Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations

Abstract

The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)beta in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFRbeta(-/-) and PDGFRbeta epicardial mutant hearts. We found that PDGFRbeta(-/-) hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs). This constellation of defects was consistent with a primary defect in the epicardium. To verify that these defects were specific to epicardial derivatives, we generated mice with an epicardial deletion of PDGFRbeta that resulted in reduced cVSMCs distal to the aorta. The regional absence of cVSMCs suggested that cVSMCs could arise from 2 sources, epicardial and nonepicardial, and that both were dependent on PDGFRbeta. In the absence of PDGFRbeta signaling, epicardial cells adopted an irregular actin cytoskeleton, leading to aberrant migration of epicardial cells into the myocardium in vivo. In addition, PDGF receptor stimulation promoted epicardial cell migration, and PDGFRbeta-driven phosphoinositide 3'-kinase signaling was critical for this process. Our data demonstrate that PDGFRbeta is required for the formation of 2 distinct cVSMC populations and that loss of PDGFRbeta-PI3K signaling disrupts epicardial cell migration.

Figure 1. PDGFRβ expression is required for proper coronary artery development

Whole mount (A) E14.5 and (B) E17.5 hearts stained for PECAM (endothelial cells) by immunohistochemistry. Asterisks indicate sinusoidal PECAM staining.

Figure 2. Loss of cVSMC in the absence of PDGFRβ

Whole mount (A) E14.5 and (B) E17.5 hearts stained for β-galactosidase activity (cVSMC) of the XlacZ4 transgene. Arrows indicate scattered β-galactosidase⁺ cells.

Figure 3. Decreased presence of epicardial derivatives in PDGFRβ^-/- hearts

(A, B) Cells of the epicardium were identified using capsulin^lacZ+ driven-expression of β-galactosidase (blue). (C-H) Sections stained for WT1 of E14.5 (C,D) and E15.5 (E-H) hearts. Figures in C'-F' are higher magnification views of boxed areas in C-F.

Figure 4. PDGFRβ^-/- hearts express the mesenchymal marker, vimentin

(A-F) Vimentin expression in the indicated genotypes. e; epicardium, and edm; epicardial-derived mesenchyme.

Figure 5. PDGFRβ stimulation induces epicardial cell migration

(A) Quantification of WT1⁺ cells in right and left lateral ventricles of E15.5 hearts from the indicated genotypes. Numbers in parentheses indicate the number of independent hearts quantified. All mutant alleles exhibited a reduction in WT1⁺ cells within the myocardium compared to those present in wild type myocardium. (One- way ANOVA, *p<0.001). See text for allele description. (B-E) Migration of epicardial cells by CCFSE dye tracing. Inset in B shows a time zero E13.5 heart labeled with CCFSE. Arrowheads mark the epicardial boundary. F. Quantification of adeno-viral GFP-labeled epicardial cells that have migrated into the myocardium. Numbers in parentheses indicate the number of hearts assayed. LY=LY294002. All samples were compared to wild type cell migration. (Two-way ANOVA, *p<0.001)

Figure 6. Aberrant actin organization and epicardial cell migration in PDGFRβ signaling mutant hearts

(A-D) Actin localization in control and PDGFRβ mutant hearts using fluorescent-phalloidin. (A,B) Confocal imaging of whole mount actin localization of E14.5 hearts. Arrowheads indicate cells with cortical actin distribution. (C, D) Confocal imaging of E14.5 sectioned hearts. Figures indicated by the prime have DAPI staining to permit identification of epicardial cells on the exterior of the heart. Asterisks indicate cells that have cortical actin localization. (E) Quantification of cells possessing cortical actin at the specified ages in the indicated genotype compared to controls at the same age. (Two-way ANOVA *p<0.001) (F, G) Thick sections of E14.5 hearts processed for TEM and imaged on a light microscope. (H,I) Representative TEM images of thin sections.

Figure 7. Disruption of PDGF signaling results in failure to activate and localize cortactin

(A) Western blot analysis of primary epicardial cell lysates from the indicated genotypes and treatments were probed for phosphorylated (top panel) and total protein (bottom panel). (B) Cortactin localization on wounded epicardial cells. Cells were stimulated with 50ng/ml PDGFDD. Arrowhead indicates abundant cortactin localization to lamellipodia.