The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors

Abstract

The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

Fig. 1.

Epicardial development in Tcf21 null hearts. (A,B) Whole-mount images of E18.5 wild-type (A) and Tcf21 null (B) hearts. Arrows and asterisks indicate the absence of a distinct apex and an enlarged right atrium, respectively, in the null hearts. (C-F) Epicardial-specific gene expression at E12.5. Arrowheads indicate regions of epicardial detachment in null hearts and arrows point to atrioventricular cushion region.

Fig. 2.

Defective epicardial EMT in Tcf21 null hearts. (A-H) β-Galactosidase staining in heart sections of the indicated stages and genotypes. A-F show high magnification images of the left ventricle (LV). Arrowheads in H point to null cells in the epicardium. RV, right ventricle. (I) Quantification of lacZ⁺ cells in the myocardial wall. A 20× field of view, spanning the entire LV (three sections per heart) was quantified for total number of lacZ⁺ cells. n values are indicated in parentheses. (J-O) Wild-type primary epicardial cultures were transduced with either Adβgal (J-L) or AdTcf21 (M-O) and stained for adherens junctions using β-catenin (red), and actin cytoskeleton using phalloidin (green). Nuclei were stained with DAPI (blue). (P) EMT-related gene expression changes in E12.5 primary epicardial cultures. Fold change in gene expression with respect to wild type (normalized to 1) is shown. (Q) Gene expression changes upon overexpression of Tcf21 (AdTcf21) in primary epicardial cultures. AdGFP served as control (normalized to 1). Data are mean ± s.d.

Fig. 3.

Tcf21 null hearts form cVSMCs. (A-D) Immunohistochemistry for β-galactosidase (green) in E17.5 Tcf21^lacZ/+ hearts co-stained (in red) for PECAM (A) or Pdgfrβ (C). Nuclear counterstain with DAPI (blue) for images A and C is shown in B and D, respectively. Arrowheads point to vessels. (E,F) Development of coronary vasculature detected using whole-mount β-galactosidase staining of wild-type (E) and null (F) E18.5 hearts carrying the XlacZ4 transgene. (G-J) Immunohistochemistry for SMC marker expression in wild-type (G,I) and Tcf21^–/– (H,J) hearts. E18.5 hearts were co-stained for SMC (Pdgfrβ or SM22α in green) and endothelial (isolectin B4 or PECAM in red) markers to detect vessels. Arrowheads point to vessels.

Fig. 4.

Tcf21 null hearts lack epicardial-derived cardiac fibroblasts. (A-B′) Periostin immunohistochemistry on wild-type (A) and Tcf21^–/– (B) hearts. Inset images (A′,B′) show staining of the atrioventricular valve region. Arrowheads indicate valves. (C-F) Expression of Col1GFP transgene (green) in wild-type (C,E) and null (D,F) hearts. Hearts were co-stained for Pdgfrβ (red). E and F represent higher magnification LV images of C and D, respectively. Arrows and arrowheads indicate epicardium and valves, respectively. (G-L) In situ hybridization of wild-type (G,I,K) and Tcf21 null (H,J,L) hearts. Expression of cardiac fibroblast specific genes Col1a1 (G,H), Col3a1 (I,J) and Pdgfra (K,L) is shown. Corresponding higher magnification images of the LV are shown to the right. (M) Percentage of epicardial-derived cardiac fibroblasts (Thy1⁺:YFP⁺/YFP⁺) from wild-type, Tcf21^+/– and Tcf21^–/– hearts. CD31⁺ endothelial cells were excluded from the analysis. n values are indicated in parentheses. (N) Quantitative RT-PCR showing expression of cardiac fibroblast (CF), vascular smooth muscle cell (SMC), and cardiomyocyte (CM) differentiation markers in Tcf21^–/– hearts relative to wild-type littermate controls. Fold change with respect to control (normalized to 1) is shown. Data represented are mean ± s.d. (O) Immunoblots comparing cardiac fibroblast (periostin) and VSMC (SM22α) protein expression in control versus Tcf21^–/– hearts. Protein amounts were normalized using β-tubulin. All experiments were performed using E18.5 hearts.

Fig. 5.

Lineage tracing with Tcf21^iCre labels cardiac fibroblasts. (A-J) Lineage tracing using R26R^YFP (green; A,B,F,G) and R26R^tdT (red; C-E,H-J) in E18.5 Tcf21^iCre embryonic hearts induced at E14.5 (A-E) or E10.5 (F-J). PECAM labels endothelial cells, Pdgfrβ and SM22α label smooth muscle cells, and Pdgfrα^GFP and Col1GFP label cardiac fibroblasts. Arrowheads in D, E, I and J show examples of cells that are positive for both GFP and R26R^tdT reporter activity. Asterisks indicate the lumen of vessels. (K) Quantitative gene expression profiling for cardiac fibroblast markers following FAC sorting of Tcf21^iCre/+:R26R^tdT lineage traced, Pdgfra^GFP/+ and Col1GFP transgenic hearts. Flow sorted tomato⁺ cells from Tcf21^iCre/+:R26R^tdT lineage-traced hearts derived from embryos induced at E14.5 are compared by qRT-PCR with sorted GFP⁺ cardiac fibroblast population from Pdgfrα^GFP/+ and Col1GFP transgenic hearts. Fold change in gene expression relative to unsorted population (normalized to 1) is shown. (L) Quantitative gene expression profiling of sorted cell populations shown in K for vascular smooth muscle cell (VSMC), cardiomyocyte (CM) and endothelial cell (EC) marker genes. Fold change in gene expression compared with the unsorted population (normalized to 1) is shown.

Fig. 6.

Tcf21^iCre lineage-traced epicardial cells do not give rise to cardiomyocytes. (A-L) Representative images of lineage-traced E18.5 Tcf21^iCre;R26T^tdT embryonic (A-H) or adult (I-L) hearts induced at E14.5 (A-D), at E10.5 (E-H) or in the adult (I-L). Co-stain is cardiomyocyte marker, Acta1 (in green).

Fig. 7.

Lineage tracing with Tcf21^iCre in null hearts. (A-H′) Lineage tracing in E18.5 control (Tcf21^iCre/+) and null (Tcf21^iCre/lacZ) embryonic hearts following Cre induction at indicated ages. Panels A′-H′ represent lineage-traced cells showing R26R^YFP activity (in green), and co-staining with the VSMC marker Pdgfrβ (red) is shown in panels A-H. (I) Quantification of percentage of migrated lineage-traced cells that are Pdgfrβ⁺ in A-H. Percentage of overlap between YFP⁺ (green) and Pdgfrβ⁺ (red) area was determined relative to the total Pdgfrβ⁺ area using Image J software. The epicardium was excluded from this analysis. An average of four sections per heart were utilized for the quantification and n values are indicated on the bars. Data shown is mean ± s.d. and P values represent statistical significance of differences between E14.5 and each of the earlier time points indicated for the same genotype. Differences noted between control and null hearts of the same time point were not statistically significant.

Fig. 8.

Proposed function of Tcf21 in epicardial progenitor cell fate determination. Prior to epicardial EMT (E10.5), Tcf21-expressing progenitors (in green) can develop into either cardiac fibroblasts (CFs) or SMCs. As Tcf21 is essential for EMT of the cardiac fibroblast progenitors, null hearts lack cardiac fibroblasts, but Tcf21^iCre reporter-labeled cVSMCs are detected. Around E13.5, Tcf21 expression is downregulated in smooth muscle cell progenitors (SMCPs) but is maintained in cardiac fibroblast progenitors (CFPs). Labeled cells in wild-type hearts form predominantly cardiac fibroblasts, but in Tcf21 null hearts the cardiac fibroblast progenitors remain in the epicardium. SMC, smooth muscle cell.