Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells

Abstract

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.

Figure 1.

RGS5 expression in brain pericytes. Whole mount in situ hybridization of flat-mounted wild-type hindbrain (ventricular view of vascular plexus in subventricular zone; ml, midline) at E12.5 (A and B) and in situ hybridization on sections from wild-type embryos at E14.5 (C and D) shows RGS5 expression (arrows) in tight association with microvessels in the brain. Double staining with isolectin B4 (arrowheads), which labels endothelial cells, indicates that RGS5 is expressed by pericytes and not in endothelial cells.

Figure 2.

Comparison of RGS5, PDGFRβ, and NG2 expression in E14.5 mouse embryos. In the brain, RGS5 (A) is expressed in cells in the capillary wall and this mimics the pattern of PDGFRβ (B) and NG2 (C) expression. Similar expression of the three markers was also seen in larger arteries (D–F) and in mesangial cells in the kidney (G–I). In the lung and gut, the expression patterns of RGS5 and PDGFRβ and NG2 are not completely overlapping. In the lung, PDGFRβ- and NG2-positive cells can be found scattered in the mesenchyme at areas corresponding to ongoing angiogenesis (K and L, asterisk), whereas RGS5-positive cells are mainly found around developing airways, in cells that are also positive for NG2 expression (J and L, arrowheads). All three markers are expressed in VSMCs surrounding the developing lung arterial branches (J–L, arrows). In the gut, PDGFRβ- and NG2-positive cells are found in mesenchymal cells immediately subjacent to the epithelium (N and O, arrows), whereas RGS5 is expressed by cells located more peripherally in the gut wall at the expected site of the developing SMC layers (M, arrowhead). NG2 is also expressed by these developing SMC layers (O, arrowheads). a, artery; v, vein; p, podocytes; e, endothelial layer; M, mesangial core.

Figure 3.

RGS5, PDGFRβ, and NG2 expression in PDGF-B−/− embryos. The developing CNS of E14.5 PDGFB−/− embryos exhibits loss of RGS5-positive cells in comparison with PDGF-B+/− embryos (B). This loss correlates with the expression pattern of PDGFRβ and NG2 seen in PDGFB−/− (D and F) mice, confirming the pericyte-specific expression of RGS5 in the brain. A small number of RGS5-positive pericytes appear to persist in PDGF-B−/− embryonic brain (a single blue spot is seen in B), which would be in agreement with previous studies demonstrating some residual pericytes in PDGF-B−/− brain. To confirm that the rare spots of RGS5 staining corresponded to pericytes, double labeling for RGS5 and endothelial cells was performed. These stainings showed that the residual RGS5-positive cells had pericytic location in the capillary wall (H, arrow). Using this method, the reduction in number, but not complete absence, of RGS5-positive VSMC was also observed in developing arteries (compare I and J; a residual RGS5-positive VSMC in the PDGF-B−/− artery is indicated by an arrow).