Epicardium-derived cells (EPDCs) invade the myocardium and differentiate into fibroblasts and vascular smooth muscle (SM) cells, which support the coronary vessels. The transcription factor Pod1 (Tcf21) is expressed in subpopulations of the epicardium and EPDCs in chicken and mouse embryonic hearts, and the transcription factors WT1, NFATC1, and Tbx18 are expressed in overlapping and distinct subsets of Pod1-expressing cells. Expression of Pod1 and WT1, but not Tbx18 or NFATC1, is activated with all-trans-retinoic acid (RA) treatment of isolated chick EPDCs in culture. In intact chicken hearts, RA inhibition leads to decreased Pod1 expression while RA treatment inhibits SM differentiation. The requirements for Pod1 in differentiation of EPDCs in the developing heart were examined in mice lacking Pod1. Loss of Pod1 in mice leads to epicardial blistering, increased SM differentiation on the surface of the heart, and a paucity of interstitial fibroblasts, with neonatal lethality. Epicardial epithelial-to-mesenchymal transition (EMT) and endothelial differentiation of coronary vessels are relatively unaffected. On the surface of the myocardium, expression of multiple SM markers is increased in Pod1-deficient EPDCs, demonstrating premature SM differentiation. Increased SM differentiation also is observed in Pod1-deficient lung mesenchyme. Together, these data demonstrate a critical role for Pod1 in controlling mesenchymal progenitor cell differentiation into SM and fibroblast lineages during cardiac development.

During embryonic heart development, the transcription factors Tcf21, Wt1, and Tbx18 regulate activation and differentiation of epicardium-derived cells, including fibroblast lineages. Expression of these epicardial progenitor factors and localization of cardiac fibrosis was examined in mouse models of cardiovascular disease and in human diseased hearts. Following ischemic injury in mice, epicardial fibrosis is apparent in the thickened layer of subepicardial cells that express Wt1, Tbx18, and Tcf21. Perivascular fibrosis with predominant expression of Tcf21, but not Wt1 or Tbx18, occurs in mouse models of pressure overload or hypertensive heart disease, but not following ischemic injury. Areas of interstitial fibrosis in ischemic and hypertensive hearts actively express Tcf21, Wt1, and Tbx18. In all areas of fibrosis, cells that express epicardial progenitor factors are distinct from CD45-positive immune cells. In human diseased hearts, differential expression of TCF21, WT1, and TBX18 also is detected with epicardial, perivascular, and interstitial fibrosis, indicating conservation of reactivated developmental mechanisms in cardiac fibrosis in mice and humans. Together, these data provide evidence for distinct fibrogenic mechanisms that include Tcf21, separate from Wt1 and Tbx18, in different fibroblast populations in response to specific types of cardiac injury.

SUMMARYEpicardium-derived cells (EPDCs) contribute to formation of coronary vessels and fibrous matrix of the mature heart. Nuclear factor of activated T-cells cytoplasmic 1 (NFATC1) is expressed in cells of the proepicardium (PE), epicardium and EPDCs in mouse and chick embryos. Conditional loss of NFATC1 expression in EPDCs in mice causes embryonic death by E18.5 with reduced coronary vessel and fibrous matrix penetration into myocardium. In osteoclasts, calcineurin-mediated activation of NFATC1 by receptor activator of NFkB ligand (RANKL) signaling induces cathepsin K (CTSK) expression for extracellular matrix degradation and cell invasion. RANKL/NFATC1 pathway components also are expressed in EPDCs, and loss of NFATC1 in EPDCs causes loss of CTSK expression in the myocardial interstitium in vivo. Likewise, RANKL treatment induces Ctsk expression in PE-derived cell cultures via a calcineurin-dependent mechanism. In chicken embryo hearts, RANKL treatment increases the distance of EPDC invasion into myocardium, and this response is calcineurin dependent. Together, these data demonstrate a crucial role for the RANKL/NFATC1 signaling pathway in promoting invasion of EPDCs into the myocardium by induction of extracellular matrixdegrading enzyme gene expression.

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4(-/-) embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4(-/-) lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.

Proper lumen morphogenesis during pancreas development is critical to endocrine and exocrine cell fate. Recent studies showed that a central network of lumens (termed core), but not the surrounding terminal branches (termed periphery), produces most islet endocrine cells. To date, it remains unclear how pancreatic lumens form and remodel and which aspects of lumen morphogenesis influence cell fate. Importantly, models testing the function of the central lumen network as an endocrine niche are lacking. Here, we identify mechanisms underlying lumen formation and remodeling and show that central lumen network morphogenesis impacts pancreatic endocrine mass. We show that loss of the scaffolding protein Afadin disrupts de novo lumenogenesis and lumen continuity in the tip epithelium. Codepletion of the actomyosin regulator RhoA and Afadin results in defects in the central lumens and arrests lumen remodeling. This arrest leads to prolonged perdurance of the central lumen network over developmental time and expansion of the endocrine progenitor population and, eventually, endocrine mass. Our study uncovers essential roles of Afadin and RhoA in pancreatic central lumen morphogenesis, which subsequently determines endocrine cell mass.

Epicardial derivatives, including vascular smooth muscle cells and cardiac fibroblasts, are crucial for proper development of the coronary vasculature and cardiac fibrous matrix, both of which support myocardial integrity and function in the normal heart. Epicardial formation, epithelial-to-mesenchymal transition (EMT), and epicardium-derived cell (EPDC) differentiation are precisely regulated by complex interactions among signaling molecules and transcription factors. Here we review the roles of critical transcription factors that are required for specific aspects of epicardial development, EMT, and EPDC lineage specification in development and disease. Epicardial cells and subepicardial EPDCs express transcription factors including Wt1, Tcf21, Tbx18, and Nfatc1. As EPDCs invade the myocardium, epicardial progenitor transcription factors such as Wt1 are downregulated. EPDC differentiation into SMC and fibroblast lineages is precisely regulated by a complex network of transcription factors, including Tcf21 and Tbx18. These and other transcription factors also regulate epicardial EMT, EPDC invasion, and lineage maturation. In addition, there is increasing evidence that epicardial transcription factors are reactivated with adult cardiac ischemic injury. Determining the function of reactivated epicardial cells in myocardial infarction and fibrosis may improve our understanding of the pathogenesis of heart disease.

The majority of cardiac fibroblasts in a mature mammalian heart are derived from the epicardium during prenatal development and reactivate developmental programs during the progression of fibrotic disease. In addition, epicardial activation, proliferation, and fibrosis occur with ischemic, but not hypertensive injury. Here we review cellular and molecular mechanisms that control epicardium-derived cell lineages during development and disease with a focus on cardiac fibroblasts. This article is part of a special issue entitled “Fibrosis and Myocardial Remodeling”.

The Hippo pathway directs cell differentiation during organogenesis, in part by restricting proliferation. How Hippo signaling maintains a proliferation-differentiation balance in developing tissues via distinct molecular targets is only beginning to be understood. Our study makes the unexpected finding that Hippo suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling in pancreatic progenitors to permit cell differentiation and epithelial morphogenesis. We find that pancreas-specific deletion of the large tumor suppressor kinases 1 and 2 ( Lats1/2 PanKO ) from mouse progenitor epithelia results in failure to differentiate key pancreatic lineages: acinar, ductal, and endocrine. We carried out an unbiased transcriptome analysis to query differentiation defects in Lats1/2 PanKO . This analysis revealed increased expression of NFκB activators, including the pantetheinase vanin1 ( Vnn1 ). Using in vivo and ex vivo studies, we show that VNN1 activates a detrimental cascade of processes in Lats1/2 PanKO epithelium, including (1) NFκB activation and (2) aberrant initiation of epithelial-mesenchymal transition (EMT), which together disrupt normal differentiation. We show that exogenous stimulation of VNN1 or NFκB can trigger this cascade in wild-type (WT) pancreatic progenitors. These findings reveal an unexpected requirement for active suppression of NFκB by LATS1/2 during pancreas development, which restrains a cell-autonomous deleterious transcriptional program and thereby allows epithelial differentiation.