Genetic Lineage Traces the Differentiation Fate of Epicardial Cells during Heart Development

Research Article

Austin J Clin Cardiolog. 2023; 9(2): 1107.

Genetic Lineage Traces the Differentiation Fate of Epicardial Cells during Heart Development

FR Lu*; XJ Yang

College of Life Science and Technolog, Jinan University, Guangzhou, 510632, Guangdong, China

*Corresponding author: FR Lu College of Life Science and Technolog, Jinan University, Guangzhou, 510632, Guangdong, China. Tel: +8618337319163 Email: [email protected]

Received: November 06, 2023 Accepted: December 11, 2023 Published: December 18, 2023


Objective: The genetic lineage tracing method was used to examine the Epithelial-Mesenchymal Transition (EMT) process and the contribution of epicardial cells to mesenchymal cells at various stages of fetal heart development.

Methods: In Wt1-CreER; R26-tdTomato transgenic mice, tamoxifen was utilized to promote the tagging of epicardial cells with tdTomato fluorescence at E10. At E11.5, E12.5, and E16.5, embryonic hearts were harvested and photographed using confocal fluorescence microscopy and stereomicroscopy.

Results: According to the findings, the tdTomato+ cells at E11.5 were still in the epicardium and had not yet moved into the myocardium. Epicardial cells began to separate from the epicardium and give rise to epicardial-derived cells at embryonic day 12.5 (E12.5). On the valve primordium, fibroblasts generated from epicardium have been found. By E16.5, many epicardial cells had moved into the myocardium and formed fibroblasts, mesenchymal cells, vascular smooth muscle cells, as well as migrated into the ventricular septum and valves, contributing to their growth and creation.

Conclusions: The contribution of epicardial cells to mesenchymal cells during development is shown by genetic lineage tracing, opening up possibilities and offering references for creating relevant treatment approaches based on epicardial cells.

Keywords: Lineage tracing; Epithelial-to-mesenchymal transition; Valve; Heart


Heart diseases, including heart failure, myocardial infarction, and coronary artery disease, have become significant global health issues [1-2]. Following heart damage, activated epicardium-derived cells (EPDCs) can invade the injury site, promoting the proliferation of coronary artery endothelial cells, regenerating myocardial cells, and forming fibroblasts to maintain normal heart function [3]. Embryonic epicardial cells possess greater differentiation potential compared to adult epicardial cells, and transplantation of epicardial cells derived from embryonic stem cells has emerged as a promising therapy for various heart diseases. Therefore, the use of lineage tracing systems to track the fate of embryonic epicardial cells holds significant potential for the treatment of heart disease [4].

The Cre-loxP system is a widely used, powerful technology for mammalian gene editing. Concerning the mechanism of the Cre-loxP system, a single Cre recombinase recognizes two repeated loxP site, then the Cre excises the loxP-flanked DNA [5]. To achieve more accurate genetic functional studies and clinical applications using the Cre-loxP system,a more sophisticated technique was required that controled the Cre activation at a precise time and in an specific cell. An inducible Cre system is controlled by cell-specific regulatory elements (promoters and enhancers) and inducible way by exogenous inducers such as tam [6].

EMT refers to epithelial cells losing their epithelial features and acquiring mesenchymal characteristics with migratory potential. EMT has been shown to be essential for both developmental and pathological processes, including embryo morphogenesis, wound healing, tissue fibrosis, and cancer. Heart formation involves a series of epithelial-mesenchymal transition and reverse EMT processes, with almost all cardiac and non-cardiac cells generated through EMT. During heart development, EPDCS can migrate into the myocardium and differentiate into various cell types, including smooth muscle cells that comprise the coronary artery, acute fibroblasts, and so on [7]. EPDCs also contribute to the development of the atrioventricular valves and ventricular septum, with fibroblasts derived from the epicardium preferentially participating in the formation and development of the wall leaflets of the atrioventricular valve [8-9].

The epicardium is typically distinguished by the expression of transcription factors T-box18 (Tbx18),Wilm tumor gene1(Wt1), and Transcription factor 21(Tcf21). While Tbx18 is known to be involved in heart development, it is not essential for the formation of cardiac chambers and coronary vessels and may also be expressed in the myocardium, potentially confounding experimental results [10]. Tcf21, on the other hand, plays an important role in cell differentiation and growth processes and is expressed in cardiac mesenchymal cells, making it a commonly used marker in zebrafish studies [11]. In this study, we focused on characterizing the fate of epicardial-derived cells during the epithelial-to-mesenchymal transition in embryonic heart development, using Wt1 to drive the Cre-LoxP system. Wt1 promotes epicardial EMT through Wnt and retinoic acid signaling pathways, and its inactivation can lead to the abnormal formation of EPDCs and affect normal heart development. Our findings provide valuable insights into the role of epicardial cells in the development of valvular and ventricular septa.

Materials and Methods

Animal Studies and Ethics Statement

All Animals were used in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The study was approved by the Institutional Animal Care and Use Committee of Jinan University (approval number: 20220613-16). The Wt1-CreER;R26-tdTomato knock-in mouse line was generated by homologous recombination using CRISPR/Cas9 methods. Wt1-CreER; Rosa26-tdTomato mice lines were maintained on a C129/C57BL6/J-mixed background.

We administered 5mg tam (Sigma) by gavage to pregnant mice at E10.5 to induce Cre.

Tissue Collection and Immunostaining

Mouse embryonic hearts were collected in PBS and then washed gently to remove blood. They were then fixed in 4% PFA for 15-60 min at 4°C depending on tissue size. After washing several times in PBS, we used fluorescence microscopy (Leica M205 FA/DFC 7000T) to take photographs of embryonic heart with indicated fluorescence reporters.After that, the embryonic hearts were dehydrated in 30% sucrose/PBS for several hours or overnight at 4°C, and then embedded in OCT (Sakura) the next day. Frozen sections of 6-10 μm were collected on slides. For immunostaining, tissue sections were first washed in PBS to remove OCT, then blocked in DAPI for 30 min at room temperature and then incubated with primary antibodies overnight at 4°C. Primary antibodies against the following proteins and dilution were used:Wt1 (Abcam,ab89901;1:200), a-smooth muscle actin (aSMA) (Abcam, ab5694; 1:500), platelet-derived growth factor receptor β (PDGFRβ) (eBioscience, 14-1402-82; 1:2000), platelet-derived growth factor receptor a (PDGFRa) (R&D, AF062; 1:1000), RFP Antibody pre-adsorbed(Rockland, 600-401-379; 1:500) and ImmPRESS Horse Anti-Rabbit IgG (Vector lab, MP-7401; 1:1). The next day, sections were washed with PBS several times and then incubated with Alexa fluorescence secondary antibodiesat room temperature for 30 min in dark.After washing several times in PBS, all slides were mounted with mounting medium. For weak signals, HRP-conjugated secondary antibodies were used to amplify the signals. Images were acquired using an Olympus confocal microscopy system (FV3000).

Genomic PCR

Genomic DNA was extracted from mouse tail,Tissues were lysed in lysis buffer (100 μg/ml proteinase K) overnight at 55°C and the mixture was centrifuged at the maximum speed of 20,000 g for 8 min to obtain supernatant with genomic DNA the next day. DNA was precipitated using isopropanol, and then washed in 70% ethanol by centrifugation at 20,000g for 3 min.

All the mice or embryos were genotyped using genomic PCR to distinguish knock-in allele from wild-type allele.For the R26-tdToamto line, primers 5'-TCCCGACAAAACCGAAAATCTGTGG-3' and 5'-TGCATCGCATTGTCTGAGTAGG-3' were used to detect the R26-tdToamt positive allele, and 5'-TCCCGACAAAACCGAAAATCTGTGG-3',5'-GGGGCGTGCTGAGCCAGACCTCCAT-3' were

used to detect the wild-type allele.For the Wt1-Cre line, primers 5'-GGCTTAAAGGCTAACCTGGTGTG-3' and 5'-GGAGCGGGAGAAATGGATATG-3' were used to

detect the Wt1 positive allele, and 5'-CCAAGTCCAGCGCCGAGAAT-3' and 5'-TGTCCATCAGGTTCTTGCGA-3' were used to detect the wild-type allele.

Statistical Analysis

All data were obtained from three to more independent experiments, as indicated in each figure legend and were presented as mean±Standard Deviation .Two-tailed Student’ s t tests were used to calculate statistical significance with p values. P value<0.05 was considered statistically significant, ns is non-statistically significant. Graphs were generated using GraphPad Prism 9.0.0 software.


The Cre-LoxP System Functions After Tamoxifen

The Cre-LoxP system is commonly used for tracing mammalian cell lineages and has been widely employed in lineage tracing studies of various organs and cell types [12-13]. Notably, Cre recombinase is dependent on tamoxifen induction and only translocates into the nucleus to mediate recombination under tamoxifen-induced conditions (Figure 1A,1B).