Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling

Abstract

Rationale: In early heart development, platelet-derived growth factor (PDGF) receptor expression in the heart ventricles is restricted to the epicardium. Previously, we showed that PDGFRβ is required for coronary vascular smooth muscle cell (cVSMC) development, but a role for PDGFRα has not been identified. Therefore, we investigated the combined and independent roles of these receptors in epicardial development.

Objective: To understand the contribution of PDGF receptors in epicardial development and epicardial-derived cell fate determination.

Methods and results: By generating mice with epicardial-specific deletion of the PDGF receptors, we found that epicardial epithelial-to-mesenchymal transition (EMT) was defective. Sox9, an SRY-related transcription factor, was reduced in PDGF receptor-deficient epicardial cells, and overexpression of Sox9 restored epicardial migration, actin reorganization, and EMT gene expression profiles. The failure of epicardial EMT resulted in hearts that lacked epicardial-derived cardiac fibroblasts and cVSMC. Loss of PDGFRα resulted in a specific disruption of cardiac fibroblast development, whereas cVSMC development was unperturbed.

Conclusions: Signaling through both PDGF receptors is necessary for epicardial EMT and formation of epicardial-mesenchymal derivatives. PDGF receptors also have independent functions in the development of specific epicardial-derived cell fates.

Figure 1. PDGFR^EKO epicardial cells fail to migrate into the myocardium

(A) Whole mount images showing regions of epicardial detachment and hemorrhaging (Arrows). (B) R26R^YFP IHC was used to examine epicardial cell migration into subepicardial mesenchyme (brackets) from indicated genotypes induced with tamoxifen at E12.5. Arrowheads point to migrated cells within the subepicardial mesenchyme. (C) Quantification of the R26R^YFP fluorescent area in (B). N values are indicated in parentheses. (*) p<0.005 (D) Quantification of GFP⁺ cells within myocardium of E12.5 hearts transduced with an adenovirus expressing GFP and stimulated with hTGFβ₁ or bFGF (n=3 for each genotype/condition). Data are represented as mean ± SD. (*) p<0.001 (compared to vehicle treated control) (LV – left ventricle, RV – right ventricle, EKO – epicardial knockout)

Figure 2. PDGFR^EKO epicardial cells fail to undergo EMT

(A) qPCR analysis of gene expression in PDGFR^EKO whole hearts (atria and conotruncal regions removed) for transcriptional EMT markers (Snail, Slug, Sox9) and epicardial markers (Tbx18, WT1, PDGFRα, PDGFRβ). Data were compared to control littermates and are representative of three independent experiments. (*) p<0.001 (B) βcatenin and phalloidin localization in primary epicardial cultures after 48h of stimulation with hTGFβ₁ and PDGFBB. (C–E) qPCR on primary cultures for expression of epithelial, transcriptional, and mesenchymal markers. Data were compared to vehicle treated control epicardial cultures (represented by a baseline of 1.0). Data are represented as mean ± SD. N values are indicated in parentheses.

Figure 3. Sox9 rescues PDGF receptor mutant phenotypes

(A) qPCR for Sox9 expression in primary epicardial cultures stimulated with PDGFBB for 24h. (*) p<0.001 (B) βcatenin and phalloidin localization of control and PDGFR^EKO cultures stimulated with hTGFβ₁ and PDGFBB in the presence of adenoviral LacZ or Sox9. (C–E) qPCR analysis of epithelial, mesenchymal, and EMT transcription factors of primary epicardial cultures transduced with adenoviral Sox9. Data were compared to vehicle treated, GFP adenoviral transduced control epicardial cultures (Online Figure III, D–F) (represented as a baseline of 1.0). Data are represented as mean ± SD. N values are indicated in parentheses. (*) p<0.001 (n.s. – no significant difference)

Figure 4. Epicardial expression of Sox9

(A) Sox9 expression in hearts of indicated genotypes. Images to the right of each frame are higher magnification of left ventricle. Arrows point to examples of Sox9⁺ cells. Arrowheads indicate Sox9⁺ cells in the valves. (B) Quantification of Sox9⁺ cells within the myocardial ventricular wall at E14.5. Valves, epicardium, and septum were excluded from analysis. (C) Quantification of GFP⁺ cells within myocardium of E12.5 hearts transduced with indicated viruses and/or stimulation with hTGFβ₁. Data are represented as mean ± SD. N values are indicated in parentheses. (*) p<0.001 (compared to vehicle treated AdGFP/AdLacZ control)

Figure 5. PDGFRα epicardial phenotype

(A–B) GFP fluorescence was used to follow PDGFRα expressing cells in the indicated genotypes. (C) Confocal images of R26R^tdT and PDGFRα^GFP fluorescence of the indicated genotypes induced with tamoxifen at E12.5. (D) Quantification of R26R^tdT fluorescent area in (C). (*) p<0.001 (E) IHC for coronary endothelial cells (Isolectin B₄) and cVSMC (SMMHC and SM22α). (epi – epicardium, myo – myocardium Arrowheads denote coronary vessels).

Figure 6. PDGFRα is required for epicardial derived cardiac fibroblast formation

(A) Percentage of epicardially-derived cardiac fibroblasts (Gata5cre^Tg; R26R^YFP) from P21–P28 PDGF receptor mutant hearts. There was a subpopulation of Thy1⁺ CD31^+, cells that were excluded from our analysis. (B) β-galactosidase activity (blue) in passage 1 primary cardiac fibroblast cultures using P21–P28 Gata5cre^Tg; R26R^LacZ hearts to follow EPDCs. Nuclear fast red was used as a counterstain. (C) Quantification of blue cells in (B). Data are represented as mean ± SD. N values are indicated in parentheses. (*) p<0.001 (compared to control)

Figure 7. PDGFRα is required for cardiac fibroblast development

(A) Col1a1 in situ hybridization of the indicated genotypes and ages. Insets represent higher magnifications of left ventricle. (B) Quantification of interstitial col1a1 expression in (A) as described in online methods. (*) p<0.005 (C) Prolyl-4-hydroxylase β IHC and (D) quantification in the left ventricle of the indicated genotypes. (*) p<0.001. (E) qPCR RNA expression levels in the left ventricle of the indicated genotypes. (n=2 for each genotype) (*) p<0.005 (F) IHC for a fibroblast marker, periostin in E18.5 hearts (upper panel). (pm - papillary muscle) Arrowheads indicate staining present in the ventricular wall. (Lower panel) Western blot of total periostin expression from individual P1 hearts (without aorta, atria, or valves) of indicated genotypes. The positive control is whole cell lysate from differentiated prechondrocyte MC3T3-E1 cells. (G) Masson Trichrome staining (MTC) of coronary arteries in 8–10 week old adult hearts (upper panel). Perivascular collagen deposition is stained blue (arrows). (Lower panel) Quantification of perivascular collagen as seen by MTC. N values are indicated in parentheses. (*) p<0.001 (compared to control)