Look Who’s Talking
FGFs and BMPs in the Proepicardium
See related article, pages 431–441
Given the increasing prevalence of patients with congestive heart failure, there is a strong interest in identifying and isolating cardiac progenitor cells capable of differentiating into cardiomyocytes that could be used to repair a damaged, failing heart. Recent work in the zebrafish model system has suggested that the epicardium, the nonmyocyte epithelial layer of cells covering the heart, may play a critical role in the regeneration of injured myocardium.1 The epicardium has a distinct embryological origin from both the myocardium and endocardium. It arises from a mass of mesothelial cells termed the proepicardium, located on the wall of the embryonic pericardial cavity just dorsal and caudal to the developing heart.2 During early embryonic development, cells of the proepicardium migrate onto the heart at the atrioventricular junction and then spread over the surface of the heart to form a primitive epicardium. Some cells of the epicardium then undergo a mesenchymal transformation and invade into the subepicardial space. There these epicardial-derived cells further differentiate into cells that form the coronary vasculature as well as cardiac fibroblasts. Through this multistep differentiation pathway, the proepicardium serves as an important source for the noncardiomyocyte cellular component of the heart.
Over the last few years, the pathways that regulate the development and differentiation of the proepicardium have begun to be revealed. First, the formation of the proepicardium is critically dependent on the transcription factor GATA4 and inductive signals from the developing liver bud.3,4 Once formed, cells of the proepicardium express the transcription factors Tbx18 and Wt-1. They may also become specified to a particular cell fate (ie, fibroblast, smooth muscle cell, endothelial cell) before beginning their migration to the developing heart, as suggested by proepicardial retroviral labeling studies in chick.5 The proepicardium has been receiving increasing attention as recent work has suggested that these cells have the potential to differentiate into cardiomyocytes as well as cells of the epicardium. Indeed, lineage-tracing experiments using cre-lox technology with cre expressed under the control of the Tbx18 or WT-1 gene promoters have suggested that a fraction of these proepicardial cells become cardiomyocytes in the developing heart.6,7 However, this conclusion is somewhat controversial as some cardiomyocytes may express low levels of these markers later in development, complicating the interpretation of these lineage tracing experiments.8
Additional support for the ability of proepicardial cells to differentiate into cardiomyocytes has come from in vitro culture experiments of proepicardial explants. Both bone morphogenetic protein (BMP)-2 and BMP-4 were found to induce cardiomyocyte formation from proepicardial explant cultures.9,10 In contrast, fibroblast growth factor (FGF)-2 was found to promote the differentiation of proepicardial cells along noncardiomyocyte cardiac lineages. Further, FGF-2 also blocks the ability of BMP-2 to induce cardiomyocyte differentiation in these cultures, suggesting an interaction between these two signaling pathways during proepicardial differentiation.
In this issue of Circulation Research, van Wijk et al provide further evidence for the potential of proepicardial cells to differentiate into cardiomyocytes.11 Using vital cell labeling with DiI, they show that cells within a field of the chick proepicardium can become cardiomyocytes in the inflow tract myocardium, supporting the notion that proepicardial cells have cardiomyocyte potential. However, these experiments are unable to determine whether cells within this field are truly bipotential (ie, having both myocardial and epicardial potential), or whether this field of cells contains 2 distinct populations, one with solely myocardial potential and the other with epicardial potential. Further work will be necessary to resolve this question.
van Wijk et al also take an important step forward in our understanding of the interplay of signaling pathways regulating the differentiation of cells within the proepicardium (see the Figure).11 Consistent with previous work, they show that BMP-2 can induce cardiomyocyte differentiation in proepicardial cultures and FGF-2 can block it, but in this report, they extend these observations by defining the downstream signaling pathways activated by each of these ligands. They show that FGF-2 activates a kinase cascade that results in the phosphorylation of extracellular signal-regulated kinase (ERK), whereas BMP-2 signaling is mediated through phosphorylation of SMAD1/5/8. Interestingly, immunohistochemistry of the proepicardium using anti–phospho-ERK or anti–phospho-SMAD antibodies reveals significant heterogeneity within this structure, because some proepicardial cells express phospho-SMADs, others express phospho-ERK, and still others express both. This observation is consistent with the idea that the proepicardium is not a homogenous field of cells and thus may contain cells with distinct developmental potentials.
The ability of FGF-2 to block BMP-induced cardiomyocyte differentiation of proepicardial cells is also explained in this report through the demonstration that activation of FGF signaling in proepicardial cells attenuates BMP-induced SMAD phosphorylation and nuclear localization. This presumably results in a failure to transcriptionally activate SMAD-dependent target genes important for the induction of the cardiomyocyte cell fate. Furthermore, the ability of FGF-2 to block BMP-induced differentiation is mediated through a mitogen-activated protein kinase kinase (MEK)/ERK-dependent pathway as the MEK inhibitor U0126 blocked these effects. Consistent with their in vitro results, van Wijk et al show that treatment of whole chick embryos in ovo with BMP2+U0126 blocked the migration of the proepicardium onto the developing heart tube and enhanced myocardial formation in the venous pole of the heart, whereas treatment with FGF-2 led to enhanced epicardial formation. Together, these results suggest that the there is crosstalk between the FGF and BMP signaling pathways in the differentiation of cells of the proepicardium into myocardial versus nonmyocardial cardiac lineages (see the Figure).
Although this work is a step forward in our understanding of the pathways regulating proepicardial and epicardial development, important questions remain. First, what are the downstream transcriptional targets of FGF and BMP signaling that modulate proepicardial differentiation? Second, what signaling pathways modulate proepicardial lineage decisions leading to a cardiac fibroblast, smooth muscle, or endothelial cell fate? Third, are cells of the proepicardium truly committed to a particular lineage before leaving the proepicardium, or do they receive additional inductive cues on their arrival to the epicardium or subepicardial space? Finally, can mature epicardium be reprogrammed into a proepicardial-like state for potential use in myocardial regeneration strategies? It would not be surprising to find that a number of “conversations” are occurring in the proepicardium to direct these lineage decisions. The challenge for the field will be to identify whom is talking to whom.
Sources of Funding
Supported by NIH grant HL71063.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Ishii Y, Langberg JD, Hurtado R, Lee S, Mikawa T. Induction of proepicardial marker gene expression by the liver bud. Development. 2007; 134: 3627–3637.
Watt AJ, Battle MA, Li J, Duncan SA. GATA4 is essential for formation of the proepicardium and regulates cardiogenesis. Proc Natl Acad Sci U S A. 2004; 101: 12573–12578.
Mikawa T, Fischman DA. Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels. Proc Natl Acad Sci U S A. 1992; 89: 9504–9508.
van Wijk B, van den Berg G, Abu-Issa R, Barnett P, van der Velden S, Schmidt M, Ruijter JM, Kirby ML, Moorman AFM, van den Hoff MJB. Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein– and fibroblast growth factor–signaling pathways. Circ Res. 2009; 105: 431–441.