Identification of mesenchymal-to-epithelial transition during heart regeneration through genetic lineage tracing.

The epicardium is the important outermost mesothelial/epithelial layer of the heart that serves as a signaling center for cardiac development and repair. During heart development, epicardial cells undergo a process known as epithelial-to-mesenchymal transition to form diverse mesenchymal cell lineages, such as fibroblasts, coronary vascular smooth muscle cells, and pericytes. However, it is not clear whether the reverse process, mesenchymal-to-epithelial transition (MET), takes place in the mammalian heart. In this study, we performed apical resection on neonatal hearts and used Fap-CreER;Ai9 labeling to track activated fibroblasts in the injured cardiac regions. We found that these fibroblasts underwent MET to generate epicardial cells during heart regeneration. To our knowledge, this is the first report of MET occurring in vivo during heart development and regeneration. Our findings suggest that it is feasible to directly convert fibroblasts into epicardial cells, providing a novel approach to generate epicardial cells.

Functions and origins of cardiac fat.

Triglyceride droplets can be stored within cardiac adipocytes (CAs) and cardiomyocytes in the heart. Cardiac adipocytes reside in three distinct regions: pericardial, epicardial, and intramyocardial adipose tissues. In healthy individuals, cardiac adipose tissues modulate cardiovascular functions and energy partitioning, which are, thus, protective. However, ectopic deposition of cardiac adipose tissues turns them into adverse lipotoxic, prothrombotic, and pro-inflammatory tissues with local and systemic contribution to the development of cardiovascular disorders. Accumulation of triglyceride droplets in cardiomyocytes may lead to lipotoxic injury of cardiomyocytes and contribute to the development of cardiac hypertrophy and dysfunction. Here, we summarize the roles of CAs and myocardial triglyceride droplets under physiological and pathological conditions and review the cellular sources of CAs in heart development and diseases. Understanding the functions and cellular origins of cardiac fat will provide clues for future studies on pathophysiological processes and treatment of cardiovascular diseases.

Generation and characterization of a Myh6-driven Cre knockin mouse line.

Gene deletion by the Cre-Loxp system has facilitated functional studies of many critical genes in mice, offering important insights and allowing deeper understanding on the mechanisms underlying organ development and diseases, such as heart development and diseases. In this study, we generated a Myh6-Cre knockin mouse model by inserting the IRES-Cre-wpre-polyA cassette between the translational stop codon and the 3' untranslated region of the endogenous Myh6 gene. By crossing knockin mice with the Rosa26 reporter lines, we found that Myh6-Cre targeted cardiomyocytes at the embryonic and postnatal stages. In addition, we were able to inactivate the desmosome gene Desmoplakin (Dsp) by breeding Myh6-Cre mice with a conditional Dspflox knockout mouse line, which resulted in embryonic lethality during the mid-term pregnancy. These results suggest that the new Myh6-Cre mouse line can serve as a robust tool to dissect the distinct roles of genes involved in heart development and function.

PDGFRb+ mesenchymal cells, but not NG2+ mural cells, contribute to cardiac fat.

Understanding cellular origins of cardiac adipocytes (CAs) can offer important implications for the treatment of fat-associated cardiovascular diseases. Here, we perform lineage tracing studies by using various genetic models and find that cardiac mesenchymal cells (MCs) contribute to CAs in postnatal development and adult homeostasis. Although PDGFRa+ and PDGFRb+ MCs both give rise to intramyocardial adipocytes, PDGFRb+ MCs are demonstrated to be the major source of intramyocardial adipocytes. Moreover, we find that PDGFRb+ cells are heterogenous, as PDGFRb is expressed not only in pericytes and smooth muscle cells (SMCs) but also in some subendocardial, pericapillary, or adventitial PDGFRa+ fibroblasts. Dual-recombinase-mediated intersectional genetic lineage tracing reveals that PDGFRa+PDGFRb+ double-positive periendothelial fibroblasts contribute to intramyocardial adipocytes. In contrast, SMCs and NG2+ pericytes do not contribute to CAs. These in vivo findings demonstrate that PDGFRb+ MCs, but not NG2+ coronary vascular mural cells, are the major source of intramyocardial adipocytes.

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

RATIONALE: Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration. OBJECTIVE: To examine the contribution of EMPs to intraembryonic endothelial cells. METHODS AND RESULTS: We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc)+ circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development. CONCLUSIONS: Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.

Dual lineage tracing identifies intermediate mesenchymal stage for endocardial contribution to fibroblasts, coronary mural cells, and adipocytes.

Early embryonic endocardium undergoes endothelial-to-mesenchymal transition to form cardiac cushion mesenchymal cells (MCs). Embryonic endocardium also gives rise to fibroblasts, intramyocardial adipocytes, and coronary mural cells, including smooth muscle cells and pericytes, in development. Whether endocardial cells directly differentiate into fibroblasts, coronary mural cells, and adipocytes or indirectly via an intermediate stage of endocardial-derived cushion MCs remains unknown. In addition to endocardium, epicardium and neural crest also contribute to cardiac cushion MCs. Given the developmental heterogeneity of cushion MCs and the lack of specific markers for endocardial-derived cushion MCs, conventional genetic lineage tracing utilizing Cre recombinase driven by one specific regulatory element is not sufficient to examine the fates of endocardial-derived cushion MCs. Intersectional genetic targeting approaches, which combine regulatory elements from two or more genes, have been employed to increase the specificity of cell targeting. Here, we developed a dual-recombinase intersectional targeting approach using Nfatc1-Dre, Sox9-CreER, and Cre/Dre double-dependent reporter Ai66 to specifically label endocardial-derived cushion MCs. Taking advantage of intersectional lineage tracing, we found that a subset of cardiac cells including fibroblasts, coronary mural cells, and intramyocardial adipocytes in adult hearts were derived from endocardial-derived cushion MCs. Our study suggests that embryonic endocardium contributes to cushion MCs first, and then endocardial-derived cushion MCs migrate into myocardium and differentiate into fibroblasts, coronary mural cells, and adipocytes in development. Understanding developmental origins of cardiac cell lineages will provide us more insights into cardiac development, regeneration, and diseases.