGFP mice. Some of eGFP expressing cells express also alpha-smooth muscle actin (α-SMA, in red). The white arrows and arrowhead in insert point to the PC in the capillaries and PC/VSMC in the arteriole, respectively. Yellow arrows point to the vein covered by eGFP positive cells. The scale bars in top and lower panels are 50 μm and 10 μm, respectively. (C) Pdgfrb-eGFP positive cells in the cerebral cortex of Pdgfrb-eGFP mouse are expressing PDGFRβ (in red). The scale bar is 50 μm. (D) Pdgfrb-eGFP and NG2-DsRed expression in cerebral capillaries of Pdgfrb-eGFP/NG2-DsRed mice. The Pdgfrb-eGFP-positive cells surrounding capillaries are also positive for NG2-DsRed. The scale bar is 30 μm. (E) Pdgfrb-eGFP expressing cells are positive for PC marker CD13 (in red). E’ shows a higher magnification image. The scale bars in E and E’ are 100 μm and 10 μm, respectively. (F) Expression pattern of Pdgfrb-eGFP and NG2-DsRed in the retina of Pdgfrb-eGFP/NG2-DsRed mice. White arrows indicate Pdgfrb-eGFP-positive cells with weak or undetectable NG2-DsRed expression. The scale bars in left panels and the right enlarged panels are 50 μm and 10 μm, respectively.
Figure 2
Figure 2. FACS of mural cells from brains of Pdgfrb-eGFP/NG2-DsRed mice.
Dot blots showing the intensity of eGFP and DsRed channel of analyzed cells from (A) a negative control (C57BL6) and (B) Pdgfrb-eGFP/NG2-DsRed mice. The sorted, double-positive cell population is indicated by the rectangle in the upper right quadrant.
Figure 3
Figure 3. Venn diagram showing the overlap of identified mural cell genes between different datasets.
The studies where these datasets have been reported are listed in Table 2.
Figure 4
Figure 4. Validation of novel brain pericyte markers by in situ hybridization and immunohistochemistry.
RNA in situ hybridization of 2-month old C57BL6 mouse cerebral cortex. (A) Vitronectin (Vtn, in red) is expressed in PC, identified by Pdgfrb expression (in green). While Pdgfrb expression is restricted to the cell body, Vtn can be found also in PC processes. Arrowhead points to a PC, arrow points to a vascular smooth muscle cell. In magnified inset is seen an overlap of Pdgfrb and Vtn expression in a PC. Cell nuclei are visualized by DAPI (in blue) and EC are visualized by Pecam1 expression (in white). (B) Interferon induced transmembrane protein 1 (Ifitm1, in red) is expressed in PC, identified by Pdgfrb expression (in green). In inserts, cell nuclei are encircled with dotted lines. Arrow points to a PC nucleus, arrowhead points to an EC nucleus. Cell nuclei are visualized by DAPI (in blue) and EC are visualized by Pecam1 expression (in white). (C) Immunohistochemical detection of vitronectin (in red) in PC (in green, CD13). The higher magnification image (C’) shows a strong expression of vitronectin in PC cell body and processes. The scale bars in top and lower panels are 20 μm (A,B), 50 μm (C) and 10 μm (A,B and C’), respectively.
Figure 5
Figure 5. Protein interaction groups among brain mural cell-enriched genes.
Proteins are represented with nodes and interactions between them are indicated with lines. The proteins are grouped according to their subcellular location (i.e cytoplasmic, membrane or extracellular). Proteins discussed in the text are indicated in red.
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